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LncRNA HOTAIR regulates HIF-1α/AXL signaling through inhibition of miR-217 in renal cell carcinoma

LncRNA HOTAIR regulates HIF-1α/AXL signaling through inhibition of miR-217 in renal cell carcinoma Citation: Cell Death and Disease (2017) 8, e2772; doi:10.1038/cddis.2017.181 OPEN Official journal of the Cell Death Differentiation Association www.nature.com/cddis LncRNA HOTAIR regulates HIF-1α/AXL signaling through inhibition of miR-217 in renal cell carcinoma 1 1 1 2 2 1 1 2 ,1 Quan Hong ,Ou Li , Wei Zheng , Wen-zhen Xiao , Lu Zhang ,Di Wu , Guang-yan Cai , John Cijiang He and Xiang-mei Chen* Long non-coding RNA HOTAIR was regarded as an oncogene in multiple cancers. Previous studies have shown that HOTAIR is involved in the proliferation and tumorigenesis of renal carcinoma cells, while microRNA (miR)-217 functions as a tumor suppressor in renal cell carcinoma (Rcc). However, the underlying molecular mechanism of HOTAIR in Rcc, especially in association with miR-217, has not been studied. In this study, we first demonstrated that HOTAIR expression was upregulated, which was correlated with tumor progression, and miR-217 downregulated in Rcc tissues and cells. Importantly, HOTAIR expression was negatively correlated with miR-217 expression in Rcc tissues. Gain- and loss-of-function of HOTAIR revealed that HOTAIR functioned as a ceRNA for miR-217 to facilitate HIF-1α expression and then upregulated AXL level promoting Rcc proliferation, migration, and EMT process, and inhibiting apoptosis. Furthermore, HOTAIR knockdown suppressed tumor growth and reduced the expression of proliferation antigen ki-67, HIF-1α, and AXL, but upregulated the expression of miR-217 in vivo. Finally, with AXL inhibitor BGB324, we confirmed that HOTAIR promoted Rcc activity through AXL signaling both in vitro and in vivo. In conclusion, these results suggest that HOTAIR promotes Rcc tumorigenesis via miR-217/HIF-1α/AXL signaling, which may provide a new target for the diagnosis and therapy of Rcc disease. Cell Death and Disease (2017) 8, e2772; doi:10.1038/cddis.2017.181; published online 11 May 2017 Renal cell carcinoma (Rcc) represents more than 90% of silencing HOTAIR expression inhibits the proliferation and cases of kidney cancer, which is currently the 9th most tumorigenicity of Rcc cells. However, the underlying molecular common cancer in men and the 14th in women all over the mechanism of HOTAIR in Rcc has not been fully understood. world. Rcc is mainly classified into three histological Recent reports have suggested that lncRNAs function subtypes: clear cell (70–80%), papillary (10–15%), and as competing endogenous RNAs (ceRNAs) for microRNA (miRNA) to mediate mRNAs expression at post-transcriptional chromophobe (5–10%) Rcc. The incidence of Rcc varies level. HOTAIR regulates cyclin J expression via inhibition of between countries and is still increased worldwide. Despite miR-205 expression in bladder cancer. HOTAIR modulates advances in imaging techniques, the diagnosis of early stage HER2 expression through sponging miR-331-3p to promote kidney cancer is very poor. In addition, the resistance to the tumorigenesis of gastric cancer. miR-217 is a tumor traditional therapies results in poor prognosis in Rcc 4,5 suppressor in many cancers including Rcc. Recently, patients. Therefore, it is urgent to find an effective method studies showed that the interaction between HOTAIR and for early diagnosis and treatment of Rcc. PIK3R3 is mediated through miR-217 in ovarian cancer Long non-coding RNAs (lncRNAs), a heterogeneous class of cells, but the interaction between HOTAIR and miR-217 in transcripts longer than 200 nucleotides, are pervasively tran- Rcc has not been reported before. scribed in the genome and associated with physiological and In this study, we hypothesized that HOTAIR might promote pathological processes. lncRNAs cannot code protein but can Rcc progression through inhibition of miR-217 expression. In the regulate gene expression at the transcriptional and post- 5,6 present study, we first detected the expression of HOTAIR and transcriptional levels. Increasing evidence indicates that miR-217 in tumor tissues from clear cell Rcc patients as well as lncRNAs are involved in the modulation of cancer cell behavior, in Rcc cells. Furthermore, the underlying mechanism of HOTAIR such as proliferation, metastasis, epithelial-mesenchymal transi- in the development of Rcc was analyzed both in vitro and in vivo. tion (EMT), apoptosis, and drug resistance. HOX transcript This study may provide a new target for Rcc treatment. antisense intergenic RNA (HOTAIR) is a lncRNA encoded by the human HOXC locus on chromosome 12q13.13. Increased expression of HOTAIR has been involved in the increased Results 8,9 metastasis of breast, cervical, and colorectal cancer. Further- more, HOTAIR is associated with the poor prognosis of HOTAIR expression is negatively correlated with miR-217 8,10 pancreatic, breast, liver, and cervical cancer. Recent studies in human renal cancer tissues. To verify whether HOTAIR reported that HOTAIR expression is elevated in Rcc cells, and is differently expressed in Rcc tissues, HOTAIR expression Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Fuxing Road 28, Haidian District, Beijing 100853, China and Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA *Corresponding author: X-m Chen, Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Fuxing Road 28, Haidian District, Beijing 100853, China. Tel: +86 1068797416; Fax: +86 1068797416; E-mail: xmchen301@126.com Received 18.1.17; revised 16.3.17; accepted 17.3.17; Edited by R Mantovani HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al Figure 1 The expression of HOTAIR and miR-217 in Rcc tissues and cells and the relationship between HOTAIR and miR-217. qRT-PCR for the expression of HOTAIR (a) and miR-217 (b) in Rcc tissues and adjacent histological normal tissues. Data were analyzed by paired Student’s t-test. The expression levels of HOTAIR (c) and miR-217 (d) were assayed in Rcc cells (769-P and ACHN) and normal kidney cells (HK-2). The expression of HOTAIR and miR-217 was normalized to that in HK-2. The differences between groups were analyzed by unpaired Student’s t-test. (e) Bivariate correlation analysis of the relationship between HOTAIR expression and miR-217 level. (f) The putative miR-217 binding sequence of the wild type and mutation sequence of HOTAIR. (g) Relative luciferase assays. Statistical analysis was conducted using unpaired Student’s t-test. *Po0.05 was determined in 86 paired Rcc samples and adjacent Table 1 Correlation between HOTAIR expression and patient characteristics histological normal tissues. As shown in Figure 1a, compared Characteristic Number (%) HOTAIR P-value with normal control, HOTAIR expression was notably expression upregulated in Rcc tissues. Interestingly, the elevated Age 0.544 HOTAIR expression was positively correlated with the TNM ⩽ 60 53 (61.6) 6.86± 4.96 stage (Table 1). Also, we detected miR-217 expression and 460 33 (38.4) 7.56± 5.58 found it was reduced in Rcc tissues (Figure 1b). Surprisingly, miR-217 expression was inversely correlated with HOTAIR Gender 0.796 Male 61 (70.9) 7.22± 5.12 levels in 86 Rcc tissue samples (r = 0.4155, Po0.0001) Female 25 (29.1) 6.90± 5.43 (Figure 1e). Moreover, HOTAIR level was higher and miR-217 was lower in Rcc cell lines, including 769-P and ACHN cells, Tumor stage o0.001 T1 36 (41.9) 5.30± 3.97 than in normal renal cells (HK-2; Figures 1c and d). T2 25 (29.0) 8.12± 5.08 T3 17 (19.8) 10.45± 3.02 HOTAIR is a target of miR-217. To clarify the underlying T4 8 (9.3) 19.13± 5.59 relationship between HOTAIR and miR-217, we performed Lymph nodes metastasis o0.001 bioinformatics analysis by miRcode (http://www.mircode.org). N0 65 5.56± 3.28 The data showed that HOTAIR contains one conserved target N1 21 12.56± 6.79 site of miR-217 (Figure 1f), which is consistent with previous Metastasis o0.001 study. Moreover, miR-217 reduced the luciferase activity of M0 63 5.89± 4.13 pGL3-HOTAIR but not pGL3-HOTAIR-MUTANT (Figure 1g). M1 23 11.69± 7.56 These results indicate that miR-217 could bind directly to HOTAIR at the miRNA recognition sites. HOTAIR modulates Rcc proliferation, migration, apoptosis, migration, and the expression of Vimentin and Snail, but and EMT via negative regulation of miR-217. To further reduced E-cadherin expression in ACHN cells; however, this investigate whether HOTAIR functions through miR-217, effect could be ablated by miR-217 mimic (Figures 2a, c, f, HOTAIR expression was upregulated/downregulated and and h). In addition, HOTAIR downregulation significantly intervened with miR-217 mimic or anti-217 in Rcc cells. reduced the proliferation, migration, and the expression of HOTAIR overexpression increased the proliferation, Vimentin and Snail induced by TGF-β1, but facilitated Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al Figure 2 HOTAIR modulates Rcc proliferation, migration, apoptosis, and EMT via negative regulation of miR-217. Cell viability was determined by MTT assay in ACHN (a) and 769-P (b) cells. The inset graph illustrates the efficiency of si-HOTAIR on HOTAIR expression in 769-P cells. Transwell assays were used to analyze the migration of ACHN (c) and 769-P (d) cells. (e) Flow cytometry for apoptosis in 769-P cells. (f) qRT-PCR assay for the mRNA expression of E-cadherin, Vimentin, and Snail in ACHN cells. (g) The mRNA levels of EMT-related genes in 769-P cells. (h) The protein levels of EMT-related genes in Rcc cells. *Po0.05 versus NC group, Po0.05 versus HOTAIR or si-HOTAIR group, Po0.05 versus TGF-β group. Data were analyzed using One-way ANOVA E-cadherin expression and the apoptosis of 769-P cells, HOTAIR and HIF-1α share the same response elements of which could be counteracted by anti-217 (Figures 2b, d, e, g, miR-217 (Figures 1f and 3a). Hence, HOTAIR may act as a and h). These data suggest that HOTAIR promotes Rcc cell ceRNA for miR-217 to mediate HIF-1α expression in Rcc proliferation, migration, EMT, and inhibits apoptosis via development. To test this hypothesis, we first determined downregulation of miR-217. miR-217 expression after downregulation of HOTAIR and found si-HOTAIR significantly upregulated miR-217 levels in HOTAIR functions as a ceRNA for miR-217 to facilitate Rcc cells (Figure 3e). Then, we performed an RIP assay on HIF-1α expression. Through searching TargetScan (http:// Ago2, which is the vital component of the RNA-induced www.targetscan.org), we found that miR-217 could bind to silencing complex (RISC). As shown in Figure 3f, the 3′UTR of HIF-1α (Figure 3a). Moreover, miR-217 mimic overexpression of HOTAIR led to the increased enrich- significantly suppressed the mRNA and protein level of ment of Ago2 on HOTAIR but substantially decreased HIF-1α in ACHN cells, while downregulation of miR-217 enrichment on HIF-1α transcripts. In parallel, knockdown of increased HIF-1α expression in 769-P cells (Figures 3b HOTAIR had the contrary effects. These results indicate that and c). In addition, miR-217 remarkably dampened the HOTAIR could compete with HIF-1α transcripts for the luciferase activity of cells transfected with pGL3-HIF-1α but Ago2-based RISC. not pGL3-HIF-1α-MUTANT, suggesting a direct interaction Moreover, we evaluated whether the HOTAIR-mediated between miR-217 and the 3′UTR of HIF-1α (Figure 3d). sequestration of miR-217 was responsible for the upregulation Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al Figure 3 HOTAIR functions as a ceRNA for miR-217 to facilitate HIF-1α expression. (a) The putative miR-217 binding 3′UTR of HIF-1α (HIF-1α-WT) and HIF-1α mutation sequence (HIF-1α-MUT). The protein (b) and mRNA (c) levels of HIF-1α in ACHN and 769-P cells. *Po0.05 versus NC group. (d) Luciferase activity assay. *Po0.05 versus NC- mimic group. (e) The expression of miR-217 in 769-P cell transfected with si-NC or si-HOTAIR. *Po0.05 versus NC group. Data were analyzed by unpaired Student’s t-test. (f) RIP assay of the enrichment of Ago2 on HOTAIR and HIF-1α transcripts relative to IgG in cells transfected with pLVX-HOTAIR or si-HOTAIR. (g) Luciferase activity of pGL3 reporters which contained wild type or mutant HIF-1α 3′UTR with indicated treatment in Rcc cells. (h) qRT-PCR analysis for HIF-1α mRNA expression in ACHN and 769-P cells. # # *Po0.05 versus pLVX group, Po0.05 versus HOTAIR group in ACHN cells; *Po0.05 versus si-NC group, Po0.05 versus si-HOTAIR group in 769-P cells. Statistical analysis was conducted using One-way ANOVA of HIF-1α. The luciferase activity of HIF-1α wild-type reporters HOTAIR promotes Rcc tumorigenesis through HIF-1α/ but not the mutant one was significantly increased after AXL signaling both in vitro and in vivo. Studies have HOTAIR upregulation, whereas miR-217 mimic abolished this indicated that HIF-1 could bind directly to AXL and activate its effect. And HOTAIR siRNA showed the reversed effect on the expression; hence, to further investigate the mechanisms of luciferase activity of HIF-1α, which was rescued by anti-217 HOTAIR, we detected AXL expression in Rcc cells. HOTAIR (Figure 3g). In addition, these results were further confirmed at overexpression increased the mRNA and protein levels of HIF-1α mRNA level (Figure 3h). Collectively, these results AXL, whereas downregulation of HOTAIR notably decreased suggest that HOTAIR functions as a molecular sponge for their expression (Figures 4a and b). To determine whether miR-217 to facilitate HIF-1α expression. HOTAIR promotes Rcc activity through AXL signaling, an Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al Figure 4 HOTAIR regulates Rcc activity through AXL signaling in vitro. The mRNA (a) and protein (b) expression of AXL in ACHN and 769-P cells. Statistical analysis was conducted by unpaired Student’s t-test. (c) ACHN cells stably transfected with HOTAIR were treated with BGB324 (0.1, 0.5, 1 nM) for 72 h to examine the cell viability. Data were analyzed using One-way ANOVA. (d) The protein expression of E-cadherin, Vimentin, and Snail in ACHN cells stably transfected with HOTAIR and treated with different concentrations of BGB324 were assayed by western blot. The mRNA expression of HIF-1α (e) and AXL (f) in 86 paired Rcc tissues and adjacent histological normal tissues. Data were analyzed paired Student’s t-test. *Po0.05 versus control group, Po0.05 versus HOTAIR treated only group AXL inhibitor BGB324 was used. The results showed that levels (Figures 5c–f). In addition, the role of AXL in HOTAIR- BGB324 suppressed the effect of HOTAIR upregulation on mediated Rcc tumorigenesis was further confirmed by the proliferation and EMT process of ACHN cells in a dose- oral administration of BGB324. BGB324 notably decreased dependent manner (Figures 4c and d). Also, the mRNA levels HOTAIR-induced tumor growth and EMT process (Figures 5g and h). Collectively, these data show that HOTAIR promotes of HIF-1α and AXL were detected in 86 Rcc samples and Rcc tumorigenesis via activating HIF-1/AXL signaling. adjacent histological normal tissues. As shown in Figures 4e and f, the mRNA expression of HIF-1α and AXL in Rcc tissues was dramatically upregulated compared with normal Discussion control tissues. Furthermore, the role of HOTAIR in Rcc tumorigenesis was analyzed in vivo and the results showed Studies have revealed that lncRNAs play vital roles in Rcc that knockdown of HOTAIR significantly inhibited tumor pathogenesis. However, the role and mechanism of HOTAIR growth and the staining intensity of proliferation antigen in Rcc has not been fully understood. In this study, we found ki-67 (Figures 5a and b). HOTAIR downregulation reduced that HOTAIR expression was significantly increased in Rcc the expression of HIF-1α and AXL, but elevated miR-217 tissue samples compared with their corresponding non-tumor Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al Figure 5 HOTAIR promotes Rcc cell growth via AXL signaling in vivo.(a) Tumor volume was evaluated for 12–30 days. The inset graph illustrates the efficiency of si-HOTAIR on HOTAIR expression. (b) Representative ki-67 staining (×100). mRNA expression of miR-217 (c), HIF-1α (d), and AXL (e) in tumor tissues. Statistical analysis was conducted by unpaired Student’s t-test. *Po0.05 versus si-NC group (f) Western blot analysis for the protein levels of HIF-1α and AXL in tumor tissues. (g) Mice were subcutaneously injected with ACHN cells stably transfected with HOTAIR and orally administrated with BGB324 twice daily. Tumor volume was examined. (h) The expression of E-cadherin, Vimentin, and Snail was determined by Western blot. Data were analyzed using One-way ANOVA. n= 6 per group, *Po0.05 versus pLVX group, Po0.05 versus HOTAIR group. (i) Schematic of the proposed mechanism of HOTAIR in Rcc. HOTAIR functions as a ceRNA to ‘sponge’ miR-217 and upregulates the expression of HIF-1α as well as the HIF-1α downstream effector that promotes Rcc progression tissues, and the elevated expression was positively correlated The results of bioinformatics analysis showed HOTAIR with the TNM stage (Figure 1). Also, HOTAIR expression was possesses one conserved target site for miR-217 binding. And higher in Rcc cells. Studies have shown that HOTAIR acts as a dual-luciferase reporter assay demonstrated that miR-217 ceRNA to sponge miRNAs to modulate the de-repression of could reduce the luciferase activity of pGL3-HOTAIR 13,14 miRNA targets in bladder and gastric cancer. Hence, we (Figure 1). In addition, HOTAIR overexpression markedly speculated that HOTAIR acts as a ceRNA in Rcc. miR-217 increased the proliferation, migration, and EMT process, plays a suppression role in the growth of various types of which were ablated by miR-217 mimic. Contrarily, knockdown tumors including Rcc. We further investigated miR-217 of HOTAIR remarkably reduced the proliferation, migration, expression and found it was remarkably downregulated in Rcc EMT, and promoted the apoptosis of 769-P cells, which could tissues and cells. Furthermore, miR-217 expression was be counteracted by anti-217 (Figure 2). Taken together, these negatively correlated with HOTAIR level in Rcc tissues results indicate that HOTAIR promotes Rcc development via (Figure 1). negative regulation of miR-217. Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al HIF-1α is a transcription factor that is frequently stabilized pathway can be regulated directly by miR-217 targeting PTEN 20 29 and active in human clear cell Rcc. It is largely responsible (independent of AXL), which may account for the role of for activating the transcription of target genes that are widely HOTAIR in our study. Taken together, these results indicate 20,21 involved in the malignant features of tumors. High levels of that HOTAIR promotes Rcc tumorigenesis partly by activating HIF-1α are associated with the poor prognosis of clear cell HIF-1α/AXL signaling. Rcc. It has previously been reported that miR-217 increased In conclusion, our study revealed that HOTAIR is an HIF-1α levels by targeting SIRT1 in rat glomerular mesangial oncogene in Rcc. Elevated level of HOTAIR is positively cells cultured with high glucose. However, in our study, the correlated with tumor progression. HOTAIR functions as a result of bioinformatics analysis revealed HIF-1α is a potential ceRNA for miR-217 to facilitate Rcc progression partly via target of miR-217. And miR-217 decreased HIF-1α expres- HIF-1α/AXL signaling (Figure 5i). Thus, our study sion, which could be upregulated by anti- 217. Also, miR-217 provides further insight into the molecular mechanism of notably dampened the luciferase activity of pGL3-HIF-1α. HOTAIR in Rcc tumorigenesis, which may promote the These results indicated that HIF-1α was a direct target of development of lncRNA-directed diagnosis and therapy for miR-217. An RIP assay on Ago2 showed HOTAIR over- this disease. expression increased the enrichment on HOTAIR but sig- nificantly decreased the enrichment on HIF-1α transcripts and Material and Methods knockdown of HOTAIR had the reversed effect. These results Patients and samples. 86 clear cell Rcc tissue samples and neighboring indicated that HOTAIR could compete with HIF-1α transcripts non-cancerous tissues (collected postoperatively from June 2011 to January 2014) used in this study were obtained from Chinese PLA General Hospital. All patients for the Ago2-based RISC. Furthermore, the luciferase activity provided informed consent and had not undergone any previous therapy. Tumors of HIF-1α reporters and HIF-1α mRNA expression were were classified according to the tumor-node-metastasis (TNM) system of increased after HOTAIR overexpression, but reversed by classification (2010 version). This study was approved by the Human Research miR-217 mimic; however, they were reduced when HOTAIR Ethics Committee of Chinese PLA General Hospital (Beijing, China). The clinical was downregulated, which could be rescued by anti-217 data of patients were shown in Table 1. (Figure 3). Taken together, these results suggest that HOTAIR functions as a ceRNA for miR-217 to facilitate the expression Cell lines. The human Rcc cell lines (769-P and ACHN) and normal kidney cell of HIF-1α. line (kidney proximal tubular cells, HK-2) were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and cultured in a humidified Rankin et al. demonstrated that HIF-1 bound directly to incubator at 37 °C with an atmosphere of 5% CO . 769-P, ACHN, and HK-2 cells AXL to activate its expression. AXL, a member of the TAM were maintained in RPMI-1640, MEM, and K-SFM medium, respectively. family of receptor tyrosine kinases (RTKs), has been identified as an essential mediator of cancer metastasis including Rcc. Plasmid constructs and transfection. HOTAIR cDNA was amplified from Binding of its ligand, growth arrest-specific protein 6 (Gas6), human Rcc tissue with primers: 5′-CCGCTCGAGACATTCTGCCCTGATTTCCG- AXL receptor is dimerized and autophosphorylated at its GAACC-3′ (forward) and 5′-CGCGGATCCCCACCACACACACACAACCTACAC-3′ tyrosine resides, which subsequently triggers a cascade of (reverse). cDNA was then cloned into the XhoI and BamHI sites of pLVX-IRES-Neo vector (Invitrogen) to construct the pLVX-HOTAIR vector. The vector was then intracellular signaling, involving PI3K/Akt, ERK, NF-κB, p38, transfected into HEK293T cells to package lentivirus using Lipofectamine 2000 Rho family proteins, JAK-STAT, and Src family kinases. AXL (Invitrogen). ACHN cells were infected by the lentivirus and stably transfected cells oncogenic signaling promotes cancer cell survival, prolifera- were selected by G418 (0.5 mg/ml, Sigma-Aldrich, St Louis, MO, USA). To tion, migration, invasion, and the EMT phenotype, as well as construct luciferase reporter vectors, HIF-1α 3′-untranslated regions (UTR) and 25,26 resisting tumor cell apoptosis. In addition, AXL mediates a HOTAIR cDNA fragment containing the predicted potential miR-217 binding sites or resistance to sunitinib treatment in Rcc. AXL is highly mutant sites were amplified by PCR, and then cloned to into the XhoI and KpnI sites of pGL3 luciferase reporter vector (Promega, Madison, WI, USA). expressed in Rcc tissues and cells, and knockdown of AXL HOTAIR siRNA, control siRNA, miR-217 mimic, control mimic (NC-mimic), miR-217 reduces cell viability. To evaluate whether HOTAIR regulates antagomirs (anti-217), and control antagomirs (anti-NC) were synthesized by Rcc activity through HIF-1α/AXL signaling, we detected AXL GenePharma Co. (Shanghai, China). The sequences were listed in Supplementary expression after gain- and loss-of-function of HOTAIR in Rcc Table S1. To construct si-HOTAIR vector, the self-complementary hairpin DNA cells. Our results revealed that overexpression of HOTAIR oligonucleotides were annealed and subcloned into the pEGFP-N1 plasmid vector. A notably increased while downregulation of HOTAIR decreased negative control was named as si-NC vector. The vectors were transfected into 769-P cells with Lipofectamine 2000 and stable cell lines were established by G418 selection. AXL expression. Similarly, the expression of HIF-1α and AXL Stable transfected cells were grown in 6-well plates and transfected using Lipofectamine in Rcc tissues was higher than in normal tissues in the clinical 2000 according to manufacturer’s instructions. Cells were collected for real-time PCR or specimens. Moreover, BGB324 inhibited the effect of HOTAIR western bolt 48 h after transfection. The final concentrations of miRNAs or plasmids upregulation on the proliferation and EMT process of ACHN used in this study were as follows: HOTAIR/negative control 50 nM/ml, HOTAIR siRNA/ cells in a dose-dependent manner (Figure 4). In addition, control siRNA 30 nM/ml, miR-217 mimic/NC mimic 120 nM/ml, and anti-217/ anti-NC HOTAIR knockdown significantly suppressed tumor growth 200 nM/ml. and ki-67 expression, increased miR-217 levels and decreased the expression of HIF-1α and AXL, and BGB324 Proliferation assay. Proliferation of cells transfected with indicated vector was measured by MTT assay kit at 24, 48, and 72 h, respectively. In addition, ACHN abrogated the effect of HOTAIR on tumor growth and EMT cells with stable HOTAIR upregulation were treated with BGB324 (0.1, 0.5, 1 nM), process in vivo (Figure 5). However, si-HOTAIR treatment which also known as R428 (Selleck, Houston, TX, USA), for 72 h and the cell resulted in a 450% decrease in tumor volume after 30 days, viability was detected. whereas treatment with the AXL inhibitor resulted in a much more modest reduction of o20%, strongly implying Transwell analysis. Cells, transfected with the designated vector for 24 h, that other AXL-independent pathways make important were seeded in the upper chambers in 200 μl serum-free medium at a density of contributions. Previous study has reported that the PI3K/Akt 2× 10 /well. The lower chambers were filled with 500 μl medium containing 10% Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al FBS for inducing cell migration. After 48 h incubation, the cells attached to the lower Conflict of Interest surface were fixed with 4% paraformaldehyde, stained with crystal violet, and then The authors declare no conflict of interest. examined under a microscope. Flow cytometry. Apoptosis was assayed through dual staining with annexin Acknowledgements. This work was supported by Chinese National Natural V-FITC and propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, USA). In Sciences Foundation (No. 81470949), Major State Basic Research Development brief, cells were harvested 48 h after transfection; Annexin V-FITC and PI were Program of China (No. 2013CB530800 and No. 2014CBA02005). added to the cellular suspension according to the manufacturer’s instructions. Then, samples were analyzed by a FACSCalibur flow cytometer (BD, San Jose, CA, USA). 1. Znaor A, Lortet-Tieulent J, Laversanne M, Jemal A, Bray F. International variations and trends in renal cell carcinoma incidence and mortality. Eur Urol 2015; 67: 519–530. 2. Silva-Santos RM, Costa-Pinheiro P, Luis A, Antunes L, Lobo F, Oliveira J et al. 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Cell Death and Disease http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death & Disease Springer Journals

LncRNA HOTAIR regulates HIF-1α/AXL signaling through inhibition of miR-217 in renal cell carcinoma

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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
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10.1038/cddis.2017.181
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Citation: Cell Death and Disease (2017) 8, e2772; doi:10.1038/cddis.2017.181 OPEN Official journal of the Cell Death Differentiation Association www.nature.com/cddis LncRNA HOTAIR regulates HIF-1α/AXL signaling through inhibition of miR-217 in renal cell carcinoma 1 1 1 2 2 1 1 2 ,1 Quan Hong ,Ou Li , Wei Zheng , Wen-zhen Xiao , Lu Zhang ,Di Wu , Guang-yan Cai , John Cijiang He and Xiang-mei Chen* Long non-coding RNA HOTAIR was regarded as an oncogene in multiple cancers. Previous studies have shown that HOTAIR is involved in the proliferation and tumorigenesis of renal carcinoma cells, while microRNA (miR)-217 functions as a tumor suppressor in renal cell carcinoma (Rcc). However, the underlying molecular mechanism of HOTAIR in Rcc, especially in association with miR-217, has not been studied. In this study, we first demonstrated that HOTAIR expression was upregulated, which was correlated with tumor progression, and miR-217 downregulated in Rcc tissues and cells. Importantly, HOTAIR expression was negatively correlated with miR-217 expression in Rcc tissues. Gain- and loss-of-function of HOTAIR revealed that HOTAIR functioned as a ceRNA for miR-217 to facilitate HIF-1α expression and then upregulated AXL level promoting Rcc proliferation, migration, and EMT process, and inhibiting apoptosis. Furthermore, HOTAIR knockdown suppressed tumor growth and reduced the expression of proliferation antigen ki-67, HIF-1α, and AXL, but upregulated the expression of miR-217 in vivo. Finally, with AXL inhibitor BGB324, we confirmed that HOTAIR promoted Rcc activity through AXL signaling both in vitro and in vivo. In conclusion, these results suggest that HOTAIR promotes Rcc tumorigenesis via miR-217/HIF-1α/AXL signaling, which may provide a new target for the diagnosis and therapy of Rcc disease. Cell Death and Disease (2017) 8, e2772; doi:10.1038/cddis.2017.181; published online 11 May 2017 Renal cell carcinoma (Rcc) represents more than 90% of silencing HOTAIR expression inhibits the proliferation and cases of kidney cancer, which is currently the 9th most tumorigenicity of Rcc cells. However, the underlying molecular common cancer in men and the 14th in women all over the mechanism of HOTAIR in Rcc has not been fully understood. world. Rcc is mainly classified into three histological Recent reports have suggested that lncRNAs function subtypes: clear cell (70–80%), papillary (10–15%), and as competing endogenous RNAs (ceRNAs) for microRNA (miRNA) to mediate mRNAs expression at post-transcriptional chromophobe (5–10%) Rcc. The incidence of Rcc varies level. HOTAIR regulates cyclin J expression via inhibition of between countries and is still increased worldwide. Despite miR-205 expression in bladder cancer. HOTAIR modulates advances in imaging techniques, the diagnosis of early stage HER2 expression through sponging miR-331-3p to promote kidney cancer is very poor. In addition, the resistance to the tumorigenesis of gastric cancer. miR-217 is a tumor traditional therapies results in poor prognosis in Rcc 4,5 suppressor in many cancers including Rcc. Recently, patients. Therefore, it is urgent to find an effective method studies showed that the interaction between HOTAIR and for early diagnosis and treatment of Rcc. PIK3R3 is mediated through miR-217 in ovarian cancer Long non-coding RNAs (lncRNAs), a heterogeneous class of cells, but the interaction between HOTAIR and miR-217 in transcripts longer than 200 nucleotides, are pervasively tran- Rcc has not been reported before. scribed in the genome and associated with physiological and In this study, we hypothesized that HOTAIR might promote pathological processes. lncRNAs cannot code protein but can Rcc progression through inhibition of miR-217 expression. In the regulate gene expression at the transcriptional and post- 5,6 present study, we first detected the expression of HOTAIR and transcriptional levels. Increasing evidence indicates that miR-217 in tumor tissues from clear cell Rcc patients as well as lncRNAs are involved in the modulation of cancer cell behavior, in Rcc cells. Furthermore, the underlying mechanism of HOTAIR such as proliferation, metastasis, epithelial-mesenchymal transi- in the development of Rcc was analyzed both in vitro and in vivo. tion (EMT), apoptosis, and drug resistance. HOX transcript This study may provide a new target for Rcc treatment. antisense intergenic RNA (HOTAIR) is a lncRNA encoded by the human HOXC locus on chromosome 12q13.13. Increased expression of HOTAIR has been involved in the increased Results 8,9 metastasis of breast, cervical, and colorectal cancer. Further- more, HOTAIR is associated with the poor prognosis of HOTAIR expression is negatively correlated with miR-217 8,10 pancreatic, breast, liver, and cervical cancer. Recent studies in human renal cancer tissues. To verify whether HOTAIR reported that HOTAIR expression is elevated in Rcc cells, and is differently expressed in Rcc tissues, HOTAIR expression Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Fuxing Road 28, Haidian District, Beijing 100853, China and Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA *Corresponding author: X-m Chen, Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Fuxing Road 28, Haidian District, Beijing 100853, China. Tel: +86 1068797416; Fax: +86 1068797416; E-mail: xmchen301@126.com Received 18.1.17; revised 16.3.17; accepted 17.3.17; Edited by R Mantovani HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al Figure 1 The expression of HOTAIR and miR-217 in Rcc tissues and cells and the relationship between HOTAIR and miR-217. qRT-PCR for the expression of HOTAIR (a) and miR-217 (b) in Rcc tissues and adjacent histological normal tissues. Data were analyzed by paired Student’s t-test. The expression levels of HOTAIR (c) and miR-217 (d) were assayed in Rcc cells (769-P and ACHN) and normal kidney cells (HK-2). The expression of HOTAIR and miR-217 was normalized to that in HK-2. The differences between groups were analyzed by unpaired Student’s t-test. (e) Bivariate correlation analysis of the relationship between HOTAIR expression and miR-217 level. (f) The putative miR-217 binding sequence of the wild type and mutation sequence of HOTAIR. (g) Relative luciferase assays. Statistical analysis was conducted using unpaired Student’s t-test. *Po0.05 was determined in 86 paired Rcc samples and adjacent Table 1 Correlation between HOTAIR expression and patient characteristics histological normal tissues. As shown in Figure 1a, compared Characteristic Number (%) HOTAIR P-value with normal control, HOTAIR expression was notably expression upregulated in Rcc tissues. Interestingly, the elevated Age 0.544 HOTAIR expression was positively correlated with the TNM ⩽ 60 53 (61.6) 6.86± 4.96 stage (Table 1). Also, we detected miR-217 expression and 460 33 (38.4) 7.56± 5.58 found it was reduced in Rcc tissues (Figure 1b). Surprisingly, miR-217 expression was inversely correlated with HOTAIR Gender 0.796 Male 61 (70.9) 7.22± 5.12 levels in 86 Rcc tissue samples (r = 0.4155, Po0.0001) Female 25 (29.1) 6.90± 5.43 (Figure 1e). Moreover, HOTAIR level was higher and miR-217 was lower in Rcc cell lines, including 769-P and ACHN cells, Tumor stage o0.001 T1 36 (41.9) 5.30± 3.97 than in normal renal cells (HK-2; Figures 1c and d). T2 25 (29.0) 8.12± 5.08 T3 17 (19.8) 10.45± 3.02 HOTAIR is a target of miR-217. To clarify the underlying T4 8 (9.3) 19.13± 5.59 relationship between HOTAIR and miR-217, we performed Lymph nodes metastasis o0.001 bioinformatics analysis by miRcode (http://www.mircode.org). N0 65 5.56± 3.28 The data showed that HOTAIR contains one conserved target N1 21 12.56± 6.79 site of miR-217 (Figure 1f), which is consistent with previous Metastasis o0.001 study. Moreover, miR-217 reduced the luciferase activity of M0 63 5.89± 4.13 pGL3-HOTAIR but not pGL3-HOTAIR-MUTANT (Figure 1g). M1 23 11.69± 7.56 These results indicate that miR-217 could bind directly to HOTAIR at the miRNA recognition sites. HOTAIR modulates Rcc proliferation, migration, apoptosis, migration, and the expression of Vimentin and Snail, but and EMT via negative regulation of miR-217. To further reduced E-cadherin expression in ACHN cells; however, this investigate whether HOTAIR functions through miR-217, effect could be ablated by miR-217 mimic (Figures 2a, c, f, HOTAIR expression was upregulated/downregulated and and h). In addition, HOTAIR downregulation significantly intervened with miR-217 mimic or anti-217 in Rcc cells. reduced the proliferation, migration, and the expression of HOTAIR overexpression increased the proliferation, Vimentin and Snail induced by TGF-β1, but facilitated Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al Figure 2 HOTAIR modulates Rcc proliferation, migration, apoptosis, and EMT via negative regulation of miR-217. Cell viability was determined by MTT assay in ACHN (a) and 769-P (b) cells. The inset graph illustrates the efficiency of si-HOTAIR on HOTAIR expression in 769-P cells. Transwell assays were used to analyze the migration of ACHN (c) and 769-P (d) cells. (e) Flow cytometry for apoptosis in 769-P cells. (f) qRT-PCR assay for the mRNA expression of E-cadherin, Vimentin, and Snail in ACHN cells. (g) The mRNA levels of EMT-related genes in 769-P cells. (h) The protein levels of EMT-related genes in Rcc cells. *Po0.05 versus NC group, Po0.05 versus HOTAIR or si-HOTAIR group, Po0.05 versus TGF-β group. Data were analyzed using One-way ANOVA E-cadherin expression and the apoptosis of 769-P cells, HOTAIR and HIF-1α share the same response elements of which could be counteracted by anti-217 (Figures 2b, d, e, g, miR-217 (Figures 1f and 3a). Hence, HOTAIR may act as a and h). These data suggest that HOTAIR promotes Rcc cell ceRNA for miR-217 to mediate HIF-1α expression in Rcc proliferation, migration, EMT, and inhibits apoptosis via development. To test this hypothesis, we first determined downregulation of miR-217. miR-217 expression after downregulation of HOTAIR and found si-HOTAIR significantly upregulated miR-217 levels in HOTAIR functions as a ceRNA for miR-217 to facilitate Rcc cells (Figure 3e). Then, we performed an RIP assay on HIF-1α expression. Through searching TargetScan (http:// Ago2, which is the vital component of the RNA-induced www.targetscan.org), we found that miR-217 could bind to silencing complex (RISC). As shown in Figure 3f, the 3′UTR of HIF-1α (Figure 3a). Moreover, miR-217 mimic overexpression of HOTAIR led to the increased enrich- significantly suppressed the mRNA and protein level of ment of Ago2 on HOTAIR but substantially decreased HIF-1α in ACHN cells, while downregulation of miR-217 enrichment on HIF-1α transcripts. In parallel, knockdown of increased HIF-1α expression in 769-P cells (Figures 3b HOTAIR had the contrary effects. These results indicate that and c). In addition, miR-217 remarkably dampened the HOTAIR could compete with HIF-1α transcripts for the luciferase activity of cells transfected with pGL3-HIF-1α but Ago2-based RISC. not pGL3-HIF-1α-MUTANT, suggesting a direct interaction Moreover, we evaluated whether the HOTAIR-mediated between miR-217 and the 3′UTR of HIF-1α (Figure 3d). sequestration of miR-217 was responsible for the upregulation Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al Figure 3 HOTAIR functions as a ceRNA for miR-217 to facilitate HIF-1α expression. (a) The putative miR-217 binding 3′UTR of HIF-1α (HIF-1α-WT) and HIF-1α mutation sequence (HIF-1α-MUT). The protein (b) and mRNA (c) levels of HIF-1α in ACHN and 769-P cells. *Po0.05 versus NC group. (d) Luciferase activity assay. *Po0.05 versus NC- mimic group. (e) The expression of miR-217 in 769-P cell transfected with si-NC or si-HOTAIR. *Po0.05 versus NC group. Data were analyzed by unpaired Student’s t-test. (f) RIP assay of the enrichment of Ago2 on HOTAIR and HIF-1α transcripts relative to IgG in cells transfected with pLVX-HOTAIR or si-HOTAIR. (g) Luciferase activity of pGL3 reporters which contained wild type or mutant HIF-1α 3′UTR with indicated treatment in Rcc cells. (h) qRT-PCR analysis for HIF-1α mRNA expression in ACHN and 769-P cells. # # *Po0.05 versus pLVX group, Po0.05 versus HOTAIR group in ACHN cells; *Po0.05 versus si-NC group, Po0.05 versus si-HOTAIR group in 769-P cells. Statistical analysis was conducted using One-way ANOVA of HIF-1α. The luciferase activity of HIF-1α wild-type reporters HOTAIR promotes Rcc tumorigenesis through HIF-1α/ but not the mutant one was significantly increased after AXL signaling both in vitro and in vivo. Studies have HOTAIR upregulation, whereas miR-217 mimic abolished this indicated that HIF-1 could bind directly to AXL and activate its effect. And HOTAIR siRNA showed the reversed effect on the expression; hence, to further investigate the mechanisms of luciferase activity of HIF-1α, which was rescued by anti-217 HOTAIR, we detected AXL expression in Rcc cells. HOTAIR (Figure 3g). In addition, these results were further confirmed at overexpression increased the mRNA and protein levels of HIF-1α mRNA level (Figure 3h). Collectively, these results AXL, whereas downregulation of HOTAIR notably decreased suggest that HOTAIR functions as a molecular sponge for their expression (Figures 4a and b). To determine whether miR-217 to facilitate HIF-1α expression. HOTAIR promotes Rcc activity through AXL signaling, an Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al Figure 4 HOTAIR regulates Rcc activity through AXL signaling in vitro. The mRNA (a) and protein (b) expression of AXL in ACHN and 769-P cells. Statistical analysis was conducted by unpaired Student’s t-test. (c) ACHN cells stably transfected with HOTAIR were treated with BGB324 (0.1, 0.5, 1 nM) for 72 h to examine the cell viability. Data were analyzed using One-way ANOVA. (d) The protein expression of E-cadherin, Vimentin, and Snail in ACHN cells stably transfected with HOTAIR and treated with different concentrations of BGB324 were assayed by western blot. The mRNA expression of HIF-1α (e) and AXL (f) in 86 paired Rcc tissues and adjacent histological normal tissues. Data were analyzed paired Student’s t-test. *Po0.05 versus control group, Po0.05 versus HOTAIR treated only group AXL inhibitor BGB324 was used. The results showed that levels (Figures 5c–f). In addition, the role of AXL in HOTAIR- BGB324 suppressed the effect of HOTAIR upregulation on mediated Rcc tumorigenesis was further confirmed by the proliferation and EMT process of ACHN cells in a dose- oral administration of BGB324. BGB324 notably decreased dependent manner (Figures 4c and d). Also, the mRNA levels HOTAIR-induced tumor growth and EMT process (Figures 5g and h). Collectively, these data show that HOTAIR promotes of HIF-1α and AXL were detected in 86 Rcc samples and Rcc tumorigenesis via activating HIF-1/AXL signaling. adjacent histological normal tissues. As shown in Figures 4e and f, the mRNA expression of HIF-1α and AXL in Rcc tissues was dramatically upregulated compared with normal Discussion control tissues. Furthermore, the role of HOTAIR in Rcc tumorigenesis was analyzed in vivo and the results showed Studies have revealed that lncRNAs play vital roles in Rcc that knockdown of HOTAIR significantly inhibited tumor pathogenesis. However, the role and mechanism of HOTAIR growth and the staining intensity of proliferation antigen in Rcc has not been fully understood. In this study, we found ki-67 (Figures 5a and b). HOTAIR downregulation reduced that HOTAIR expression was significantly increased in Rcc the expression of HIF-1α and AXL, but elevated miR-217 tissue samples compared with their corresponding non-tumor Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al Figure 5 HOTAIR promotes Rcc cell growth via AXL signaling in vivo.(a) Tumor volume was evaluated for 12–30 days. The inset graph illustrates the efficiency of si-HOTAIR on HOTAIR expression. (b) Representative ki-67 staining (×100). mRNA expression of miR-217 (c), HIF-1α (d), and AXL (e) in tumor tissues. Statistical analysis was conducted by unpaired Student’s t-test. *Po0.05 versus si-NC group (f) Western blot analysis for the protein levels of HIF-1α and AXL in tumor tissues. (g) Mice were subcutaneously injected with ACHN cells stably transfected with HOTAIR and orally administrated with BGB324 twice daily. Tumor volume was examined. (h) The expression of E-cadherin, Vimentin, and Snail was determined by Western blot. Data were analyzed using One-way ANOVA. n= 6 per group, *Po0.05 versus pLVX group, Po0.05 versus HOTAIR group. (i) Schematic of the proposed mechanism of HOTAIR in Rcc. HOTAIR functions as a ceRNA to ‘sponge’ miR-217 and upregulates the expression of HIF-1α as well as the HIF-1α downstream effector that promotes Rcc progression tissues, and the elevated expression was positively correlated The results of bioinformatics analysis showed HOTAIR with the TNM stage (Figure 1). Also, HOTAIR expression was possesses one conserved target site for miR-217 binding. And higher in Rcc cells. Studies have shown that HOTAIR acts as a dual-luciferase reporter assay demonstrated that miR-217 ceRNA to sponge miRNAs to modulate the de-repression of could reduce the luciferase activity of pGL3-HOTAIR 13,14 miRNA targets in bladder and gastric cancer. Hence, we (Figure 1). In addition, HOTAIR overexpression markedly speculated that HOTAIR acts as a ceRNA in Rcc. miR-217 increased the proliferation, migration, and EMT process, plays a suppression role in the growth of various types of which were ablated by miR-217 mimic. Contrarily, knockdown tumors including Rcc. We further investigated miR-217 of HOTAIR remarkably reduced the proliferation, migration, expression and found it was remarkably downregulated in Rcc EMT, and promoted the apoptosis of 769-P cells, which could tissues and cells. Furthermore, miR-217 expression was be counteracted by anti-217 (Figure 2). Taken together, these negatively correlated with HOTAIR level in Rcc tissues results indicate that HOTAIR promotes Rcc development via (Figure 1). negative regulation of miR-217. Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al HIF-1α is a transcription factor that is frequently stabilized pathway can be regulated directly by miR-217 targeting PTEN 20 29 and active in human clear cell Rcc. It is largely responsible (independent of AXL), which may account for the role of for activating the transcription of target genes that are widely HOTAIR in our study. Taken together, these results indicate 20,21 involved in the malignant features of tumors. High levels of that HOTAIR promotes Rcc tumorigenesis partly by activating HIF-1α are associated with the poor prognosis of clear cell HIF-1α/AXL signaling. Rcc. It has previously been reported that miR-217 increased In conclusion, our study revealed that HOTAIR is an HIF-1α levels by targeting SIRT1 in rat glomerular mesangial oncogene in Rcc. Elevated level of HOTAIR is positively cells cultured with high glucose. However, in our study, the correlated with tumor progression. HOTAIR functions as a result of bioinformatics analysis revealed HIF-1α is a potential ceRNA for miR-217 to facilitate Rcc progression partly via target of miR-217. And miR-217 decreased HIF-1α expres- HIF-1α/AXL signaling (Figure 5i). Thus, our study sion, which could be upregulated by anti- 217. Also, miR-217 provides further insight into the molecular mechanism of notably dampened the luciferase activity of pGL3-HIF-1α. HOTAIR in Rcc tumorigenesis, which may promote the These results indicated that HIF-1α was a direct target of development of lncRNA-directed diagnosis and therapy for miR-217. An RIP assay on Ago2 showed HOTAIR over- this disease. expression increased the enrichment on HOTAIR but sig- nificantly decreased the enrichment on HIF-1α transcripts and Material and Methods knockdown of HOTAIR had the reversed effect. These results Patients and samples. 86 clear cell Rcc tissue samples and neighboring indicated that HOTAIR could compete with HIF-1α transcripts non-cancerous tissues (collected postoperatively from June 2011 to January 2014) used in this study were obtained from Chinese PLA General Hospital. All patients for the Ago2-based RISC. Furthermore, the luciferase activity provided informed consent and had not undergone any previous therapy. Tumors of HIF-1α reporters and HIF-1α mRNA expression were were classified according to the tumor-node-metastasis (TNM) system of increased after HOTAIR overexpression, but reversed by classification (2010 version). This study was approved by the Human Research miR-217 mimic; however, they were reduced when HOTAIR Ethics Committee of Chinese PLA General Hospital (Beijing, China). The clinical was downregulated, which could be rescued by anti-217 data of patients were shown in Table 1. (Figure 3). Taken together, these results suggest that HOTAIR functions as a ceRNA for miR-217 to facilitate the expression Cell lines. The human Rcc cell lines (769-P and ACHN) and normal kidney cell of HIF-1α. line (kidney proximal tubular cells, HK-2) were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and cultured in a humidified Rankin et al. demonstrated that HIF-1 bound directly to incubator at 37 °C with an atmosphere of 5% CO . 769-P, ACHN, and HK-2 cells AXL to activate its expression. AXL, a member of the TAM were maintained in RPMI-1640, MEM, and K-SFM medium, respectively. family of receptor tyrosine kinases (RTKs), has been identified as an essential mediator of cancer metastasis including Rcc. Plasmid constructs and transfection. HOTAIR cDNA was amplified from Binding of its ligand, growth arrest-specific protein 6 (Gas6), human Rcc tissue with primers: 5′-CCGCTCGAGACATTCTGCCCTGATTTCCG- AXL receptor is dimerized and autophosphorylated at its GAACC-3′ (forward) and 5′-CGCGGATCCCCACCACACACACACAACCTACAC-3′ tyrosine resides, which subsequently triggers a cascade of (reverse). cDNA was then cloned into the XhoI and BamHI sites of pLVX-IRES-Neo vector (Invitrogen) to construct the pLVX-HOTAIR vector. The vector was then intracellular signaling, involving PI3K/Akt, ERK, NF-κB, p38, transfected into HEK293T cells to package lentivirus using Lipofectamine 2000 Rho family proteins, JAK-STAT, and Src family kinases. AXL (Invitrogen). ACHN cells were infected by the lentivirus and stably transfected cells oncogenic signaling promotes cancer cell survival, prolifera- were selected by G418 (0.5 mg/ml, Sigma-Aldrich, St Louis, MO, USA). To tion, migration, invasion, and the EMT phenotype, as well as construct luciferase reporter vectors, HIF-1α 3′-untranslated regions (UTR) and 25,26 resisting tumor cell apoptosis. In addition, AXL mediates a HOTAIR cDNA fragment containing the predicted potential miR-217 binding sites or resistance to sunitinib treatment in Rcc. AXL is highly mutant sites were amplified by PCR, and then cloned to into the XhoI and KpnI sites of pGL3 luciferase reporter vector (Promega, Madison, WI, USA). expressed in Rcc tissues and cells, and knockdown of AXL HOTAIR siRNA, control siRNA, miR-217 mimic, control mimic (NC-mimic), miR-217 reduces cell viability. To evaluate whether HOTAIR regulates antagomirs (anti-217), and control antagomirs (anti-NC) were synthesized by Rcc activity through HIF-1α/AXL signaling, we detected AXL GenePharma Co. (Shanghai, China). The sequences were listed in Supplementary expression after gain- and loss-of-function of HOTAIR in Rcc Table S1. To construct si-HOTAIR vector, the self-complementary hairpin DNA cells. Our results revealed that overexpression of HOTAIR oligonucleotides were annealed and subcloned into the pEGFP-N1 plasmid vector. A notably increased while downregulation of HOTAIR decreased negative control was named as si-NC vector. The vectors were transfected into 769-P cells with Lipofectamine 2000 and stable cell lines were established by G418 selection. AXL expression. Similarly, the expression of HIF-1α and AXL Stable transfected cells were grown in 6-well plates and transfected using Lipofectamine in Rcc tissues was higher than in normal tissues in the clinical 2000 according to manufacturer’s instructions. Cells were collected for real-time PCR or specimens. Moreover, BGB324 inhibited the effect of HOTAIR western bolt 48 h after transfection. The final concentrations of miRNAs or plasmids upregulation on the proliferation and EMT process of ACHN used in this study were as follows: HOTAIR/negative control 50 nM/ml, HOTAIR siRNA/ cells in a dose-dependent manner (Figure 4). In addition, control siRNA 30 nM/ml, miR-217 mimic/NC mimic 120 nM/ml, and anti-217/ anti-NC HOTAIR knockdown significantly suppressed tumor growth 200 nM/ml. and ki-67 expression, increased miR-217 levels and decreased the expression of HIF-1α and AXL, and BGB324 Proliferation assay. Proliferation of cells transfected with indicated vector was measured by MTT assay kit at 24, 48, and 72 h, respectively. In addition, ACHN abrogated the effect of HOTAIR on tumor growth and EMT cells with stable HOTAIR upregulation were treated with BGB324 (0.1, 0.5, 1 nM), process in vivo (Figure 5). However, si-HOTAIR treatment which also known as R428 (Selleck, Houston, TX, USA), for 72 h and the cell resulted in a 450% decrease in tumor volume after 30 days, viability was detected. whereas treatment with the AXL inhibitor resulted in a much more modest reduction of o20%, strongly implying Transwell analysis. Cells, transfected with the designated vector for 24 h, that other AXL-independent pathways make important were seeded in the upper chambers in 200 μl serum-free medium at a density of contributions. Previous study has reported that the PI3K/Akt 2× 10 /well. The lower chambers were filled with 500 μl medium containing 10% Cell Death and Disease HOTAIR as a ceRNA for miR-217 to promote Rcc Q Hong et al FBS for inducing cell migration. After 48 h incubation, the cells attached to the lower Conflict of Interest surface were fixed with 4% paraformaldehyde, stained with crystal violet, and then The authors declare no conflict of interest. examined under a microscope. 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Axl receptor tyrosine kinase is a potential otherwise in the credit line; if the material is not included under the therapeutic target in renal cell carcinoma. Br J Cancer 2015; 113: 616–625. Creative Commons license, users will need to obtain permission from 29. Kato M, Putta S, Wang M, Yuan H, Lanting L, Nair I et al. TGF-beta activates Akt kinase the license holder to reproduce the material. To view a copy of this through a microRNA-dependent amplifying circuit targeting PTEN. Nat Cell Biology 2009; 11: 881–889. license, visit http://creativecommons.org/licenses/by/4.0/ 30. Liu Q, Jin J, Ying J, Cui Y, Sun M, Zhang L et al. Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target. Oncotarget 2015; 6: 22706–22723. r The Author(s) 2017 Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis). Cell Death and Disease

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