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MicroRNA-100/99a, deregulated in acute lymphoblastic leukaemia, suppress proliferation and promote apoptosis by regulating the FKBP51 and IGF1R/mTOR signalling pathways

MicroRNA-100/99a, deregulated in acute lymphoblastic leukaemia, suppress proliferation and... FULL PAPER British Journal of Cancer (2013) 109, 2189–2198 | doi: 10.1038/bjc.2013.562 Keywords: acute lymphoblastic leukaemia; miRNA; FKBP51 signalling; IGF1R/mTOR signalling pathway; apoptosis; proliferation MicroRNA-100/99a, deregulated in acute lymphoblastic leukaemia, suppress proliferation and promote apoptosis by regulating the FKBP51 and IGF1R/mTOR signalling pathways 1,3 2,3 1 1 1 ,1 X-J Li , X-Q Luo , B-W Han , F-T Duan , P-P Wei and Y-Q Chen Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, School of Life Science, Sun Yat-sen University, Guangzhou 510275, China and The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China Background: MicroRNAs alter multiple cell processes and thus influence tumour carcinogenesis and progression. MiR-100 and miR-99a have been reported to be aberrantly expressed in acute leukaemia. In this study, we focused on their functions in acute lymphoblastic leukaemia (ALL) and the molecular networks in which they are involved. Methods: MiR-100 and miR-99a expression levels were measured in acute leukaemia patients by qRT–PCR. Kaplan  Meier analysis and log-rank tests were used to calculate the survival rate. Three human ALL cell lines were studied. Apoptosis and proliferation were analysed using siRNA transfection, western blot and flow cytometry. Results: In vivo, miR-100 and miR-99a were down-regulated in 111 ALL patients, especially in high-risk groups; their expression levels were correlated with the patient’s 5-year survival. In vitro, the restoration of miR-100 and miR-99a in ALL cells suppressed cell proliferation and increased dexamethasone-induced cell apoptosis. Ectopic expression of miR-100 and miR-99a targeted FK506- binding protein 51 (FKBP51) and, in turn, influenced glucocorticoid receptor (GR) activity. Meanwhile, miR-100 and miR-99a overexpression inhibited the expression of IGF1R and mTOR and their downstream oncogene MCL1. Conclusion: MiR-100 and miR-99a have critical roles in altering cellular processes by targeting both the FKBP51 and IGF1R/mTOR signalling pathways in vitro and might represent a potential novel strategy for ALL treatment. Childhood acute lymphoblastic leukaemia (ALL) is a commonly should be elucidated. In patients with ALL, several cell signalling occurring malignancy characterised by the uncontrolled prolifera- pathways that control cancer occurrence and development have tion of immature lymphoid cells (Pui et al, 2004). Although been shown to be altered (Weng et al, 2004; Hirsch and Chiarle, treatment has improved remarkably over the past decade, B10% 2012); however, the specific mechanisms responsible for the of ALL patients fail to reach long-term remission (Kraszewska et al, development and treatment of this disease remain unknown. 2012). To better understand this disease, the intricate regulatory Hence, the discovery of more important molecules might further networks among molecular interactions and signalling pathways elucidate the networks and facilitate new therapeutic strategies. In *Correspondence: Dr Y-Q Chen; E-mail: lsscyq@mail.sysu.edu.cn These authors contributed equally to this work. Received 5 July 2013; revised 19 August 2013; accepted 22 August 2013; published online 12 September 2013 & 2013 Cancer Research UK. All rights reserved 0007 – 0920/13 www.bjcancer.com | DOI:10.1038/bjc.2013.562 2189 BRITISH JOURNAL OF CANCER MiR-100/99a in acute lymphoblastic leukaemia the past decade, aberrant microRNA (miRNA) expression has been patients, and the study was approved by the Ethics Committee of shown to have an essential role in the pathogenesis of leukaemia, the First Affiliated Hospital of Sun Yat-sen University. and miRNAs have been considered as potential therapeutic targets Cell culture and cell treatment. The ALL cell lines, CCRF-CEM, for the disease (Fernando et al, 2012). CEM/C1, and Jurkat, and the HEK-293T cell line were purchased MicroRNAs are 20–22 nucleotides long and exert their from American Type Culture Collection (ATCC, Manassas, VA, regulatory effects by directly binding to the 3 untranslated regions USA). All cell lines were grown with 10% fetal bovine serum (UTRs) of their target genes, thus repressing the target gene at the (HyClone, Logan, UT, USA). Rapamycin (Beyotime, Haimen, level of translation (Esquela-Kerscher and Slack, 2006). Recent China) was dissolved in dimethyl sulfoxide (Sigma, St Louis, MO, studies have demonstrated that miRNAs have important roles in USA) and used at a concentration of 10-nM. Dexamethasone cancer pathogenesis and development, and subsequently, in (Sigma) was dissolved in ethanol and used at concentrations of treatment and prognosis (Hwang and Mendell, 2006). In ALL, 1-mM and 100-nM. The final concentrations of dimethyl sulfoxide miR-125b has an important role in early hematopoietic develop- and ethanol in the culture media were 0.05% and 0.1%, ment (Shaham et al, 2012); miR-124a contributes to the abnormal respectively. proliferation of ALL cells (Agirre et al, 2009), and knockdown of miR-17-92 clusters has been shown to increase glucocorticoid- RNA isolation and real-time quantitative RT  PCR. Total RNA mediated apoptosis in ALL cell lines (Molitoris et al, 2011). was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, MiR-100 and miR-99a belong to the same family and have USA) according to the manufacturer’s guidelines. Real-time PCR similar roles in a variety of cancers (Doghman et al, 2010; Mueller was performed to quantify mRNA expression using ExTaq SYBR et al, 2012; Chen et al, 2012b). Our previous study showed that Green Supermix (Bio-Rad, Hercules, CA, USA) according to the both miR-100 and miR-99a are up-regulated in childhood acute manufacturer’s instructions (details are provided in the supporting myeloid leukaemia (AML) and that overexpression of miR-100 information, Supplementary Table S2). arrested the differentiation of human granulocyte and monocyte Proliferation and apoptosis assays. Cell proliferation was cells and promoted cell survival (Zhang et al, 2009; Zheng et al, assessed using the Cell Counting Kit-8 (CCK-8). After transfection, 2012). In contrast, miR-100 and miR-99a have very low expression 1*10 cells per well were plated in 96-well sterile plastic culture levels in ALL patients (Zhang et al, 2009), suggesting that these two plates, and the CCK-8 assay (Dojindo Molecular Technologies, miRNAs have different roles in myeloid cell and lymphocyte Shanghai, China) was performed after 0, 24, 48, 72, and 96 h. To pathogenesis. However, the molecular basis of how miR-100 and assess the rate of apoptosis, transfected cells were harvested and miR-99a are involved in lymphoblastic leukaemogenesis and washed twice with cold PBS, and the Annexin V-PI Kit (Nanjing progression has not yet been elucidated. Keygen, Nanjing, China) was used according to the manufacturer’s Based on a bioinformatics analysis, FK506-binding protein 51 guidelines. The detection was performed with a FACS Calibur (FKBP51) was predicted to be a target of miR-100 and miR-99a using CellQuest software (BDIS, San Jose, CA, USA). (Bhushan and Kandpal, 2011). The evidence collected to date shows that FKBP51 is an immunophilin that is physiologically Transient transfection. For the luciferase reporter assay, HEK- expressed in lymphocytes and is involved in sustaining cell growth, 293T cells were transfected with Lipofectamine 2000 (Invitrogen) malignancy, and resistance to therapy (Li et al, 2011; Schmidt et al, following the manufacturer’s protocol. CCRF-CEM, CEM/C1, and 2012). Furthermore, IGF1R and mTOR are mutual targets of miR- Jurkat cells were transfected with the small molecular RNAs using 100 and miR-99a in several cancer cells (Doghman et al, 2010; the Neon Transfection System (Invitrogen) according to the Lerman et al, 2011; Sun et al, 2011), and they have been implicated manufacturer’s guidelines (details are provided in the Supporting in the initiation and progression of several malignant neoplasms Information Online, Supplementary Materials and Methods). (Petroulakis et al, 2006), including ALL (Cardoso et al, 2009; Dual luciferase activity assay. Luciferase constructs were estab- Medyouf et al, 2011). Thus, we hypothesised that FKBP51, IGF1R, lished by inserting oligonucleotides containing the wild-type or and mTOR are involved in the functioning of miR-100 and miR-99a mutant putative target site of the FKBP51 3 -UTR into the multi- in ALL and serve as their targets. cloning site of the psiCHECK2 vector (Promega, Fitchburg, WI, In this study, we found that miR-100 and miR-99a were down- USA) (see Supporting Information Online, Supplementary regulated in childhood ALL patients and that their expression Materials and Methods for more details). levels were related to the prognosis of the ALL patients. In vitro investigation further demonstrated that miR-100 and miR-99a Western blot assay. Western blot analyses were performed as participated in the regulation of cell proliferation and dexametha- described previously (Zheng et al,2012).Nuclear–cytoplasmic sone (Dex)-induced apoptosis in ALL cell lines. Our data revealed fractionation was conducted using the NE-PER Nuclear and that FKBP51 is a novel target of miR-100 and miR-99a and, in Cytoplasmic Extraction Reagents kit (Pierce, Rockford, IL, USA) turn, influences its downstream gene glucocorticoid receptor (GR). according to the manufacturer’s protocol. Transfected cells were We also proved that miR-100 and miR-99a overexpression assayed with anti-FKBP51 (Epitomics, Zhejiang, China); anti-H3 inhibited IGF1R and mTOR and down-regulated MCL1. The (Abcam, Cambridge, UK); anti-MCL1 and anti-GR (Santa Cruz, results of the present study suggest that miR-100 and miR-99a act Paso Robles, CA, USA); and anti-IGF1R, anti-mTOR, anti-phospho- as tumour suppressors and that their restoration might be a mTOR (Ser-2448), anti-phospho-GR (Ser-211), and anti-GAPDH possible therapeutic strategy for patients with ALL. (Cell Signalling Technology, Boston, MA, USA). After a 1-h incubation with anti-rabbit (Sigma-Aldrich) HRP-conjugated sec- ondary antibody, the protein level was detected using a luminal reagent (Millipore). X-ray films were acquired and quantified with MATERIALS AND METHODS the densitometric software Quantity One (Bio-Rad). The value under each sample indicates the fold change of protein amounts that Patients. This study included 71 AML patients, 111 ALL patients, normalised with the corresponding GAPDH or H3 in each sample. and 10 healthy donors from the First Affiliated Hospital of Sun Yat-sen University. Patient demographics are presented in Bioinformatic and statistical analyses. Online miRNA databases supporting information Supplementary Table S1. Bone marrow (TargetScan and PicTarget) were used to predict the miR-100 and was obtained from patients and healthy donors. Informed consent miR-99a target genes. SPSS13.0 software (Chicago, IL, USA) was to perform the biological studies was obtained from all of the used for statistical analysis. Data are expressed as the mean s.d. 2190 www.bjcancer.com | DOI:10.1038/bjc.2013.562 MiR-100/99a in acute lymphoblastic leukaemia BRITISH JOURNAL OF CANCER The differences between two groups were analysed using the expression levels of miR-100 and miR-99a in both childhood ALL Mann Whitney U test for two groups or the Kruskal Wallis test and AML patients at diagnosis using qRT  PCR. As shown in when more than two groups were compared. All tests were two- Figure 1A, both miRNAs showed significantly different expression sided. The Kruskal Wallis test was implemented for the patterns between ALL and AML compared with healthy donors. comparison of three groups, and multiple comparisons were MiR-100 and miR-99 showed very low expression levels in most performed using one-way ANOVA and a least significant difference ALL patients compared with AML patients and healthy donors. t test after the relative concentration was ranked. Differences were We next analysed their correlation with the patient characteristics. considered significant at *Po0.05, **Po0.01, and ***Po0.001. Although the expression levels of miR-100 and miR-99a had no correlation with sex, age, or therapy (data not shown), we found that the expression levels of these two miRNAs were significantly RESULTS correlated with patients who carried known high-risk prognostic factors, including white blood cell (WBC) count, ALL sub- classification (here, indicated as T-cell and B-cell ALL), the MiR-100 and miR-99a are associated with specific risk groups MLL-rearranged gene and the BCR-ABL fusion gene. As shown and survival in childhood ALL. To investigate the link between 4  3 in Figure 1B, a higher WBC count (45  10 mm ) before miR-100 and miR-99a and childhood ALL, we first measured the A miR-100 3 miR-99a B miR-100 miR-99a 2 2 *** 10 10 4 P<0.0001 ** P<0.0001 *** *** 2 *** *** 10 0 0 10 –1 10 10 –1 –1 –1 10 –2 –2 –3 –2 –3 –2 10 10 10 10 3 3 3 3 Control AML ALL Control AML ALL WBC >50 000mm <50 000mm WBC >50 000mm <50 000mm C miR-100 miR-99a D miR-100 miR-99a 10 3 10 3 P=0.0004 P=0.0004 10 1 * 2 ** 10 10 0 10 –1 –1 –1 10 10 –1 –2 10 –2 –2 10 10 –2 –3 –3 –3 10 10 10 T-ALL B-ALL T-ALL B-ALL WT MLL BCR-ABL WT MLL BCR-ABL miR-100 miR-99a miR-100 miR-99a E 100 100 100 100 80 80 80 80 60 60 60 60 40 40 40 20 20 20 20 Low miR-100 Low miR-99a Low miR-100 Low miR-99a P=0.01266 P=0.0054 P=0.0466 P=0.0233 High miR-100 High miR-99a High miR-100 High miR-99a 0 0 0 0 020 40 60 020 40 60 020 40 60 020 40 60 Time (months) Time (months) Time (months) Time (months) Figure 1. MiR-100 and miR-99a are associated with specific risk groups and the survival of patients with childhood ALL. (A) The expression levels of miR-100 (left) and miR-99a (right) were quantified by real-time PCR in 111 ALL patients, 71 AML patients and 10 healthy donors. MiR-100 and miR-99a were significantly down-regulated in ALL patients compared with AML patients (Po0.0001 and Po0.0001, respectively) and healthy donors (P ¼ 0.001 and P ¼ 0.0086, respectively). (B) miR-100 (left) and miR-99a (right) expression levels, determined by qRT  PCR, in ALL patients 4  3 4  3 with more than 5  10 mm white blood cells (WBCs) or less than 5  10 mm WBCs at diagnosis (Po0.0001 and Po0.0001, respectively). (C) Comparison of the expression of miR-100 (left) and miR-99a (right) between B-ALL (n ¼ 93) and T-ALL (n ¼ 18) patients by qRT–PCR (P ¼ 0.0004 and P ¼ 0.0004, respectively). (D) The expression levels of miR-100 (left) and miR-99a (right) were significantly down-regulated in ALL patients with the MLL-rearranged gene (n ¼ 22) and the BCR-ABL fusion gene (n ¼ 11), in comparison with the patients without fusion genes (n ¼ 78) (Po0.001 and Po0.05, respectively, for miR-99a, and Po0.05 and Po0.05, respectively, for miR-99a). Wild type (WT): patients without these two fusion genes. (E) The OS of patients with high miR-100 and miR-99a expression levels was significantly higher than that of patients with low miR-100 and miR-99a expression levels (P¼ 0.0466 and P¼ 0.0126, respectively). (F) The leukaemia-free survival (LFS) of patients with high miR-100 and miR- 99a expression levels was significantly higher than that of patients with low miR-100 and miR-99a expression levels (P¼ 0.0054 and P ¼ 0.0233, respectively). The Kruskal  Wallis test was implemented for the comparison of three groups, and multiple comparisons were performed using one-way ANOVA and a least significant difference t test after the relative concentration was ranked. A Kaplan  Meier analysis and the log-rank test were used to analyse OS and LFS. *Po0.05, **Po0.01, and ***Po0.001 vs control or WT. www.bjcancer.com | DOI:10.1038/bjc.2013.562 2191 Relative miRNA expression Relative miRNA expression Overall survival Overall survival Relative miRNA expression Relative miRNA expression Leukemia-free survival Relative miRNA expression Relative miRNA expression Leukemia-free survival Relative miRNA expression Relative miRNA expression BRITISH JOURNAL OF CANCER MiR-100/99a in acute lymphoblastic leukaemia chemotherapy was observed in patients with lower expression that miR-100 and miR-99a are associated with specific risk groups levels of miR-100 and miR-99a. Notably, all T-ALL cases showed within childhood ALL. low expression levels of miR-100 and miR-99a compared with Furthermore, the association of miR-100 and miR-99a expres- B-ALL patients (Figure 1C). We also found that patients carrying sion with the prognosis of ALL patients was investigated using the MLL-rearranged or BCR-ABL fusion gene, two crucial Kaplan  Meier analysis and a log-rank test. As shown in biological mutations associated with poor outcome, expressed Figure 1E, the 5-year overall survival (OS) of patients with high lower levels of miR-100 and miR-99 compared with patients miR-100 and miR-99a expression levels was significantly higher without these two fusion genes (Figure 1D). These results suggest than in patients with low miR-100 and miR-99a expression levels. 0.8 Control miRNA 2.0 1.5 Control miRNA Control miRNA miR-100-mimics miR-100-mimics miR-100-mimics 0.6 miR-99a-mimics * * 1.5 * * miR-99a-mimics miR-99a-mimics * * * * * * 1.0 * * * * * * 0.4 1.0 0.5 0.2 0.5 CEM/C1 CCRF-CEM Jurkat 0.0 0.0 0.0 0h 24h 48h 72h 96h 0h 24h 48h 72h 96h 0h 24h 48h 72h 96h CCRF-CEM CCRF-CEM Control miRNA miR-100-mimics miR-99a-mimics *** 26.9% 23.1% 15.7% *** 10 25 3.62% 6.02% 10 6.44% 15 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Control miRNA miR-100-mimics miR-99a-mimics Annexin Annexin Annexin CEM/C1 CEM/C1 miR-100-mimics Control miRNA miR-99a-mimics 7.90% 18.2% 16.6% *** *** 0 9.40% 15.2% 14.2% 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Control miRNA miR-100-mimics miR-99a-mimics Annexin Annexin Annexin Jurkat Jurkat miR-100-mimics miR-99a-mimics Control miRNA *** 61.0% 34.3% 69.8% *** 10 70 1 30 5.45% 12.0% 9.11% 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Control miRNA miR-100-mimics miR-99a-mimics Annexin Annexin Annexin Figure 2. MiR-100 and miR-99a inhibit cell proliferation and promote Dex-induced apoptosis in ALL cell lines and function as potential tumour suppressors. (A) Transfection of miR-100 and miR-99a slowed ALL cell proliferation. The time points after transfection with control miRNA, miR-100, or miR-99a are indicated. MiR-100 and miR-99a significantly inhibited cell proliferation in CCRF-CEM (left), CEM/C1 (middle), and Jurkat (right) cells, as determined using the CCK-8 assay. (B–D) miR-100 and miR-99a significantly promoted Dex-induced cell apoptosis. CCRF-CEM (B), CEM/C1 (C), and Jurkat (D) cells were transfected with miR-100, miR-99a, or control miRNA and cultured for 24 h; the three cell lines were exposed to 1-mM Dex for 8, 18, and 24 h, respectively. Cells were stained with PI/Annexin V and analysed by flow cytometry. A representative experiment is shown. **Po0.01 and ***Po0.001 vs control. Values are presented as the mean s.d. of at least three independent experiments performed in triplicate. 2192 www.bjcancer.com | DOI:10.1038/bjc.2013.562 OD value (450 nM/630 nM) PI PI PI OD value (450 nM/630 nM) Apoptosis (%) Apoptosis (%) Apoptosis (%) OD value (450 nM/630 nM) MiR-100/99a in acute lymphoblastic leukaemia BRITISH JOURNAL OF CANCER Moreover, a trend towards increased leukaemia-free survival was We further studied the effect of miR-100 and miR-99a on Dex- observed in patients with ALL and up-regulated miR-100 and miR-99a induced ALL cell apoptosis. After transfection of miR-100 and expression levels over the 5-year follow-up period (Figure 1F). miR-99a mimics for 24 h, cells were treated with 1-mM Dex, a Taken together, these results suggest that higher expression levels therapeutic drug that is widely used to treat ALL by triggering of miR-100 and miR-99a were associated with better survival and leukaemic cell apoptosis. Overexpression of miR-100 and miR-99a prognosis. increased Dex-induced apoptosis compared with control miRNA in CCRF-CEM, CEM/C1, and Jurkat cells (Figure 2B–D). These data suggest that the ectopic expression of miR-100 and miR-99a MiR-100 and miR-99a inhibit cell proliferation and promote inhibits cell proliferation and promotes the response to Dex- Dex-induced apoptosis in ALL cell lines and function as induced apoptosis in ALL cells. potential tumour suppressors. The observations described above suggest that both miR-100 and miR-99a are clinically relevant and FKBP51 is a novel target of miR-100 and miR-99a. To might affect leukaemic cell progression. To explore the role of miR- determine the molecular mechanism by which miR-100 and 100 and miR-99a, we investigated their influences on leukaemic miR-99a are involved in ALL pathogenesis, we further investigated cell proliferation and cell apoptosis. Three ALL cell lines, CCRF- their downstream targets. Among the nearly 100 predicted targets, CEM, CEM/C1, and Jurkat, were used to perform the in vitro FKBP51 was chosen for further investigation because this gene has functional analysis. A qRT  PCR assay was used to measure the an active role in cancer aetiology and responds to antineoplastic transfection efficiency of the miRNAs (supporting information, therapy (Baughman et al, 1995; Febbo et al, 2005; Pei et al, 2009). Supplementary Figure S1). The results of a CCK-8 assay indicated As shown in Figure 3A, to test whether FKBP51 is directly that overexpression of miR-100 and miR-99a inhibited cell regulated by miR-100 and miR-99a, a dual-luciferase assay was proliferation of all three ALL cell lines in a time-dependent performed, and the 3 UTR fragment, which was cloned into a manner (Figure 2A). reporter plasmid, was inserted into the luciferase open reading AB 3′ 3′-FKBP51-Wt 5′ 1.4 1.2 hsa-miR-100 3′ 5′ *** *** 3′-FKBP51-Wt 5′ 3′ 0.8 Control miRNA 3′ 5′ hsa-miR-99a miR-100-mimics 0.6 3′-FKBP51-Mut 5′ 3′ miR-99a-mimics 0.4 3′-FKBP51-Wt Renilla luciferase 0.2 Renilla luciferase 3′-FKBP51-Wt FKBP51 FKBP51 pSi-Check2 point Mu Wt CD 0 0 Control miR-100 miR-99a Control Anti-miR Anti-miR miRNA -mimics -mimics miRNA -100 -99a Control miR-100 miR-99a Control Anti-miR Anti-miR miRNA -mimics -mimics miRNA -100 -99a FKBP51 FKBP51 51 KDa 51 KDa 1.0 1.7 2.4 1.0 0.3 0.3 36 KDa GAPDH GAPDH 36 KDa Figure 3. MiR-100 and miR-99a target FKBP51. (A) Schematic representation of the constructs used in the luciferase assay.The sequences shown below indicate the putative miR-100 and miR-99a target site on the wild-type 3 UTR (construct FKBP51-wt), its mutated derivative (construct FKBP51-mut), and the pairing regions of miR-100 and miR-99a. (B) 293T cells were co-transfected with pSi-Check2 with either FKBP51-wt or FKBP51-mut and with miR-100, miR-99a, or empty vector (pCD6.2). Repression of luciferase activity due to the interaction between miR-100, miR-99a, and the luciferase constructs. Each Renilla luciferase reading was normalised to that obtained for the control firefly luciferase. (C, D) Jurkat cells were electroporated with 100-nmol mimics-NC (control miRNA), mimics-miR-100, or miR-99a and anti-scramble (control miRNA), anti-miR-100, or anti-miR-99a. The levels of miR-100 and miR-99a were assessed by qRT  PCR. Cell lysates were prepared for western blotting with the antibody against FKBP51, and the expression of GAPDH served as a loading control. Western blot figures are representative of at least three independent experiments. The value under each sample indicates the fold change of FKBP51 protein levels relative to that of the control. ***Po0.001. Values are presented as the mean s.d. of at least three independent experiments performed in triplicate. www.bjcancer.com | DOI:10.1038/bjc.2013.562 2193 Relative expression of miRNA Relative expression of miRNA Relative luciferase activity (%) BRITISH JOURNAL OF CANCER MiR-100/99a in acute lymphoblastic leukaemia si-FKBP51 Control si-RNA AB FKBP51 1.0 0 CEM/C1 2.0 Control si-RNA si-FKBP51 35 GAPDH 10 *** 5.63% 9.63% Control siRNA 1.5 3 si-FKBP51 10 25 1.0 10 10 0.5 6.42% 20.0% CEM/C1 10 0 1 2 3 4 0 1 2 3 4 0.0 10 10 10 10 10 10 10 10 10 10 Control si-RNA si-FKBP51 0h 24h 48h 72h 96h Annexin Annexin CD FKBP51 51 KDa FKBP51 51 KDa 51 KDa FKBP51 51 KDa FKBP51 1.0 0.6 0.7 1.0 0.7 0.6 1.0 0.7 0.7 1.0 GR GR 95 KDa 95 KDa GR GR 95 KDa 95 KDa 1.0 1.7 1.0 0.9 1.1 1.0 1.5 2.3 1.0 1.0 p-GR p-GR 95 KDa p-GR p-GR 95 KDa 95 KDa 95 KDa 1.0 1.1 0.8 1.0 2.2 5.4 1.0 2.6 1.0 1.1 H3 GAPDH 15 KDa GAPDH H3 36 KDa 36 KDa 36 KDa Cytoplasm Nucleus Nucleus Cytoplasm Figure 4. MiR-100 and miR-99a affected cell proliferation and the response to Dex by targeting FKBP51 and, in turn, influenced the activity of steroid receptor GR. (A) Transfecting CEM/C1 cells with si-FKBP51 significantly inhibited cell proliferation. Cell viability was determined using the CCK-8 assay. The time points after transfection with control siRNA or si-FKBP51 are indicated. The western blot analysis of the effect of si-FKBP51 is shown in the upper-right corner. The value under each sample indicates the fold change of FKBP51 protein levels relative to that of the control. (B)FKBP51 knockdown enhanced Dex-induced apoptosis. CEM/C1 cells were transfected with control siRNA or si-FKBP51 24 h after transfection, which was followed by 1-mM Dex treatment for 18 h. Control siRNA served as a negative control. Then, the cells were collected for apoptosis analysis by PI/Annexin V staining and flow cytometry. A representative experiment is shown. (C) FKBP51 knockdown promoted the expression of GR and phosphorylated forms of GR in the nucleus. After 48 h of transfection with control siRNA or si-FKBP51, cells were exposed to 100-nM Dex for 24 h, and then cytoplasm and nucleus extracts were prepared. Equal amounts of protein from each fraction were analysed by immunoblotting with anti-FKBP51, anti-GR (total GR protein), and anti-phospho-Ser211 (p-GR), using GAPDH as an internal control for the cytoplasm fraction and H3 for the nucleus fraction. The value under each sample indicates the fold changes of FKBP51, GR, and p-GR protein levels relative to that of the control. (D)miR-100 and miR-99a overexpression induced the expression of GR and p-GR by down-regulating FKBP51 in the nucleus. After 48 h of transfection with control miRNA, miR-100, or miR-99a, cells were exposed to 100-nM Dex for 24 h, and then cytoplasm and nucleus extracts were prepared to detect the protein levels of FKBP51, GR, p-GR, and GAPDH in the cytoplasm fraction and of H3 in the nucleus fraction. Western blot figures are representative of at least three independent experiments. ***Po0.001 vs control. Values are presented as the mean s.d. of at least three independent experiments. Figure 5. MiR-100/99a target the IGF1R/mTOR pathways in ALL. (A) IGF1R and mTOR are direct targets of miR-100 and miR-99a. Jurkat cells were electroporated with 100-nmol mimics-NC, miR-100-mimics, or miR-99a-mimics and 100 nmol Control miRNA, anti-miR-100, or anti-miR-99a. Cell lysates were prepared for western blotting with an antibody against IGF1R and mTOR, and the expression of GAPDH served as a loading control. The value under each sample indicates the fold changes of IGF1R and mTOR protein levels relative to that of the control. (B) CEM/C1 cell proliferation was significantly inhibited, and Dex-induced apoptosis (C) was significantly enhanced after the transfection of si-IGF1R and si-mTOR, as determined using the CCK-8 assay and flow cytometry. For flow cytometry, the apoptotic cells were stained by PI/Annexin V 18 h after exposure to 1 mM Dex. Control siRNA served as a negative control. A representative experiment is shown. The western blot analysis of the transfection efficiency of si-IGF1R and si-mTOR in CEM/C1 cells is shown in the upper-right corner of (B). (D) Western blot analysis of the expression level of mTOR and MCL1 in Jurkat cells following treatment with vehicle, 1-mM Dex, 10-nM Rap, or 1-mM Dex plus 10-nM Rap for 24 h. The value under each sample indicates the fold changes of mTOR and MCL1 protein levels relative to that of the control. The expression levels of miR-100 and miR-99a were detected after the treatment described above. These two miRNAs were significantly up-regulated after the combined treatment of Dex and Rap. (E) Both overexpression of miR-100/99a and knockdown of mTOR reduced the expression of MCL1. At 48 h after the transfection, Jurkat cells were treated with 1 mM Dex for 24 h, as determined by Western blot analysis. The value under each sample indicates the fold changes of mTOR and MCL1 protein levels relative to that of the control. (F) IGF1R knockdown promoted the phosphorylation of mTOR and, in turn, repressed the expression of MCL1. At 48 h after transfection, Jurkat cells were exposed to 1-mM Dex for 24 h, and the expression of IGF1R, p-mTOR (Ser-2448), and MCL1 was detected by western blot analysis. The value under each sample indicates the fold changes of IGF1R, p-mTOR, mTOR, and MCL1 protein levels relative to that of the control. (G) CEM/C1 cell proliferation was significantly inhibited, and (H) Dex-induced apoptosis was significantly enhanced after the transfection of si-MCL1. Control si-RNA served as a negative control. A representative experiment is shown. (I) Schematic shows the function of miR-100 and miR-99a in cell proliferation and cell apoptosis in ALL. (i) Upon post-transcriptional suppression, FKBP51 was inhibited by miR-100 and miR-99a, thus restraining cell proliferation and activating the GR at the phosphorylation site of Ser-211, which in turn led to apoptosis. (ii) Through inhibition of mTOR, miR-100 and miR-99a suppressed the expression of MCL1, which resulted in the blockade of cell proliferation and led to cell apoptosis. By targeting IGF1R, miR-100 and miR-99a triggered cell apoptosis and inhibited cell proliferation. In the meantime, down-regulated expression of IGF1R inhibited the activity of mTOR (phosphorylation at the site of Ser-2448), which also led to the suppression of the mTOR/MCL1 signalling pathway. Western blot figures are representative of at least three independent experiments. ***Po0.001 vs Control. Values are presented as the mean s.d. of at least three independent experiments. 2194 www.bjcancer.com | DOI:10.1038/bjc.2013.562 Control si-RNA si-FKBP51 Control si-RNA si-FKBP51 Control miRNA miR-100-mimics miR-99a-mimics Control miRNA miR-100-mimics miR-99a-mimics OD value (450 nM/630 nM) PI Apoptosis (%) *** *** MiR-100/99a in acute lymphoblastic leukaemia BRITISH JOURNAL OF CANCER frame. The interaction between the miRNAs and the target mRNA MiR-100 and miR-99a affect cell proliferation and the response sequences was further validated using a point mutation of the to Dex by targeting FKBP51 and, in turn, influencing the putative miRNA-binding site in the 3 UTR of the FKBP51 gene. activity of steroid receptor GR. Although FKBP51 has important We found that the luciferase activity of the 3 UTR of FKBP51 was roles in neoplastic diseases (Rees-Unwin et al, 2007), the function reduced nearly 50% and 70% after transfection of both miR-100 of FKBP51 in leukaemia has not yet been elucidated. To explore and miR-99a; when the predicted target sites were mutated, the the function of FKBP51, we used RNAi to investigate the role of luciferase activity was unaffected (Figure 3B). this gene in cell proliferation and apoptosis. As shown in the To further confirm that the FKBP51 protein is suppressed by upper-right corner of Figure 4A, FKBP51 expression was miR-100 and miR-99a, we performed both miR-100/99a over- remarkably reduced after the silencing of FKBP51 in CEM/C1 expression and knockdown experiments in Jurkat cells and cells. FKBP51 knockdown inhibited cell proliferation after 4 days examined FKBP51 expression. As shown in Figure 3C and D, of incubation using a CCK-8 assay (Figure 4A) and led to a the level of FKBP51 protein was significantly reduced in Jurkat significant increase in Dex-induced apoptosis in CEM/C1 cells by cells that were transfected with the miR-100 and miR-99a mimics, flow cytometry analysis (Figure 4B). Similar results were obtained whereas it was increased in cells that were transfected with the in CCRF-CEM and Jurkat cell lines (Supplementary Figure S2). antisense of miR-100 and miR-99a. These data indicate that These results show that FKBP51 knockdown has a positive effect, FKBP51 is a bona fide target of miR-100 and miR-99a in similar to that of miR-100 and miR-99a (Figure 2), suggesting that ALL cells. miR-100 and miR-99a exert their functions of inhibiting cell AB C Control miRNA miR-100 miR-99a Control si-RNA si-IGF1R Control si-RNA si-mTOR IGF1R 95 KDa IGF1R mTOR CEM/C1 1.0 0.4 0.3 1.0 0.5 1.0 0.5 mTOR GAPDH GAPDH ** * 289 KDa ** * 1.0 0.5 0.3 2.0 Control siRNA GAPDH 36 KDa 20 si-IGF1R 1.5 Control miRNA Anti-miR-100 Anti-miR-99a 15 si-mTOR IGF1R 1.0 95 KDa 10 1.0 3.0 3.7 5 0.5 mTOR 289 KDa CEM/C1 0.0 Control si-RNA si-IGF1R si-mTOR 1.0 12.7 9.4 0h 24h 48h 72h 96h GAPDH 36 KDa D E G Control Dex Rap R+D 289 KDa mTOR Control si-RNA si-MCL1 1.0 1.4 0.9 0.5 MCL1 289 KDa 42 KDa mTOR MCL1 1.0 0.1 1.0 0.4 1.0 0.8 0.2 0.02 1.0 0.6 0.4 GAPDH MCL1 42 KDa GAPDH 36 KDa 1.0 0.5 0.4 2.0 1.0 0.3 GAPDH 36 KDa 4.5 Control siRNA miR-100 *** 4 si-MCL1 miR-99a 1.5 *** 3.5 Control si-RNA si-IGF1R * 95 KDa IGF1R 1.0 2.5 1.0 0.4 289 KDa p-mTOR 0.5 1.5 1.0 0.5 CEM/C1 289 KDa mTOR 0.5 1.0 0.9 0.0 0 0h 24h 48h 72h 96h 42 KDa MCL1 Control Rapa Dex R+D 1.0 0.4 36 KDa GAPDH miR-100 miR-99a CEM/C1 Control si-RNA si-MCL1 4 P-Ser *** FKBP51 5.63% 16.2% IGF1R mTOR ii P-Ser Cell proliferation 10 GR MCL1 6.42% 10.9% Cell apoptosis 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 Control si-RNA si-MCL1 Annexin Annexin Cell apoptosis Cell proliferation www.bjcancer.com | DOI:10.1038/bjc.2013.562 2195 Control miRNA miR-100 miR-99a Control si-RNA si-mTOR Relative expression of miRNA PI OD value (450 nM/630 nM) Apoptosis (%) Apoptosis (%) OD value (450 nM/630 nM) BRITISH JOURNAL OF CANCER MiR-100/99a in acute lymphoblastic leukaemia proliferation and promoting Dex-induced cell apoptosis by with rapamycin and Dex and found significant decreases in both reducing FKBP51 expression. protein levels after exposure to both drugs. In addition, we found Glucocorticoid receptor is an essential transcription factor that that the expression levels of miR-100 and miR-99a were is involved in cellular proliferation, inflammatory responses, and significantly up-regulated after treatment with both rapamycin cell differentiation (Rogatsky and Ivashkiv, 2006; Chen et al, and Dex (Figure 5D). Therefore, miR-100 and miR-99a might 2012a), and FKBP51 is a potent inhibitor of GR activity on regulate the expression of MCL1 by suppressing mTOR. We hormone induction and influences GR nuclear translocation determined that the protein expression of MCL1 was down- (Ratajczak et al, 2003; Denny et al, 2005). Because the presence regulated in Jurkat cells with miR-100 and miR-99a overexpression of FKBP51 reflected a decreased amount of GR accumulation in and with mTOR knockdown, following the exposure of 1-mM Dex the nucleus (Wochnik et al, 2005; Binder, 2009) and phosphoryla- for 24 h (Figure 5E). Furthermore, the expression of MCL1 was tion at Ser-211 is the monophosphorylated version of the also down-regulated after the knockdown of IGF1R through the hormone-activated form of GR that accumulates in the nucleus inhibition of activated mTOR (with phosphorylation at the (Wang et al, 2002; Chen et al, 2008), we tested whether knockdown site of ser-2448) followed by the exposure to Dex (Figure 5F) of FKBP51 expression influenced the subcellular localisation of GR because mTOR is a downstream gene of IGF1R (Eroles et al, 2012). and Ser211-phosphorylated forms of GR in Jurkat cells. To address To elucidate the function of MCL1 in the miR-100- and this issue, cytosolic and nuclear extracts were prepared from miR-99a-mediated signalling pathway, we investigated the roles FKBP51-silenced and control cells following exposure to 100-nM of MCL1 in cell apoptosis and cell growth. As shown in Figure 5G Dex for 24 h. As shown in Figure 4C, after the silence of FKBP51, and H, after silencing MCL1, its influences on cell proliferation and the phosphor-GR (Ser-211) expression was increased in the cell apoptosis were similar to the findings obtained in the miR-100 nucleus by western blotting using H3 protein as an internal and miR-99a studies and further confirmed our hypothesis that control, accompanied by a slight increase of total GR expression miR-100 and miR-99a are important regulators of MCL1 in ALL. level. However, the relative levels of total GR and phosphor-GR (Ser-211) expression were unaffected in the cytosolic fraction. Furthermore, we asked whether miR-100 and miR-99a affected DISCUSSION this downstream target of FKBP51. In the nuclear fractions of miR-100/99a overexpressed cells, we observed increases in GR and phosphor-GR (Ser-211) expression, with down-regulation of Human miR-100 and miR-99a, which are encoded on different FKBP51 levels, while the expression of total GR and phosphor-GR chromosomes but share the same seed region, exhibit a similar (Ser-211) was unchanged in the cytosolic fraction (Figure 4D). A expression pattern (Hertel et al, 2012). Both miRNAs are down- similar result in which overexpression of miR-100/99a and regulated in several human cancers and act as tumour suppressors silencing of FKBP51 increased the expression of GR and via their influence on cell processes (Sun et al, 2011; Torres et al, phosphor-GR (Ser-211) in the nucleus of Jurkat cells was obtained 2012; Chen et al, 2012b). However, they are up-regulated in under the treatment of 1-mM Dex (Supplementary Figure S3). paediatric AML patients and are correlated with poor prognosis These data showed that miR-100/99a contributed to the influence (Bai et al, 2012; Zheng et al, 2012). In this study, we showed that of GR nuclear translocation and enhancement of the phosphory- both miR-100 and miR-99a were significantly down-regulated in lated forms of GR at Ser211 in the nucleus, which resemble the ALL patients and that ectopic expression of miR-100 and miR-99a FKBP51-silencing effect. This effect is, at least in part, mediated by inhibited cell proliferation and enhanced Dex-induced cell the inhibition of FKBP51. apoptosis. These findings suggest that these two miRNAs may have different roles in the adaptation of cells to the transformed MiR-100/99a target the IGF1R/mTOR pathways in ALL. Pre- state of myeloid and lymphoblastic cells. vious studies have reported that IGF1R and mTOR are direct This study provides evidence that miR-100 and miR-99a targets of miR-100 and miR-99a in several human cancers function in ALL. First, we revealed a differential expression pattern (Doghman et al, 2010; Torres et al, 2012; Chen et al, 2012b). We of miR-100 and miR-99a in different ALL subtypes. Lower thus asked whether IGF1R and mTOR are targets of miR-100 and expression levels of both miR-100 and miR-99a were found in miR-99a in ALL and whether these proteins act downstream from T-ALL patients and in patients carrying the MLL-rearrangement both miRNAs to alter cellular processes in ALL, similar to what and BCR-ABL fusion genes, and these lower expression levels occurs in FKBP51. As shown in Figure 5A, the protein levels of correlated with poor prognosis. These results indicate that the IGF1R and mTOR were reduced in Jurkat cells that were expressions of miR-100 and miR-99a are cell-type specific and transfected with miR-100/99a mimics, while they were increased suggest that both miRNAs are associated with leukemogenesis and in cells that were transfected with the miR-100/99a inhibitors the prognostic outlook of ALL. (antisense). These data confirmed that IGF1R and mTOR are Second, when addressing the molecular basis for the phenotype targets of miR-100/99a. To further unravel whether IGF1R and of miR-100 and miR-99a, FKBP51 was identified as a novel target mTOR are also involved in the miR-100- and miR-99a-mediated of both miRNAs. FKBP51 has effective roles in cell proliferation, alterations in cell proliferation and Dex-induced apoptosis in ALL, neoplastic diseases, and cell apoptosis in several tumour cell lines we performed loss-of-function analyses with RNAi and found that (Jiang et al, 2008; Li et al, 2011; Schmidt et al, 2012). However, the the growth rates of the CEM/C1 cells were dramatically decreased involvement of FKBP51 in lymphoblastic leukaemia cells has not and that the Dex-induced apoptosis rates were significantly enhanced yet been elucidated. The observation that FKBP51 knockdown after the silencing of IGF1R and mTOR (Figure 5B and C). caused the same effect as miR-100 and miR-99a overexpression MCL1 is highly suppressed during the increased Dex-induced indicates that FKBP51 contributes to the miR-100 and miR-99a apoptosis observed in ALL cells that have been treated with phenotypes. rapamycin, which is a newly FDA-approved immunosuppressant FKBP51 has the overall effect of impairing GR nuclear drug that inhibits mTOR (Wei et al, 2006). The results of this study translocation and reducing GR activity, and by down-regulating indicate that mTOR is directly involved in the miR-100- and miR- the GR signalling pathway, it exerts proliferative and anti-apoptotic 99a-mediated apoptotic response to Dex. Thus, we hypothesised properties (Denny et al, 2000; Romano et al, 2011). GR, which can that MCL1 might also be involved in miR-100- and miR-99a- continuously shuttle between the nuclear and cytoplasmic triggered apoptosis. To prove this hypothesis, we first examined the compartments, is an important ligand-dependent transcription expression of mTOR and MCL1 protein levels following induction factor that belongs to the nuclear receptor family and is also a 2196 www.bjcancer.com | DOI:10.1038/bjc.2013.562 MiR-100/99a in acute lymphoblastic leukaemia BRITISH JOURNAL OF CANCER monomeric protein that cooperates with other transcription factors Specifically, our experimental data indicate that miR-100 and to induce transcription (Bray and Cotton, 2003; Vandevyver et al, miR-99a are involved in two essential signalling pathways: 2012). Davies and colleagues revealed that the Dex-binding event (i) influencing GR signalling by targeting FKBP51 and causes a dissociation of FKBP51 and GR-complex that, in turn, (ii) suppressing the IGF1R/mTOR pathway and down-regulating control the intracellular trafficking of GR (Davies et al, 2002). In a the expression of the anti-apoptotic gene MCL1 to inhibit cell loss-of-function study, our results demonstrated that the relatively growth and initiate cell apoptosis. The modulation of multiple silenced expression of FKBP51 is responsible for the accumulation cancer-associated pathways underscores the significance of of GR expression in the nucleus after Dex induction, and miR-100 and miR-99a for future clinical treatments. Therefore, our results further revealed that miR-100/99a overexpression our study suggests a novel gene therapy strategy, namely, the influenced the translocation of GR to the nucleus. After enhancement of Dex-induced apoptosis, for patients with ALL. translocating to the nucleus, phosphorylation at Ser-211 is essential for subsequent GR-dependent signalling (Chen et al, 2008). We found that silenced FKBP51 increased the phosphor-GR (Ser-211) ACKNOWLEDGEMENTS expression in the nucleus of Jurkat cells. Furthermore, miR-100 and miR-99a overexpression decreased the FKBP51 protein levels This work was supported by grants from the National Science and and led to GR activation in the nucleus after exposure to Dex. Technology Department of China and National Science Foundation These findings suggest that, in the miR-100- and miR-99a- of China. mediated response to Dex in ALL cells, the GR signalling pathway is activated by the suppression of FKBP51. Recent studies have shown that multimeric chaperones, including hsp90, p23, FKBP52, CONFLICT OF INTEREST dynamitin, and dynein, are required for the translocation of GR (Echeverria et al, 2009), yet further research will be needed to The authors declare no conflict of interest. determine whether other importins or nucleoporins are required and to elucidate the complete details of how miR-100 and miR-99a affect GR activity through FKBP51. REFERENCES Another contribution of the study is the validation of two other important target genes, IGF1R and mTOR, which are involved in Agirre X, Vilas-Zornoza A, Jimenez-Velasco A, Martin-Subero JI, Cordeu L, the miR-100- and miR-99-regulated pathways. 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NonCommercial-Share Alike 3.0 Unported License. Supplementary Information accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc) 2198 www.bjcancer.com | DOI:10.1038/bjc.2013.562 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Cancer Springer Journals

MicroRNA-100/99a, deregulated in acute lymphoblastic leukaemia, suppress proliferation and promote apoptosis by regulating the FKBP51 and IGF1R/mTOR signalling pathways

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Springer Journals
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Copyright © 2013 by The Author(s)
Subject
Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
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0007-0920
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1532-1827
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10.1038/bjc.2013.562
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Abstract

FULL PAPER British Journal of Cancer (2013) 109, 2189–2198 | doi: 10.1038/bjc.2013.562 Keywords: acute lymphoblastic leukaemia; miRNA; FKBP51 signalling; IGF1R/mTOR signalling pathway; apoptosis; proliferation MicroRNA-100/99a, deregulated in acute lymphoblastic leukaemia, suppress proliferation and promote apoptosis by regulating the FKBP51 and IGF1R/mTOR signalling pathways 1,3 2,3 1 1 1 ,1 X-J Li , X-Q Luo , B-W Han , F-T Duan , P-P Wei and Y-Q Chen Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, School of Life Science, Sun Yat-sen University, Guangzhou 510275, China and The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China Background: MicroRNAs alter multiple cell processes and thus influence tumour carcinogenesis and progression. MiR-100 and miR-99a have been reported to be aberrantly expressed in acute leukaemia. In this study, we focused on their functions in acute lymphoblastic leukaemia (ALL) and the molecular networks in which they are involved. Methods: MiR-100 and miR-99a expression levels were measured in acute leukaemia patients by qRT–PCR. Kaplan  Meier analysis and log-rank tests were used to calculate the survival rate. Three human ALL cell lines were studied. Apoptosis and proliferation were analysed using siRNA transfection, western blot and flow cytometry. Results: In vivo, miR-100 and miR-99a were down-regulated in 111 ALL patients, especially in high-risk groups; their expression levels were correlated with the patient’s 5-year survival. In vitro, the restoration of miR-100 and miR-99a in ALL cells suppressed cell proliferation and increased dexamethasone-induced cell apoptosis. Ectopic expression of miR-100 and miR-99a targeted FK506- binding protein 51 (FKBP51) and, in turn, influenced glucocorticoid receptor (GR) activity. Meanwhile, miR-100 and miR-99a overexpression inhibited the expression of IGF1R and mTOR and their downstream oncogene MCL1. Conclusion: MiR-100 and miR-99a have critical roles in altering cellular processes by targeting both the FKBP51 and IGF1R/mTOR signalling pathways in vitro and might represent a potential novel strategy for ALL treatment. Childhood acute lymphoblastic leukaemia (ALL) is a commonly should be elucidated. In patients with ALL, several cell signalling occurring malignancy characterised by the uncontrolled prolifera- pathways that control cancer occurrence and development have tion of immature lymphoid cells (Pui et al, 2004). Although been shown to be altered (Weng et al, 2004; Hirsch and Chiarle, treatment has improved remarkably over the past decade, B10% 2012); however, the specific mechanisms responsible for the of ALL patients fail to reach long-term remission (Kraszewska et al, development and treatment of this disease remain unknown. 2012). To better understand this disease, the intricate regulatory Hence, the discovery of more important molecules might further networks among molecular interactions and signalling pathways elucidate the networks and facilitate new therapeutic strategies. In *Correspondence: Dr Y-Q Chen; E-mail: lsscyq@mail.sysu.edu.cn These authors contributed equally to this work. Received 5 July 2013; revised 19 August 2013; accepted 22 August 2013; published online 12 September 2013 & 2013 Cancer Research UK. All rights reserved 0007 – 0920/13 www.bjcancer.com | DOI:10.1038/bjc.2013.562 2189 BRITISH JOURNAL OF CANCER MiR-100/99a in acute lymphoblastic leukaemia the past decade, aberrant microRNA (miRNA) expression has been patients, and the study was approved by the Ethics Committee of shown to have an essential role in the pathogenesis of leukaemia, the First Affiliated Hospital of Sun Yat-sen University. and miRNAs have been considered as potential therapeutic targets Cell culture and cell treatment. The ALL cell lines, CCRF-CEM, for the disease (Fernando et al, 2012). CEM/C1, and Jurkat, and the HEK-293T cell line were purchased MicroRNAs are 20–22 nucleotides long and exert their from American Type Culture Collection (ATCC, Manassas, VA, regulatory effects by directly binding to the 3 untranslated regions USA). All cell lines were grown with 10% fetal bovine serum (UTRs) of their target genes, thus repressing the target gene at the (HyClone, Logan, UT, USA). Rapamycin (Beyotime, Haimen, level of translation (Esquela-Kerscher and Slack, 2006). Recent China) was dissolved in dimethyl sulfoxide (Sigma, St Louis, MO, studies have demonstrated that miRNAs have important roles in USA) and used at a concentration of 10-nM. Dexamethasone cancer pathogenesis and development, and subsequently, in (Sigma) was dissolved in ethanol and used at concentrations of treatment and prognosis (Hwang and Mendell, 2006). In ALL, 1-mM and 100-nM. The final concentrations of dimethyl sulfoxide miR-125b has an important role in early hematopoietic develop- and ethanol in the culture media were 0.05% and 0.1%, ment (Shaham et al, 2012); miR-124a contributes to the abnormal respectively. proliferation of ALL cells (Agirre et al, 2009), and knockdown of miR-17-92 clusters has been shown to increase glucocorticoid- RNA isolation and real-time quantitative RT  PCR. Total RNA mediated apoptosis in ALL cell lines (Molitoris et al, 2011). was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, MiR-100 and miR-99a belong to the same family and have USA) according to the manufacturer’s guidelines. Real-time PCR similar roles in a variety of cancers (Doghman et al, 2010; Mueller was performed to quantify mRNA expression using ExTaq SYBR et al, 2012; Chen et al, 2012b). Our previous study showed that Green Supermix (Bio-Rad, Hercules, CA, USA) according to the both miR-100 and miR-99a are up-regulated in childhood acute manufacturer’s instructions (details are provided in the supporting myeloid leukaemia (AML) and that overexpression of miR-100 information, Supplementary Table S2). arrested the differentiation of human granulocyte and monocyte Proliferation and apoptosis assays. Cell proliferation was cells and promoted cell survival (Zhang et al, 2009; Zheng et al, assessed using the Cell Counting Kit-8 (CCK-8). After transfection, 2012). In contrast, miR-100 and miR-99a have very low expression 1*10 cells per well were plated in 96-well sterile plastic culture levels in ALL patients (Zhang et al, 2009), suggesting that these two plates, and the CCK-8 assay (Dojindo Molecular Technologies, miRNAs have different roles in myeloid cell and lymphocyte Shanghai, China) was performed after 0, 24, 48, 72, and 96 h. To pathogenesis. However, the molecular basis of how miR-100 and assess the rate of apoptosis, transfected cells were harvested and miR-99a are involved in lymphoblastic leukaemogenesis and washed twice with cold PBS, and the Annexin V-PI Kit (Nanjing progression has not yet been elucidated. Keygen, Nanjing, China) was used according to the manufacturer’s Based on a bioinformatics analysis, FK506-binding protein 51 guidelines. The detection was performed with a FACS Calibur (FKBP51) was predicted to be a target of miR-100 and miR-99a using CellQuest software (BDIS, San Jose, CA, USA). (Bhushan and Kandpal, 2011). The evidence collected to date shows that FKBP51 is an immunophilin that is physiologically Transient transfection. For the luciferase reporter assay, HEK- expressed in lymphocytes and is involved in sustaining cell growth, 293T cells were transfected with Lipofectamine 2000 (Invitrogen) malignancy, and resistance to therapy (Li et al, 2011; Schmidt et al, following the manufacturer’s protocol. CCRF-CEM, CEM/C1, and 2012). Furthermore, IGF1R and mTOR are mutual targets of miR- Jurkat cells were transfected with the small molecular RNAs using 100 and miR-99a in several cancer cells (Doghman et al, 2010; the Neon Transfection System (Invitrogen) according to the Lerman et al, 2011; Sun et al, 2011), and they have been implicated manufacturer’s guidelines (details are provided in the Supporting in the initiation and progression of several malignant neoplasms Information Online, Supplementary Materials and Methods). (Petroulakis et al, 2006), including ALL (Cardoso et al, 2009; Dual luciferase activity assay. Luciferase constructs were estab- Medyouf et al, 2011). Thus, we hypothesised that FKBP51, IGF1R, lished by inserting oligonucleotides containing the wild-type or and mTOR are involved in the functioning of miR-100 and miR-99a mutant putative target site of the FKBP51 3 -UTR into the multi- in ALL and serve as their targets. cloning site of the psiCHECK2 vector (Promega, Fitchburg, WI, In this study, we found that miR-100 and miR-99a were down- USA) (see Supporting Information Online, Supplementary regulated in childhood ALL patients and that their expression Materials and Methods for more details). levels were related to the prognosis of the ALL patients. In vitro investigation further demonstrated that miR-100 and miR-99a Western blot assay. Western blot analyses were performed as participated in the regulation of cell proliferation and dexametha- described previously (Zheng et al,2012).Nuclear–cytoplasmic sone (Dex)-induced apoptosis in ALL cell lines. Our data revealed fractionation was conducted using the NE-PER Nuclear and that FKBP51 is a novel target of miR-100 and miR-99a and, in Cytoplasmic Extraction Reagents kit (Pierce, Rockford, IL, USA) turn, influences its downstream gene glucocorticoid receptor (GR). according to the manufacturer’s protocol. Transfected cells were We also proved that miR-100 and miR-99a overexpression assayed with anti-FKBP51 (Epitomics, Zhejiang, China); anti-H3 inhibited IGF1R and mTOR and down-regulated MCL1. The (Abcam, Cambridge, UK); anti-MCL1 and anti-GR (Santa Cruz, results of the present study suggest that miR-100 and miR-99a act Paso Robles, CA, USA); and anti-IGF1R, anti-mTOR, anti-phospho- as tumour suppressors and that their restoration might be a mTOR (Ser-2448), anti-phospho-GR (Ser-211), and anti-GAPDH possible therapeutic strategy for patients with ALL. (Cell Signalling Technology, Boston, MA, USA). After a 1-h incubation with anti-rabbit (Sigma-Aldrich) HRP-conjugated sec- ondary antibody, the protein level was detected using a luminal reagent (Millipore). X-ray films were acquired and quantified with MATERIALS AND METHODS the densitometric software Quantity One (Bio-Rad). The value under each sample indicates the fold change of protein amounts that Patients. This study included 71 AML patients, 111 ALL patients, normalised with the corresponding GAPDH or H3 in each sample. and 10 healthy donors from the First Affiliated Hospital of Sun Yat-sen University. Patient demographics are presented in Bioinformatic and statistical analyses. Online miRNA databases supporting information Supplementary Table S1. Bone marrow (TargetScan and PicTarget) were used to predict the miR-100 and was obtained from patients and healthy donors. Informed consent miR-99a target genes. SPSS13.0 software (Chicago, IL, USA) was to perform the biological studies was obtained from all of the used for statistical analysis. Data are expressed as the mean s.d. 2190 www.bjcancer.com | DOI:10.1038/bjc.2013.562 MiR-100/99a in acute lymphoblastic leukaemia BRITISH JOURNAL OF CANCER The differences between two groups were analysed using the expression levels of miR-100 and miR-99a in both childhood ALL Mann Whitney U test for two groups or the Kruskal Wallis test and AML patients at diagnosis using qRT  PCR. As shown in when more than two groups were compared. All tests were two- Figure 1A, both miRNAs showed significantly different expression sided. The Kruskal Wallis test was implemented for the patterns between ALL and AML compared with healthy donors. comparison of three groups, and multiple comparisons were MiR-100 and miR-99 showed very low expression levels in most performed using one-way ANOVA and a least significant difference ALL patients compared with AML patients and healthy donors. t test after the relative concentration was ranked. Differences were We next analysed their correlation with the patient characteristics. considered significant at *Po0.05, **Po0.01, and ***Po0.001. Although the expression levels of miR-100 and miR-99a had no correlation with sex, age, or therapy (data not shown), we found that the expression levels of these two miRNAs were significantly RESULTS correlated with patients who carried known high-risk prognostic factors, including white blood cell (WBC) count, ALL sub- classification (here, indicated as T-cell and B-cell ALL), the MiR-100 and miR-99a are associated with specific risk groups MLL-rearranged gene and the BCR-ABL fusion gene. As shown and survival in childhood ALL. To investigate the link between 4  3 in Figure 1B, a higher WBC count (45  10 mm ) before miR-100 and miR-99a and childhood ALL, we first measured the A miR-100 3 miR-99a B miR-100 miR-99a 2 2 *** 10 10 4 P<0.0001 ** P<0.0001 *** *** 2 *** *** 10 0 0 10 –1 10 10 –1 –1 –1 10 –2 –2 –3 –2 –3 –2 10 10 10 10 3 3 3 3 Control AML ALL Control AML ALL WBC >50 000mm <50 000mm WBC >50 000mm <50 000mm C miR-100 miR-99a D miR-100 miR-99a 10 3 10 3 P=0.0004 P=0.0004 10 1 * 2 ** 10 10 0 10 –1 –1 –1 10 10 –1 –2 10 –2 –2 10 10 –2 –3 –3 –3 10 10 10 T-ALL B-ALL T-ALL B-ALL WT MLL BCR-ABL WT MLL BCR-ABL miR-100 miR-99a miR-100 miR-99a E 100 100 100 100 80 80 80 80 60 60 60 60 40 40 40 20 20 20 20 Low miR-100 Low miR-99a Low miR-100 Low miR-99a P=0.01266 P=0.0054 P=0.0466 P=0.0233 High miR-100 High miR-99a High miR-100 High miR-99a 0 0 0 0 020 40 60 020 40 60 020 40 60 020 40 60 Time (months) Time (months) Time (months) Time (months) Figure 1. MiR-100 and miR-99a are associated with specific risk groups and the survival of patients with childhood ALL. (A) The expression levels of miR-100 (left) and miR-99a (right) were quantified by real-time PCR in 111 ALL patients, 71 AML patients and 10 healthy donors. MiR-100 and miR-99a were significantly down-regulated in ALL patients compared with AML patients (Po0.0001 and Po0.0001, respectively) and healthy donors (P ¼ 0.001 and P ¼ 0.0086, respectively). (B) miR-100 (left) and miR-99a (right) expression levels, determined by qRT  PCR, in ALL patients 4  3 4  3 with more than 5  10 mm white blood cells (WBCs) or less than 5  10 mm WBCs at diagnosis (Po0.0001 and Po0.0001, respectively). (C) Comparison of the expression of miR-100 (left) and miR-99a (right) between B-ALL (n ¼ 93) and T-ALL (n ¼ 18) patients by qRT–PCR (P ¼ 0.0004 and P ¼ 0.0004, respectively). (D) The expression levels of miR-100 (left) and miR-99a (right) were significantly down-regulated in ALL patients with the MLL-rearranged gene (n ¼ 22) and the BCR-ABL fusion gene (n ¼ 11), in comparison with the patients without fusion genes (n ¼ 78) (Po0.001 and Po0.05, respectively, for miR-99a, and Po0.05 and Po0.05, respectively, for miR-99a). Wild type (WT): patients without these two fusion genes. (E) The OS of patients with high miR-100 and miR-99a expression levels was significantly higher than that of patients with low miR-100 and miR-99a expression levels (P¼ 0.0466 and P¼ 0.0126, respectively). (F) The leukaemia-free survival (LFS) of patients with high miR-100 and miR- 99a expression levels was significantly higher than that of patients with low miR-100 and miR-99a expression levels (P¼ 0.0054 and P ¼ 0.0233, respectively). The Kruskal  Wallis test was implemented for the comparison of three groups, and multiple comparisons were performed using one-way ANOVA and a least significant difference t test after the relative concentration was ranked. A Kaplan  Meier analysis and the log-rank test were used to analyse OS and LFS. *Po0.05, **Po0.01, and ***Po0.001 vs control or WT. www.bjcancer.com | DOI:10.1038/bjc.2013.562 2191 Relative miRNA expression Relative miRNA expression Overall survival Overall survival Relative miRNA expression Relative miRNA expression Leukemia-free survival Relative miRNA expression Relative miRNA expression Leukemia-free survival Relative miRNA expression Relative miRNA expression BRITISH JOURNAL OF CANCER MiR-100/99a in acute lymphoblastic leukaemia chemotherapy was observed in patients with lower expression that miR-100 and miR-99a are associated with specific risk groups levels of miR-100 and miR-99a. Notably, all T-ALL cases showed within childhood ALL. low expression levels of miR-100 and miR-99a compared with Furthermore, the association of miR-100 and miR-99a expres- B-ALL patients (Figure 1C). We also found that patients carrying sion with the prognosis of ALL patients was investigated using the MLL-rearranged or BCR-ABL fusion gene, two crucial Kaplan  Meier analysis and a log-rank test. As shown in biological mutations associated with poor outcome, expressed Figure 1E, the 5-year overall survival (OS) of patients with high lower levels of miR-100 and miR-99 compared with patients miR-100 and miR-99a expression levels was significantly higher without these two fusion genes (Figure 1D). These results suggest than in patients with low miR-100 and miR-99a expression levels. 0.8 Control miRNA 2.0 1.5 Control miRNA Control miRNA miR-100-mimics miR-100-mimics miR-100-mimics 0.6 miR-99a-mimics * * 1.5 * * miR-99a-mimics miR-99a-mimics * * * * * * 1.0 * * * * * * 0.4 1.0 0.5 0.2 0.5 CEM/C1 CCRF-CEM Jurkat 0.0 0.0 0.0 0h 24h 48h 72h 96h 0h 24h 48h 72h 96h 0h 24h 48h 72h 96h CCRF-CEM CCRF-CEM Control miRNA miR-100-mimics miR-99a-mimics *** 26.9% 23.1% 15.7% *** 10 25 3.62% 6.02% 10 6.44% 15 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Control miRNA miR-100-mimics miR-99a-mimics Annexin Annexin Annexin CEM/C1 CEM/C1 miR-100-mimics Control miRNA miR-99a-mimics 7.90% 18.2% 16.6% *** *** 0 9.40% 15.2% 14.2% 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Control miRNA miR-100-mimics miR-99a-mimics Annexin Annexin Annexin Jurkat Jurkat miR-100-mimics miR-99a-mimics Control miRNA *** 61.0% 34.3% 69.8% *** 10 70 1 30 5.45% 12.0% 9.11% 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Control miRNA miR-100-mimics miR-99a-mimics Annexin Annexin Annexin Figure 2. MiR-100 and miR-99a inhibit cell proliferation and promote Dex-induced apoptosis in ALL cell lines and function as potential tumour suppressors. (A) Transfection of miR-100 and miR-99a slowed ALL cell proliferation. The time points after transfection with control miRNA, miR-100, or miR-99a are indicated. MiR-100 and miR-99a significantly inhibited cell proliferation in CCRF-CEM (left), CEM/C1 (middle), and Jurkat (right) cells, as determined using the CCK-8 assay. (B–D) miR-100 and miR-99a significantly promoted Dex-induced cell apoptosis. CCRF-CEM (B), CEM/C1 (C), and Jurkat (D) cells were transfected with miR-100, miR-99a, or control miRNA and cultured for 24 h; the three cell lines were exposed to 1-mM Dex for 8, 18, and 24 h, respectively. Cells were stained with PI/Annexin V and analysed by flow cytometry. A representative experiment is shown. **Po0.01 and ***Po0.001 vs control. Values are presented as the mean s.d. of at least three independent experiments performed in triplicate. 2192 www.bjcancer.com | DOI:10.1038/bjc.2013.562 OD value (450 nM/630 nM) PI PI PI OD value (450 nM/630 nM) Apoptosis (%) Apoptosis (%) Apoptosis (%) OD value (450 nM/630 nM) MiR-100/99a in acute lymphoblastic leukaemia BRITISH JOURNAL OF CANCER Moreover, a trend towards increased leukaemia-free survival was We further studied the effect of miR-100 and miR-99a on Dex- observed in patients with ALL and up-regulated miR-100 and miR-99a induced ALL cell apoptosis. After transfection of miR-100 and expression levels over the 5-year follow-up period (Figure 1F). miR-99a mimics for 24 h, cells were treated with 1-mM Dex, a Taken together, these results suggest that higher expression levels therapeutic drug that is widely used to treat ALL by triggering of miR-100 and miR-99a were associated with better survival and leukaemic cell apoptosis. Overexpression of miR-100 and miR-99a prognosis. increased Dex-induced apoptosis compared with control miRNA in CCRF-CEM, CEM/C1, and Jurkat cells (Figure 2B–D). These data suggest that the ectopic expression of miR-100 and miR-99a MiR-100 and miR-99a inhibit cell proliferation and promote inhibits cell proliferation and promotes the response to Dex- Dex-induced apoptosis in ALL cell lines and function as induced apoptosis in ALL cells. potential tumour suppressors. The observations described above suggest that both miR-100 and miR-99a are clinically relevant and FKBP51 is a novel target of miR-100 and miR-99a. To might affect leukaemic cell progression. To explore the role of miR- determine the molecular mechanism by which miR-100 and 100 and miR-99a, we investigated their influences on leukaemic miR-99a are involved in ALL pathogenesis, we further investigated cell proliferation and cell apoptosis. Three ALL cell lines, CCRF- their downstream targets. Among the nearly 100 predicted targets, CEM, CEM/C1, and Jurkat, were used to perform the in vitro FKBP51 was chosen for further investigation because this gene has functional analysis. A qRT  PCR assay was used to measure the an active role in cancer aetiology and responds to antineoplastic transfection efficiency of the miRNAs (supporting information, therapy (Baughman et al, 1995; Febbo et al, 2005; Pei et al, 2009). Supplementary Figure S1). The results of a CCK-8 assay indicated As shown in Figure 3A, to test whether FKBP51 is directly that overexpression of miR-100 and miR-99a inhibited cell regulated by miR-100 and miR-99a, a dual-luciferase assay was proliferation of all three ALL cell lines in a time-dependent performed, and the 3 UTR fragment, which was cloned into a manner (Figure 2A). reporter plasmid, was inserted into the luciferase open reading AB 3′ 3′-FKBP51-Wt 5′ 1.4 1.2 hsa-miR-100 3′ 5′ *** *** 3′-FKBP51-Wt 5′ 3′ 0.8 Control miRNA 3′ 5′ hsa-miR-99a miR-100-mimics 0.6 3′-FKBP51-Mut 5′ 3′ miR-99a-mimics 0.4 3′-FKBP51-Wt Renilla luciferase 0.2 Renilla luciferase 3′-FKBP51-Wt FKBP51 FKBP51 pSi-Check2 point Mu Wt CD 0 0 Control miR-100 miR-99a Control Anti-miR Anti-miR miRNA -mimics -mimics miRNA -100 -99a Control miR-100 miR-99a Control Anti-miR Anti-miR miRNA -mimics -mimics miRNA -100 -99a FKBP51 FKBP51 51 KDa 51 KDa 1.0 1.7 2.4 1.0 0.3 0.3 36 KDa GAPDH GAPDH 36 KDa Figure 3. MiR-100 and miR-99a target FKBP51. (A) Schematic representation of the constructs used in the luciferase assay.The sequences shown below indicate the putative miR-100 and miR-99a target site on the wild-type 3 UTR (construct FKBP51-wt), its mutated derivative (construct FKBP51-mut), and the pairing regions of miR-100 and miR-99a. (B) 293T cells were co-transfected with pSi-Check2 with either FKBP51-wt or FKBP51-mut and with miR-100, miR-99a, or empty vector (pCD6.2). Repression of luciferase activity due to the interaction between miR-100, miR-99a, and the luciferase constructs. Each Renilla luciferase reading was normalised to that obtained for the control firefly luciferase. (C, D) Jurkat cells were electroporated with 100-nmol mimics-NC (control miRNA), mimics-miR-100, or miR-99a and anti-scramble (control miRNA), anti-miR-100, or anti-miR-99a. The levels of miR-100 and miR-99a were assessed by qRT  PCR. Cell lysates were prepared for western blotting with the antibody against FKBP51, and the expression of GAPDH served as a loading control. Western blot figures are representative of at least three independent experiments. The value under each sample indicates the fold change of FKBP51 protein levels relative to that of the control. ***Po0.001. Values are presented as the mean s.d. of at least three independent experiments performed in triplicate. www.bjcancer.com | DOI:10.1038/bjc.2013.562 2193 Relative expression of miRNA Relative expression of miRNA Relative luciferase activity (%) BRITISH JOURNAL OF CANCER MiR-100/99a in acute lymphoblastic leukaemia si-FKBP51 Control si-RNA AB FKBP51 1.0 0 CEM/C1 2.0 Control si-RNA si-FKBP51 35 GAPDH 10 *** 5.63% 9.63% Control siRNA 1.5 3 si-FKBP51 10 25 1.0 10 10 0.5 6.42% 20.0% CEM/C1 10 0 1 2 3 4 0 1 2 3 4 0.0 10 10 10 10 10 10 10 10 10 10 Control si-RNA si-FKBP51 0h 24h 48h 72h 96h Annexin Annexin CD FKBP51 51 KDa FKBP51 51 KDa 51 KDa FKBP51 51 KDa FKBP51 1.0 0.6 0.7 1.0 0.7 0.6 1.0 0.7 0.7 1.0 GR GR 95 KDa 95 KDa GR GR 95 KDa 95 KDa 1.0 1.7 1.0 0.9 1.1 1.0 1.5 2.3 1.0 1.0 p-GR p-GR 95 KDa p-GR p-GR 95 KDa 95 KDa 95 KDa 1.0 1.1 0.8 1.0 2.2 5.4 1.0 2.6 1.0 1.1 H3 GAPDH 15 KDa GAPDH H3 36 KDa 36 KDa 36 KDa Cytoplasm Nucleus Nucleus Cytoplasm Figure 4. MiR-100 and miR-99a affected cell proliferation and the response to Dex by targeting FKBP51 and, in turn, influenced the activity of steroid receptor GR. (A) Transfecting CEM/C1 cells with si-FKBP51 significantly inhibited cell proliferation. Cell viability was determined using the CCK-8 assay. The time points after transfection with control siRNA or si-FKBP51 are indicated. The western blot analysis of the effect of si-FKBP51 is shown in the upper-right corner. The value under each sample indicates the fold change of FKBP51 protein levels relative to that of the control. (B)FKBP51 knockdown enhanced Dex-induced apoptosis. CEM/C1 cells were transfected with control siRNA or si-FKBP51 24 h after transfection, which was followed by 1-mM Dex treatment for 18 h. Control siRNA served as a negative control. Then, the cells were collected for apoptosis analysis by PI/Annexin V staining and flow cytometry. A representative experiment is shown. (C) FKBP51 knockdown promoted the expression of GR and phosphorylated forms of GR in the nucleus. After 48 h of transfection with control siRNA or si-FKBP51, cells were exposed to 100-nM Dex for 24 h, and then cytoplasm and nucleus extracts were prepared. Equal amounts of protein from each fraction were analysed by immunoblotting with anti-FKBP51, anti-GR (total GR protein), and anti-phospho-Ser211 (p-GR), using GAPDH as an internal control for the cytoplasm fraction and H3 for the nucleus fraction. The value under each sample indicates the fold changes of FKBP51, GR, and p-GR protein levels relative to that of the control. (D)miR-100 and miR-99a overexpression induced the expression of GR and p-GR by down-regulating FKBP51 in the nucleus. After 48 h of transfection with control miRNA, miR-100, or miR-99a, cells were exposed to 100-nM Dex for 24 h, and then cytoplasm and nucleus extracts were prepared to detect the protein levels of FKBP51, GR, p-GR, and GAPDH in the cytoplasm fraction and of H3 in the nucleus fraction. Western blot figures are representative of at least three independent experiments. ***Po0.001 vs control. Values are presented as the mean s.d. of at least three independent experiments. Figure 5. MiR-100/99a target the IGF1R/mTOR pathways in ALL. (A) IGF1R and mTOR are direct targets of miR-100 and miR-99a. Jurkat cells were electroporated with 100-nmol mimics-NC, miR-100-mimics, or miR-99a-mimics and 100 nmol Control miRNA, anti-miR-100, or anti-miR-99a. Cell lysates were prepared for western blotting with an antibody against IGF1R and mTOR, and the expression of GAPDH served as a loading control. The value under each sample indicates the fold changes of IGF1R and mTOR protein levels relative to that of the control. (B) CEM/C1 cell proliferation was significantly inhibited, and Dex-induced apoptosis (C) was significantly enhanced after the transfection of si-IGF1R and si-mTOR, as determined using the CCK-8 assay and flow cytometry. For flow cytometry, the apoptotic cells were stained by PI/Annexin V 18 h after exposure to 1 mM Dex. Control siRNA served as a negative control. A representative experiment is shown. The western blot analysis of the transfection efficiency of si-IGF1R and si-mTOR in CEM/C1 cells is shown in the upper-right corner of (B). (D) Western blot analysis of the expression level of mTOR and MCL1 in Jurkat cells following treatment with vehicle, 1-mM Dex, 10-nM Rap, or 1-mM Dex plus 10-nM Rap for 24 h. The value under each sample indicates the fold changes of mTOR and MCL1 protein levels relative to that of the control. The expression levels of miR-100 and miR-99a were detected after the treatment described above. These two miRNAs were significantly up-regulated after the combined treatment of Dex and Rap. (E) Both overexpression of miR-100/99a and knockdown of mTOR reduced the expression of MCL1. At 48 h after the transfection, Jurkat cells were treated with 1 mM Dex for 24 h, as determined by Western blot analysis. The value under each sample indicates the fold changes of mTOR and MCL1 protein levels relative to that of the control. (F) IGF1R knockdown promoted the phosphorylation of mTOR and, in turn, repressed the expression of MCL1. At 48 h after transfection, Jurkat cells were exposed to 1-mM Dex for 24 h, and the expression of IGF1R, p-mTOR (Ser-2448), and MCL1 was detected by western blot analysis. The value under each sample indicates the fold changes of IGF1R, p-mTOR, mTOR, and MCL1 protein levels relative to that of the control. (G) CEM/C1 cell proliferation was significantly inhibited, and (H) Dex-induced apoptosis was significantly enhanced after the transfection of si-MCL1. Control si-RNA served as a negative control. A representative experiment is shown. (I) Schematic shows the function of miR-100 and miR-99a in cell proliferation and cell apoptosis in ALL. (i) Upon post-transcriptional suppression, FKBP51 was inhibited by miR-100 and miR-99a, thus restraining cell proliferation and activating the GR at the phosphorylation site of Ser-211, which in turn led to apoptosis. (ii) Through inhibition of mTOR, miR-100 and miR-99a suppressed the expression of MCL1, which resulted in the blockade of cell proliferation and led to cell apoptosis. By targeting IGF1R, miR-100 and miR-99a triggered cell apoptosis and inhibited cell proliferation. In the meantime, down-regulated expression of IGF1R inhibited the activity of mTOR (phosphorylation at the site of Ser-2448), which also led to the suppression of the mTOR/MCL1 signalling pathway. Western blot figures are representative of at least three independent experiments. ***Po0.001 vs Control. Values are presented as the mean s.d. of at least three independent experiments. 2194 www.bjcancer.com | DOI:10.1038/bjc.2013.562 Control si-RNA si-FKBP51 Control si-RNA si-FKBP51 Control miRNA miR-100-mimics miR-99a-mimics Control miRNA miR-100-mimics miR-99a-mimics OD value (450 nM/630 nM) PI Apoptosis (%) *** *** MiR-100/99a in acute lymphoblastic leukaemia BRITISH JOURNAL OF CANCER frame. The interaction between the miRNAs and the target mRNA MiR-100 and miR-99a affect cell proliferation and the response sequences was further validated using a point mutation of the to Dex by targeting FKBP51 and, in turn, influencing the putative miRNA-binding site in the 3 UTR of the FKBP51 gene. activity of steroid receptor GR. Although FKBP51 has important We found that the luciferase activity of the 3 UTR of FKBP51 was roles in neoplastic diseases (Rees-Unwin et al, 2007), the function reduced nearly 50% and 70% after transfection of both miR-100 of FKBP51 in leukaemia has not yet been elucidated. To explore and miR-99a; when the predicted target sites were mutated, the the function of FKBP51, we used RNAi to investigate the role of luciferase activity was unaffected (Figure 3B). this gene in cell proliferation and apoptosis. As shown in the To further confirm that the FKBP51 protein is suppressed by upper-right corner of Figure 4A, FKBP51 expression was miR-100 and miR-99a, we performed both miR-100/99a over- remarkably reduced after the silencing of FKBP51 in CEM/C1 expression and knockdown experiments in Jurkat cells and cells. FKBP51 knockdown inhibited cell proliferation after 4 days examined FKBP51 expression. As shown in Figure 3C and D, of incubation using a CCK-8 assay (Figure 4A) and led to a the level of FKBP51 protein was significantly reduced in Jurkat significant increase in Dex-induced apoptosis in CEM/C1 cells by cells that were transfected with the miR-100 and miR-99a mimics, flow cytometry analysis (Figure 4B). Similar results were obtained whereas it was increased in cells that were transfected with the in CCRF-CEM and Jurkat cell lines (Supplementary Figure S2). antisense of miR-100 and miR-99a. These data indicate that These results show that FKBP51 knockdown has a positive effect, FKBP51 is a bona fide target of miR-100 and miR-99a in similar to that of miR-100 and miR-99a (Figure 2), suggesting that ALL cells. miR-100 and miR-99a exert their functions of inhibiting cell AB C Control miRNA miR-100 miR-99a Control si-RNA si-IGF1R Control si-RNA si-mTOR IGF1R 95 KDa IGF1R mTOR CEM/C1 1.0 0.4 0.3 1.0 0.5 1.0 0.5 mTOR GAPDH GAPDH ** * 289 KDa ** * 1.0 0.5 0.3 2.0 Control siRNA GAPDH 36 KDa 20 si-IGF1R 1.5 Control miRNA Anti-miR-100 Anti-miR-99a 15 si-mTOR IGF1R 1.0 95 KDa 10 1.0 3.0 3.7 5 0.5 mTOR 289 KDa CEM/C1 0.0 Control si-RNA si-IGF1R si-mTOR 1.0 12.7 9.4 0h 24h 48h 72h 96h GAPDH 36 KDa D E G Control Dex Rap R+D 289 KDa mTOR Control si-RNA si-MCL1 1.0 1.4 0.9 0.5 MCL1 289 KDa 42 KDa mTOR MCL1 1.0 0.1 1.0 0.4 1.0 0.8 0.2 0.02 1.0 0.6 0.4 GAPDH MCL1 42 KDa GAPDH 36 KDa 1.0 0.5 0.4 2.0 1.0 0.3 GAPDH 36 KDa 4.5 Control siRNA miR-100 *** 4 si-MCL1 miR-99a 1.5 *** 3.5 Control si-RNA si-IGF1R * 95 KDa IGF1R 1.0 2.5 1.0 0.4 289 KDa p-mTOR 0.5 1.5 1.0 0.5 CEM/C1 289 KDa mTOR 0.5 1.0 0.9 0.0 0 0h 24h 48h 72h 96h 42 KDa MCL1 Control Rapa Dex R+D 1.0 0.4 36 KDa GAPDH miR-100 miR-99a CEM/C1 Control si-RNA si-MCL1 4 P-Ser *** FKBP51 5.63% 16.2% IGF1R mTOR ii P-Ser Cell proliferation 10 GR MCL1 6.42% 10.9% Cell apoptosis 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 Control si-RNA si-MCL1 Annexin Annexin Cell apoptosis Cell proliferation www.bjcancer.com | DOI:10.1038/bjc.2013.562 2195 Control miRNA miR-100 miR-99a Control si-RNA si-mTOR Relative expression of miRNA PI OD value (450 nM/630 nM) Apoptosis (%) Apoptosis (%) OD value (450 nM/630 nM) BRITISH JOURNAL OF CANCER MiR-100/99a in acute lymphoblastic leukaemia proliferation and promoting Dex-induced cell apoptosis by with rapamycin and Dex and found significant decreases in both reducing FKBP51 expression. protein levels after exposure to both drugs. In addition, we found Glucocorticoid receptor is an essential transcription factor that that the expression levels of miR-100 and miR-99a were is involved in cellular proliferation, inflammatory responses, and significantly up-regulated after treatment with both rapamycin cell differentiation (Rogatsky and Ivashkiv, 2006; Chen et al, and Dex (Figure 5D). Therefore, miR-100 and miR-99a might 2012a), and FKBP51 is a potent inhibitor of GR activity on regulate the expression of MCL1 by suppressing mTOR. We hormone induction and influences GR nuclear translocation determined that the protein expression of MCL1 was down- (Ratajczak et al, 2003; Denny et al, 2005). Because the presence regulated in Jurkat cells with miR-100 and miR-99a overexpression of FKBP51 reflected a decreased amount of GR accumulation in and with mTOR knockdown, following the exposure of 1-mM Dex the nucleus (Wochnik et al, 2005; Binder, 2009) and phosphoryla- for 24 h (Figure 5E). Furthermore, the expression of MCL1 was tion at Ser-211 is the monophosphorylated version of the also down-regulated after the knockdown of IGF1R through the hormone-activated form of GR that accumulates in the nucleus inhibition of activated mTOR (with phosphorylation at the (Wang et al, 2002; Chen et al, 2008), we tested whether knockdown site of ser-2448) followed by the exposure to Dex (Figure 5F) of FKBP51 expression influenced the subcellular localisation of GR because mTOR is a downstream gene of IGF1R (Eroles et al, 2012). and Ser211-phosphorylated forms of GR in Jurkat cells. To address To elucidate the function of MCL1 in the miR-100- and this issue, cytosolic and nuclear extracts were prepared from miR-99a-mediated signalling pathway, we investigated the roles FKBP51-silenced and control cells following exposure to 100-nM of MCL1 in cell apoptosis and cell growth. As shown in Figure 5G Dex for 24 h. As shown in Figure 4C, after the silence of FKBP51, and H, after silencing MCL1, its influences on cell proliferation and the phosphor-GR (Ser-211) expression was increased in the cell apoptosis were similar to the findings obtained in the miR-100 nucleus by western blotting using H3 protein as an internal and miR-99a studies and further confirmed our hypothesis that control, accompanied by a slight increase of total GR expression miR-100 and miR-99a are important regulators of MCL1 in ALL. level. However, the relative levels of total GR and phosphor-GR (Ser-211) expression were unaffected in the cytosolic fraction. Furthermore, we asked whether miR-100 and miR-99a affected DISCUSSION this downstream target of FKBP51. In the nuclear fractions of miR-100/99a overexpressed cells, we observed increases in GR and phosphor-GR (Ser-211) expression, with down-regulation of Human miR-100 and miR-99a, which are encoded on different FKBP51 levels, while the expression of total GR and phosphor-GR chromosomes but share the same seed region, exhibit a similar (Ser-211) was unchanged in the cytosolic fraction (Figure 4D). A expression pattern (Hertel et al, 2012). Both miRNAs are down- similar result in which overexpression of miR-100/99a and regulated in several human cancers and act as tumour suppressors silencing of FKBP51 increased the expression of GR and via their influence on cell processes (Sun et al, 2011; Torres et al, phosphor-GR (Ser-211) in the nucleus of Jurkat cells was obtained 2012; Chen et al, 2012b). However, they are up-regulated in under the treatment of 1-mM Dex (Supplementary Figure S3). paediatric AML patients and are correlated with poor prognosis These data showed that miR-100/99a contributed to the influence (Bai et al, 2012; Zheng et al, 2012). In this study, we showed that of GR nuclear translocation and enhancement of the phosphory- both miR-100 and miR-99a were significantly down-regulated in lated forms of GR at Ser211 in the nucleus, which resemble the ALL patients and that ectopic expression of miR-100 and miR-99a FKBP51-silencing effect. This effect is, at least in part, mediated by inhibited cell proliferation and enhanced Dex-induced cell the inhibition of FKBP51. apoptosis. These findings suggest that these two miRNAs may have different roles in the adaptation of cells to the transformed MiR-100/99a target the IGF1R/mTOR pathways in ALL. Pre- state of myeloid and lymphoblastic cells. vious studies have reported that IGF1R and mTOR are direct This study provides evidence that miR-100 and miR-99a targets of miR-100 and miR-99a in several human cancers function in ALL. First, we revealed a differential expression pattern (Doghman et al, 2010; Torres et al, 2012; Chen et al, 2012b). We of miR-100 and miR-99a in different ALL subtypes. Lower thus asked whether IGF1R and mTOR are targets of miR-100 and expression levels of both miR-100 and miR-99a were found in miR-99a in ALL and whether these proteins act downstream from T-ALL patients and in patients carrying the MLL-rearrangement both miRNAs to alter cellular processes in ALL, similar to what and BCR-ABL fusion genes, and these lower expression levels occurs in FKBP51. As shown in Figure 5A, the protein levels of correlated with poor prognosis. These results indicate that the IGF1R and mTOR were reduced in Jurkat cells that were expressions of miR-100 and miR-99a are cell-type specific and transfected with miR-100/99a mimics, while they were increased suggest that both miRNAs are associated with leukemogenesis and in cells that were transfected with the miR-100/99a inhibitors the prognostic outlook of ALL. (antisense). These data confirmed that IGF1R and mTOR are Second, when addressing the molecular basis for the phenotype targets of miR-100/99a. To further unravel whether IGF1R and of miR-100 and miR-99a, FKBP51 was identified as a novel target mTOR are also involved in the miR-100- and miR-99a-mediated of both miRNAs. FKBP51 has effective roles in cell proliferation, alterations in cell proliferation and Dex-induced apoptosis in ALL, neoplastic diseases, and cell apoptosis in several tumour cell lines we performed loss-of-function analyses with RNAi and found that (Jiang et al, 2008; Li et al, 2011; Schmidt et al, 2012). However, the the growth rates of the CEM/C1 cells were dramatically decreased involvement of FKBP51 in lymphoblastic leukaemia cells has not and that the Dex-induced apoptosis rates were significantly enhanced yet been elucidated. The observation that FKBP51 knockdown after the silencing of IGF1R and mTOR (Figure 5B and C). caused the same effect as miR-100 and miR-99a overexpression MCL1 is highly suppressed during the increased Dex-induced indicates that FKBP51 contributes to the miR-100 and miR-99a apoptosis observed in ALL cells that have been treated with phenotypes. rapamycin, which is a newly FDA-approved immunosuppressant FKBP51 has the overall effect of impairing GR nuclear drug that inhibits mTOR (Wei et al, 2006). The results of this study translocation and reducing GR activity, and by down-regulating indicate that mTOR is directly involved in the miR-100- and miR- the GR signalling pathway, it exerts proliferative and anti-apoptotic 99a-mediated apoptotic response to Dex. Thus, we hypothesised properties (Denny et al, 2000; Romano et al, 2011). GR, which can that MCL1 might also be involved in miR-100- and miR-99a- continuously shuttle between the nuclear and cytoplasmic triggered apoptosis. To prove this hypothesis, we first examined the compartments, is an important ligand-dependent transcription expression of mTOR and MCL1 protein levels following induction factor that belongs to the nuclear receptor family and is also a 2196 www.bjcancer.com | DOI:10.1038/bjc.2013.562 MiR-100/99a in acute lymphoblastic leukaemia BRITISH JOURNAL OF CANCER monomeric protein that cooperates with other transcription factors Specifically, our experimental data indicate that miR-100 and to induce transcription (Bray and Cotton, 2003; Vandevyver et al, miR-99a are involved in two essential signalling pathways: 2012). Davies and colleagues revealed that the Dex-binding event (i) influencing GR signalling by targeting FKBP51 and causes a dissociation of FKBP51 and GR-complex that, in turn, (ii) suppressing the IGF1R/mTOR pathway and down-regulating control the intracellular trafficking of GR (Davies et al, 2002). In a the expression of the anti-apoptotic gene MCL1 to inhibit cell loss-of-function study, our results demonstrated that the relatively growth and initiate cell apoptosis. The modulation of multiple silenced expression of FKBP51 is responsible for the accumulation cancer-associated pathways underscores the significance of of GR expression in the nucleus after Dex induction, and miR-100 and miR-99a for future clinical treatments. Therefore, our results further revealed that miR-100/99a overexpression our study suggests a novel gene therapy strategy, namely, the influenced the translocation of GR to the nucleus. After enhancement of Dex-induced apoptosis, for patients with ALL. translocating to the nucleus, phosphorylation at Ser-211 is essential for subsequent GR-dependent signalling (Chen et al, 2008). We found that silenced FKBP51 increased the phosphor-GR (Ser-211) ACKNOWLEDGEMENTS expression in the nucleus of Jurkat cells. Furthermore, miR-100 and miR-99a overexpression decreased the FKBP51 protein levels This work was supported by grants from the National Science and and led to GR activation in the nucleus after exposure to Dex. Technology Department of China and National Science Foundation These findings suggest that, in the miR-100- and miR-99a- of China. mediated response to Dex in ALL cells, the GR signalling pathway is activated by the suppression of FKBP51. Recent studies have shown that multimeric chaperones, including hsp90, p23, FKBP52, CONFLICT OF INTEREST dynamitin, and dynein, are required for the translocation of GR (Echeverria et al, 2009), yet further research will be needed to The authors declare no conflict of interest. determine whether other importins or nucleoporins are required and to elucidate the complete details of how miR-100 and miR-99a affect GR activity through FKBP51. REFERENCES Another contribution of the study is the validation of two other important target genes, IGF1R and mTOR, which are involved in Agirre X, Vilas-Zornoza A, Jimenez-Velasco A, Martin-Subero JI, Cordeu L, the miR-100- and miR-99-regulated pathways. 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NonCommercial-Share Alike 3.0 Unported License. Supplementary Information accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc) 2198 www.bjcancer.com | DOI:10.1038/bjc.2013.562

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British Journal of CancerSpringer Journals

Published: Sep 12, 2013

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