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MicroRNAs 125a and 125b inhibit ovarian cancer cells through post-transcriptional inactivation of EIF4EBP1

MicroRNAs 125a and 125b inhibit ovarian cancer cells through post-transcriptional inactivation of... www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 8 MicroRNAs 125a and 125b inhibit ovarian cancer cells through post-transcriptional inactivation of EIF4EBP1 1,* 1,* 1 Maria Lee , Eun Jae Kim and Myung Jae Jeon Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea These authors have contributed equally to this study Correspondence to: Maria Lee, email: marialee@snu.ac.kr Keywords: microRNA, microRNA 125a, microRNA 125b, epithelial ovarian cancer, EIF4EBP1 Received: June 04, 2015 Accepted: November 25, 2015 Published: December 05, 2015 AbstrAct The aim of the present study was to identify the specific miRNAs involved in regulation of EIF4EBP1 expression in ovarian cancer and to define their biological function. miRNA mimics and miRNA inhibitors were used in quantitative PCR, western blotting, and luciferase reporter assays to assess cell migration, invasiveness, and viability. miR-125a and miR-125b were downregulated in ovarian cancer tissue and cell lines relative to healthy controls. Increased expression of miR-125a and miR- 125b inhibited invasion and migration of SKOV3 and OVCAR-429 ovarian cancer cells and was associated with a decrease in EIF4EBP1 expression. The inverse relationship between miR-125a and miR-125b was corroborated by cotransfection of a luciferase reporter plasmid. Furthermore, miR-125a and miR-125b caused apoptosis and decreased cell viability and migration in an apparently EIF4EBP1-directed manner. Collectively, these results indicate that miR-125a and miR-125b are important posttranscriptional regulators of EIF4EBP1 expression, providing rationale for new therapeutic approaches to suppress tumour invasion and migration using miR-125a, miR-125b, or their mimics for the treatment of ovarian cancer. [9, 10]. Although miRNA genes represent approximately IntroductIon 1% of the predicted genes in the genome, approximately 30% of protein-encoding genes are regulated by at least Ovarian cancer is the fifth-leading cause of death one miRNA [11, 12]. miRNAs play key roles in diverse from gynaecological malignancies [1]. Although the pathways, including those involved in developmental molecular genetics of its initiation and progression are processes and cell growth, differentiation, and apoptosis not as well-described as other cancers, it is clear that [11, 13, 14]. In ovarian cancers, some miRNAs are translation initiation is deregulated during tumourigenesis positively associated with malignancy, including facets [2]. This deregulation could be attributed to eukaryotic such as tumour progression and chemotherapy resistance translation initiation factor 4E (EIF4E), an oncogene that [15-19]. However, the full regulatory landscape of regulates the translation of a specific subset of tumour- miRNAs in the pathogenesis of ovarian cancer has not promoting mRNAs [2, 3]. Like other oncogenes, such as been fully addressed. Akt and Ras, EIF4E induces senescence and acts as an Thus, we postulated that aberrantly-expressed intrinsic barrier to cancer [4, 5]. EIF4E-binding protein 1 miRNAs—whether over-expressed tumorigenic miRNAs (EIF4EBP1) plays a critical role in the control of protein or under-expressed protective miRNAs—contribute to the synthesis and cell growth and survival, thus promoting development of ovarian cancer by upregulating EIF4EBP1 tumourigenesis [6, 7]. Cells lacking EIF4EBP1s undergo expression. The aim of the present study was to identify p53-dependent senescence and are resistant to oncogenic the specific miRNAs involved in EIF4EBP1 expression in transformation [8]. However, it is unclear how EIF4EBP1 ovarian cancer cells and to define their functional effects. transcripts are regulated in ovarian cancer. MicroRNAs (miRNAs) are small, endogenous RNA molecules that play important regulatory roles by targeting mRNAs for cleavage or translational repression www.impactjournals.com/oncotarget 8726 Oncotarget EIF4EBP1 mRNA were identified for 15 miRNAs. Of results these, the two most notable were miR-125a and miR-125b, which were significantly downregulated in ovarian cancer relative to HOSE cells on microarray analysis. Alignment expression of mir-125a and mir-125b is of the 3′-UTR of EIF4EBP1 revealed that the putative significantly decreased in ovarian cancer tissue target sequences for miR-125a and miR-125b are highly and cell lines compared to normal ovarian tissue conserved across mammalian species. The downregulation of miR-125a and miR-125b We compared miRNA expression profiles in ovarian was also observed in ovarian cancer patients (Figure 1A, cancer cell lines and human ovarian surface epithelial 1B), accompanied by a significant increase in EIF4EBP1 (HOSE) cell lines using microarray analysis (data not mRNA expression (Figure 1C). The role of miR-125a shown). In an effort to identify specific miRNAs that and miR-125b as an inhibitor of EIF4EBP1 was further might regulate EIF4EBP1, we used the biocomputational suggested by a significant, inverse correlation between prediction algorithms of three different programs the expression levels of miR-125a and miR-125b, and (miRanda, TargetScan, and PicTar). This approach is EIF4EBP1 mRNA (Pearson correlation coefficient = -0.73 known to provide a good balance of sensitivity and and -0.83, respectively; p < 0.01; Figure 1D). specificity [20]. Potential regulatory relationships with We next examined the relationship between www.impactjournals.com/oncotarget 8727 Oncotarget Figure 1: miRNA expression in ovarian cancer tissue and normal ovarian epithelial tissue. A. qPCR analyses of two miRNAs in normal ovarian tissue and cancer tissue. B. Expression of miR-125a and miR-125b was significantly decreased in women with ovarian cancer (n = 20) compared to controls (n = 7). C. 4EBP1 mRNA expression was significantly increased in ovarian cancer patients compared with controls. D. An inverse correlation was observed between the expression levels of miR-125a or miR-125b and EIF4EBP1 mRNA. Quantitative data representing the mean ± standard deviation (SD) are presented in the bar graph. *P < 0.01 compared with control expression levels. www.impactjournals.com/oncotarget 8728 Oncotarget EIF4EBP1 expression and outcome. We searched high- EIF4EBP1 is a direct target of miR-125a and grade serous epithelial ovarian carcinoma ( HGS EOC) mir-125b cases in The Cancer Genome Atlas (TCGA) for cases with EIF4EBP1 alterations using cBioPortal [21]. Overall, 316 To assess whether miR-125a and miR-125b directly ovarian cancers with genome-wide gene expression data alter EIF4EBP1 expression, we transfected SKOV3 cells were available. In keeping with our in vitro and in vivo with luciferase expression plasmids containing the full- results, we found that EIF4EBP1 mRNA expression was length wild-type 3′-UTR of the EIF4EBP1 transcript significantly higher in ovarian cancer tissue than in normal or the 3’-UTR in which the miR-125a and miR-125b ovarian surface epithelium (P < 0.001). Furthermore, binding sites had been deleted. Co-transfection of the patients whose tumours exhibited EIF4EBP1 expression plasmid containing the wild-type UTR plus a miR-125a alteration had significantly poorer disease-free survival or miR-125b mimic, but not a negative control, resulted (Figure 2A; P = 0.042) and overall survival (Figure 2B; in a significant decrease in relative luciferase activity P < 0.001). (Figure 3C). In contrast, co-transfection of the plasmid with the mutated UTR plus either mimic completely Both miR-125a and miR-125b inhibit EIF4EBP1 abolished miR-125a and miR-125b-mediated repression, mRNA and protein levels demonstrating the specificity of these miRNAs for EIF4EBP1 suppression. Because miR-125a and miR-125b mimics reduced We performed a series of functional studies to EIF4EBP1 3’-UTR-driven luciferase activity, inhibitors determine the roles of miR-125a and miR-125b in the of these two miRNAs were assessed to determine if they regulation of EIF4EBP1. First, using specific miR mimics, exerted the opposite effect. Co-transfection with the we investigated whether overexpression of miR-125a or miRNA mimics plus a miR-125a or miR-125b inhibitor miR-125b was sufficient to reduce EIF4EBP1 levels in significantly increased the luciferase activity relative to SKOV3 and OVCAR-429 ovarian cancer cells. The miR- controls (Figure 3C). Taken together, these results show 125a and miR-125b mimics repressed EIF4EBP1 mRNA that both miR-125a and miR-125b can directly influence and protein levels in both cancer cell lines (Figure 3A). EIF4EBP1 through specific binding to its 3′-UTR. Next, cultured SKOV3 cells were transfected with a miR- Furthermore, co-treatment with miR-125a or miR-125b 125a inhibitor, a miR-125b inhibitor, or a negative control. plus their inhibitors enhanced EIF4EBP1 mRNA and Treatment with an inhibitor of miR-125a or miR-125b protein levels (Figure 3D). Taken together, these results enhanced EIF4EBP1 mRNA and protein levels (Figure show that both mi miR-125a and miR-125b can directly 3B). influence EIF4EBP1 through specific binding to its 3’- UTR. Figure 2: Kaplan-Meier plots for epithelial ovarian cancer patients stratified according to EIF4EBP1 expression. Patients whose tumours showed EIF4EBP1 alteration had significantly worse disease-free survival A. (P = 0.042) and overall survival B. (P = < 0.001). www.impactjournals.com/oncotarget 8729 Oncotarget www.impactjournals.com/oncotarget 8730 Oncotarget Figure 3: Effects of overexpression and inhibition of miR-125a and miR-125b and co-transfection of miR-125a or miR-125b and miR-125a or miR-125b inhibitors on 4EBP1 expression. qRT-PCR analysis of miR-125 and miR-125b levels in SKOV3 and OVCAR-429 cells transfected with specific miRNA mimic (A., left) or miRNA inhibitor (B., left). Transfection with miR- 125a or miR-125b mimic decreased 4EBP1 mRNA and protein levels (A., right). Conversely, transfection with miR-125a or -125b inhibitor resulted in an increase in 4EBP1 levels (B., right). miR-125a or -125b overexpression significantly decreased the relative luciferase activity of the wild-type 3′-UTR, but not of the mutant 3′-UTR in which the miRNA-binding sites were deleted (C., left). Conversely, treatment of miR-125a or -125b inhibitors significantly increased the relative luciferase activity (C., right). Co-transfection of miR-125a or miR- 125b and miR-125a or miR-125b inhibitors in SKOV3 and OVCAR-429 cells enhanced 4EBP1 protein levels D.. Each experiment was conducted in triplicate. Quantitative data representing the mean ± SD are presented in the bar graph. *P < 0.01 compared with negative controls. silencing by RNA interference on invasion in SKOV3 and miR-125a and miR-125b mimics decrease cell OVCAR-429 cells. Diminished invasion abilities were invasion and migration in EOC cells present in siRNA-treated SKOV3 and OVCAR-429 cells (Figure 4D). An invasion assay was performed to evaluate To assess whether EIF4EBP1 plays a role in whether EIF4EBP1 regulation by miR-125a and miR- migration inhibited by miR-125a and miR-125b in EOC 125b prevents invasion in EOC cells. To confirm the cells, we conducted a wound-healing assay to assess possible role of miR-125a and miR-125b in cell invasion, cell motility. Treatment with miR-125a and miR-125b we evaluated the invasive potential of SKOV3 and mimics resulted in decreased motility of SKOV3 and OVCAR-429 ovarian cancer cells treated with mimics OVCAR-429 cells compared to control treatment. In of these miRNAs. Indeed, treatment with miR-125a and contrast, miR-125a and miR-125b inhibitor treatment miR-125b mimics decreased invasion relative to control increased cell motility of these cells (Figure 4E, 4F). treatment (Figure 4A, 4C). In contrast, miR-125a and Similarly, EIF4EBP1-knockdown inhibited cell motility in miR-125b inhibitor treatment increased invasion by these SKOV3 and OVCAR-429 cells (Figure 4G). E-cadherin cells (Figure 4B, 4C). expression was decreased in miR-125a- and miR-125b- We next assessed the impact of EIF4EBP1- www.impactjournals.com/oncotarget 8731 Oncotarget www.impactjournals.com/oncotarget 8732 Oncotarget www.impactjournals.com/oncotarget 8733 Oncotarget www.impactjournals.com/oncotarget 8734 Oncotarget Figure 4: Invasion and wound healing assays in miR-125a, miR-125b-treated, and knockdown of EIF4EBP1 in SKOV3 and OVCAR-429 ovarian cancer cells. Treatment with miR-125a and miR-125b mimics resulted in decreased invasion compared to controls A., C.. In contrast, treatment with miR-125a and miR-125b inhibitors increased invasion by SKOV3 and OVCAR-429 cells B.,C.. In EIF4EBP1 siRNA-treated cells, cell invasion was decreased compared to controls D.. Treatment with miR-125a and miR-125b mimics resulted in decreased motility of SKOV3 and OVCAR-429 cells compared to control cells E., F.. In contrast, treatment with miR-125a and miR-125b inhibitor increased cell motility E., F.. In EIF4EBP1 siRNA-treated cells, cell motility was decreased compared to controls G.. E-cadherin expression was decreased in miR-125a- and miR-125b-treated EOC cells and siR-EIF4EBP1 cells compared to controls H.. *P < 0.01 compared with negative controls. mimic-treated EOC cells and in siR-EIF4EBP1-treated miR-125a or miR-125b inhibits ovarian cancer SKOV3 and OVCAR-429 cells relative to control cells cell proliferation by repressing EIF4EBP1 (Figure 4H). Taken together, these results demonstrate expression that miR-125a- and miR-125b-mediated suppression of EIF4EBP1 plays an important role in cell invasion and In addition to their role in reducing invasive and mobility in ovarian cancer. migratory abilities of ovarian cancer cells, we assessed the role of miR-125a and miR-125b in cellular proliferation in www.impactjournals.com/oncotarget 8735 Oncotarget Figure 5: Cell proliferation after treatment with miR-125a or miR-125b, their inhibitors, or knockdown of EIF4EBP1. miR-125a or miR-125b overexpression significantly decreased ovarian cancer cell proliferation 48 to 72 h after transfection. Treatment with miR-125a or miR-125b inhibitor increased viability and proliferation of SKOV3 and OVCAR-429 cells A.,B.. In EIF4EBP1 siRNA-treated cells, cell proliferation was decreased compared to controls C. TUNEL assay revealed that overexpression of miR-125a or miR-125b and knockdown of EIF4EBP1 increased apoptosis in these cells, as indicated by the intense dark brown staining of the majority of SKOV3 and OVCAR-429 cells compared to control cells. Annexin V/PI-based flow cytometric analysis was conducted to check apoptotic cell death by miR-125a, miR-125b, or knockdown of EIF4EBP1 in ovarian cancer cells 24 h after treatment D.. *P < 0.01 compared with negative controls. www.impactjournals.com/oncotarget 8736 Oncotarget SKOV3 and OVCAR-429 cells. miR-125a and miR-125b by miRNA-mediated downregulation of EIF4EBP1 overexpression significantly decreased proliferation of expression was caused by apoptotic cell death, we ovarian cancer cells within 48 to 72 h of treatment (Figure measured the levels of apoptosis in SKOV3 and 5A, 5B), as did the knockdown of EIF4EBP1 (Figure OVCAR-429 cells stably transfected with miR-125a or 5C).Conversely, treatment with a miR-125a or miR-125b miR-125b, and in control cells. TUNEL assay revealed inhibitor increased proliferation (Figure 5A, 5B). that overexpression of miR-125a or miR-125b increased the rate of apoptosis relative to control cells (Figure To explore the possibility that suppression of cell growth 5D). Cellular apoptosis was also increased in EIF4EBP- Figure 6: Effects of overexpression and inhibition of miR-125a or miR-125b and EIF4EBP1 knockdown on expression of 4EBP1, VEGF, and mTOR. VEGF expression was downregulated in miR-125a- or miR-125b-overexpressing and EIF4EBP1 knockdown SKOV3 and OVCAR-429 cells. However, no significant difference in mTOR expression was observed between miR-125a- or miR-125b-overexpressing cells and cells in which miR-125a or miR-125b was inhibited. In knockdown of EIF4EBP1 cells, no significant difference in mTOR expression was observed relative to controls. www.impactjournals.com/oncotarget 8737 Oncotarget knockdown cells relative to the siRNA-control group another member of the miR-125a family, the isoform (Figure 5D). miR-125a-3p, is downregulated in non-small cell lung cancer cells; its expression is negatively correlated with pathological stage and metastasis [30]. miR-125a-3p is miR-125a and miR-125b regulates the expression also suspected to regulate genes encoding chemokine of EIF4EBP1 and VEGF ligand 4 (CCL4) and IGF2. Both promote tumour cell migration, implying a role for miR-125a-3p in To explore the molecular mechanism underlying tumour suppression [30-32]. In the current study, miR- miR-125a or miR-125b inhibition of cell viability and 125b was also downregulated in ovarian cancer tissue. proliferation, we examined the expression levels of mTOR Overexpression of miR-125b inhibits tumour-induced and VEGF, as well as EIF4EBP1, by western blotting angiogenesis associated with HER2 and HER3 expression (Figure 6). Interestingly, VEGF expression was markedly in ovarian cancer cells [33]. On the other hand, poly-A downregulated in miR-125a or miR-125b-overexpressing binding protein-1 (PAPB-1), known to promote translation SKOV3 and OVCAR-429 cells. However, no significant of capped mRNAs, may also be targeted by miR-125b difference in mTOR expression was observed between [34]. A previous study reported that loss of miR-125b either the cells overexpressing miR-125a or miR-125b, or is associated with increased EIF4EBP1 protein levels in the cells treated with miR-125a or miR-125b inhibitor and breast cancers [35]. their respective control cells. These results were consistent EIF4EBP1 was significantly elevated in ovarian with those of EIF4EBP1-knockdown cells (Figure 6). cancer cells relative to control cells. EIF4EBP1 mRNA levels were also elevated. Our results indicated that dIscussIon EIF4EBP1 was a direct target of miR-125a and miR- 125b, and that reduction of miR-125a and miR-125b In this study, we elucidated the post-transcriptional levels using miR-125a and miR-125b inhibitors partially inhibition of EIF4EBP1-mediated pathways in ovarian restored EIF4EBP1 expression, as well as invasiveness cancer cells by the miRNAs miR-125a and miR-125b. We and migratory capabilities in SKOV3 and OVCAR429 showed a direct interaction between miR-125a, miR-125b, cells. EIF4EBP1 siRNA decreased cell proliferation and and EIF4EBP1; overexpression of miR-125a and miR- invasion in SKOV3 and OVCAR429 cells, indicating that 125b was associated with suppression of luciferase activity these abilities were EIF4EBP1-dependent. under control of the EIF4EBP1 3’-UTR. Furthermore, In case studies, EIF4EBP1 alteration was strongly we demonstrated that overexpression of miR-125a and related to survival in ovarian cancer patients. In keeping miR-125b caused significant downregulation of both with our in vitro findings, we found that EIF4EBP1 EIF4EBP1 mRNA and protein. expression was significantly elevated in ovarian cancer Translation factors functionally interact with tissue and cell lines and that protein levels were strongly oncogenes and underlie most human cancers. The associated with prognosis of patients with ovarian cancer. increased or reduced expression of these factors is Moreover, miR-125a and miR-125b downregulates VEGF associated with the development of specific cancers and and EIF4EBP1 levels, suggesting that suppression of these crucially affects disease progression. eIF4E mediates tumorigenic targets may be the mechanism by which these phenotypic changes by selectively enhancing translation of miRNAs suppress tumour growth. a limited pool of mRNAs that code for proteins involved In conclusion, we found that miR-125a and miR- in malignancy. As such, enhanced eIF4E can contribute to 125b were frequently downregulated in ovarian cancer every aspect of malignant progression [4]. EIF4EBP1, a cells. Ectopic expression of miR-125a and miR-125b protein that can inhibit the translation initiation of capped inhibited ovarian cancer cell proliferation, migration, mRNAs, is a downstream target of the AKT/mTOR and invasion in vitro. Further experiments revealed that pathway, and when EIF4EBP1 is phosphorylated, its EIF4EBP1 was a direct and functional target of miR-125a inhibitory impacts decrease, allowing increased translation and miR-125b in ovarian cancer cells. Our functional initiation [6]. Indeed, in a previous study at our institution, analysis of these two miRNAs, therefore, suggests that an elevated level of p-eIF4EBP1 was associated with miR-125a and miR-125b may represent both diagnostic advanced stage, higher histologic grade, shorter disease- markers and therapeutic targets in the treatment of ovarian free survival rate, and chemoresistance [22].The increase cancer patients. in eIF4EBP1 mRNA is somewhat surprising because such an increase would inhibit translation of capped mRNAs that can promote tumour progression. miR-125a is a tumour-suppressing miRNA downregulated in several cancers [23-27]. In the present study, we found that miR-125a was downregulated in ovarian cancer tissue relative to normal tissue. Similarly, www.impactjournals.com/oncotarget 8738 Oncotarget Biosystems, Carlsbad, CA, USA), and the two siRNAs MATERIAlS AND METHODS to each targeted gene were (EIF4EBP1 #1 sense: 5’CGAACCCUUCCUUCCGAAUtt-3’, antisense 5’AUUCGGAAGGAAGGGUUCGtt-3’; EIF4EBP1 #2 Tissue collection sense: 5’GAUCAUCUAUGACCGGAAAtt-3’, antisense 5’UUUCCGGUCAUAGAUGAUCct-3’). Cells were All experiments were performed with the approval plated to 40-60% confluency and transfected with 10 of the review board for human research of Seoul National nM EIF4EBP1-siRNA using Lipofectamin 3000 reagent University Hospital. Samples were collected between July (Invitrogen, Carlsbad, CA, USA) according to the 2012 and July 2014 from 20 ovarian cancer patients who manufacturer’s instructions. Knockdown efficiency was underwent surgery and a control group of seven patients con firmed by RT-qPCR. The transfected cells were used with benign gynaecologic disease. Informed consent was for various assays 48-72 h after transfection to allow for obtained from all of the participating women. Ovarian the effective knockdown of EIF4EBP1. tissue samples 1 × 1 cm in size were collected at the time of surgery. The samples were immediately snap-frozen in Quantitative real-time PCR analysis (qPCR) liquid nitrogen and kept at -80°C until RNA extraction was performed. Total RNA was extracted from cell lines and tissue samples using the mirVana miRNA Isolation Cell culture Kit (Ambion, Austin, TX, USA). cDNA was generated using the GoScript Reverse Transcription system SKOV3 and OVCAR429 ovarian cancer cell lines (Promega, Madison, WI, USA) according to the were purchased from Korean Cell Line Bank (KCLB, manufacturer’s protocol. PCR amplification was carried Seoul, Korea) and six types of ovarian cancer cell lines out with the following primers: EIF4EBP1, forward (SNU840, OVCAR3, TOV112D, YDOV-151, YDOV-161 5′-ATGTCCGGG GGCAGCAGCTGCAGCCAG-3′ and and YDOV-139) were provided by Korea Gynecologic reverse 5’-ACAGGUGAGGUUCUUGGGAACU-3′; and Cancer Bank through Bio & Medical Technology GAPDH, forward 5′-GTCGGAGTCAACGGATTTGG-3′ Development Program of the MSIP, Korea. Cell lines and reverse 5′-AAAAGCAGCCC TGGTGACC-3′. were maintained in Dulbecco’s modified Eagle medium Reaction mixtures were prepared using the SYBR (DMEM; Sigma-Aldrich, St Louis, MO, USA) at 37°C in Premix Ex Taq (TaKaRa, Tokyo, Japan) according to the an atmosphere of 5% CO . Culture medium was replaced manufacturer’s protocol. Each 20-μL reaction mixture with fresh medium every 2-3 days. Cells were used contained10 μL 2× SYBR Premix Ex Taq, 0.4 μL 50× between passages 5 and 10. ROX Reference Dye, 4 μL 10 μM forward and reverse primer mixture, 3.6 μL nuclease-free water, and 2 μL Transfection of miRNAs cDNA. PCR was carried out under conditions of 95°C for 5 sec followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 34 sec, and extension at 95°C SKOV3 and OVCAR429 cells were plated at a 5 for 15 sec, 60°C for 1 min, and 95°C for 15 sec. Reverse density of 5 × 10 cells per 100-mm culture dish, cultured transcription and miRNA quantification were carried in DMEM containing 10% foetal bovine serum (FBS) out using TaqMan miRNA Assays (Applied Biosystems, without antibiotic or antimycotic, and incubated at 37°C Carlsbad, CA, USA). PCR amplification was conducted in a humidified atmosphere of 5% CO until the cells using the TaqMan Universal PCR Master Mix according reached 40-50% confluence. Cells were then transfected to the manufacturer’s protocol. The samples were with synthetic precursor miRNA, miR-125a, or miR-125b analysed using the 7500 real-time PCR system (Applied at a final concentration of 100 nM using Lipofectamine Biosystems). All PCRs were performed in triplicate, 3000 (Invitrogen, Carlsbad, CA, USA). Forty-eight hours and the specificity of each reaction was determined after transfection, cell lysates were collected for RNA by melting curve analysis at the dissociation stage. For or protein isolation. Expression levels based on reporter relative quantification, the qPCR data were analysed using Cy3 fluorescence revealed high transfection efficiencies, -ΔΔCt the 2 method, where β-actin and U6B were used as exceeding 50% in some experiments. internal controls for EIF4EBP1 and miRNAs, respectively. Knockdown of EIF4EBP1 Western blot analysis Small interfering RNAs (siRNA) were used to Cells were lysed in 200 µl RIPA buffer (150 mM inhibit endogenous EIF4EBP1 in ovarian cancer cell sodium chloride, 1% NP 40, 0.5% sodium deoxycholate, lines. Scrambled siRNA (universal nega tive control 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl [pH siRNA), EIF4EBP1-siRNA was purchased (Applied www.impactjournals.com/oncotarget 8739 Oncotarget 8.0], 100 mM PMSF) and centrifuged at 14,000 g for 10 (Promega). Firefly luciferase activity was normalized to min at 4°C. The supernatant was mixed with denaturing Renilla luciferase expression level for each sample. Each sample buffer (1:1) and boiled for 5 min at 94°C. Equal experiment was conducted in triplicate. amounts of protein (50 µg) were loaded and separated by 10% SDS-polyacrylamide gel electrophoresis Invasion and wound healing assays and blotted onto nitrocellulose membranes (BioRad, Hercules, CA, USA). The membranes were blocked in Invasion by tumour cells was analysed using Cell Tris-buffered saline with Tween 20 (TBST) containing Invasion Assay Kit (8-μm pore size; Chemicon, Billerica, 5% non-fat dry milk for 1 h at 4°C and incubated with MA, USA) according to the manufacturer’s protocol. anti-4E-BP1 (1:2000), anti-phospho-4E-BP1 (1:1000), After transfection, cells were suspended in serum-free anti-mTOR (1:2000), anti-E-cadherin (1:1000) (all from medium and plated at a density of 5 × 10 cells per well Cell Signalling Technologies, Danvers, MA, USA), or in the upper chamber. The lower chamber was filled with anti-VEGF (1:2000; Santa Cruz Biotechnology, Santa culture medium supple mented with 10% FBS as the Cruz, CA, USA) antibodies overnight at 4°C. Anti-β-actin chemoattractant. After 48 h, the non-invading cells on the antibody (1:5000; Sigma-Aldrich) was used as a control. upper side of the membrane were gently removed with a Membranes were washed in TBST and incubated with cotton swab. The cells that had invaded the lower surface horseradish peroxidase-conjugated secondary antibodies of the membrane were stained, air-dried, photographed, (Jackson Immunoresearch, West Grove, PA, USA) for 1 and counted under a light microscope. For quantification, h at room temperature and washed again in TBST. The the stained cells were dissolved with 10% acetic acid, signal was detected using an enhanced chemiluminescence and absorbance was measured at 560 nm. The assay was kit (Thermo Scientific, Rockford, IL, USA), and intensity performed in triplicate. was quantified using ImageJ software. The wound healing assay was performed with a Cytoselect 24-Well Cell Invasion Assay Kit (Cell Biolabs, luciferase reporter assay San Diego, CA, USA) according to the manufacturer’s protocol. Transfected cells were added to either side of To validate the 4EBP1 3′-untranslated region (3′- the open end at the top of the insert. When the cells had UTR) as a target of miR-125a and miR-125b, in vitro formed a monolayer, the insert was removed to gener ate a assays used the miTarget miRNA 3′-UTR target clones consistent 0.9-mm gap wound in the mid dle. To determine (HmiT004676-MT01; Genecopoeia, Rockville, MD, migration distance, the size of the wound was measured at USA). These miRNA target clones consisted of the each time point. At multiple time points, cells were fixed pEZX-MT01 vector containing the coding sequences and stained with methylene blue and photographed. of both firefly and Renilla luciferase; the full 3′-UTR of the EIF4EBP1 transcript (GenBank accession number: Cell viability assay NM_004095) was inserted downstream of the firefly luciferase sequence. TargetScan (www.targetscan.org) An equal number of cells (1 × 10 ) transfected with predicted that the miR-125a and miR-125b binding sites miR-125a and miR-125b were seeded in 96-well plates are located at nucleotides 142 to 148. For mutagenesis and incubated for 48 h. The number of viable cells was assays, these two miRNA-binding sites within the 3′- determined using a Cell Counting Kit (CCK; Dojindo, UTR of the 4EBP1 transcript were deleted. After heat- Kumamoto, Japan). CCK reagents were added to cultures shock transformation in competent Escherichia coli and incubated for 2 h; measurement of absorbance of each cells (One Shot TOP 10 competent cells; Invitrogen), well at 540 nm with a micro-ELISA reader (Molecular the plasmids were amplified in Luria-Bertani medium Devices; Sunnyvale, CA, USA) was then performed. supplemented with 50 μg/ml kanamycin (Bio Basic, Markham, ON, Canada). Plasmid DNA was prepared on TUNEl assay columns (NucleoBond PC 500; Macherey-Nagel, Düren, Germany), and the identities of the amplified plasmids were confirmed by capillary sequencing (ABI 3730XL, An equal number of cells (5 × 10 ) transfected Applied Biosystems) using the sequencing primers with miR-125a and miR-125b were seeded in a Lab- 5′-CUCACUCAGGGCACCUGC-3′ (forward) and Tek II Chamber Slide w/Cover RS Glass Slide Sterile 5′-UUCAAUCCCAGAGUCCCU-3′ (reverse). SKOV3 and incubated for 48 h. The TUNEL assay was and OVCAR429 cells were plated at a density of 1 × 10 performed using the ApopTag Kit S7100 (Millipore, cells per well in 96-well plates. A total of 100 ng plasmid Billerica, MA, USA) according to the manufacturer’s DNA was co-transfected with miRNA mimic, miRNA instructions. Colour development was carried out inhibitors, or negative controls, as described above. using a 3,3’-diaminobenzidine solution, and sections Luciferase assays were performed 48 h after transfection were counterstained with methyl green. Negative using the Dual-Luciferase Reporter Assay System www.impactjournals.com/oncotarget 8740 Oncotarget control sections, processed in the absence of terminal 5. Wendel HG, De Stanchina E, Fridman JS, Malina A, Ray S, deoxynucleotidyl transferase, showed no staining. For Kogan S, Cordon-Cardo C, Pelletier J, Lowe SW. Survival measurement of apoptotic cell death in ovarian cancer signalling by Akt and eIF4E in oncogenesis and cancer cells, we performed flow cytometric analysis using therapy. Nature. 2004; 428:332-337. Annexin-V and PI staining (BD Pharmingen, CA) 6. Heesom KJ, Gampel A, Mellor H, Denton RM. Cell cycle- according to the manufacturer’s protocol. dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1). Curr Biol. 2001; Statistical analysis 11:1374-1379. 7. Topisirovic I, Ruiz-Gutierrez M, Borden KL. Phosphorylation of the eukaryotic translation initiation Statistical analyses were performed using SPSS 19.0 factor eIF4E contributes to its transformation and mRNA for Windows (SPSS, Chicago, IL, USA). The normality transport activities. Cancer Res. 2004; 64:8639-8642. of the data was assessed using the Shapiro-Wilk test. 8. 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The microRNA miR-196 acts upstream of Hoxb8 and Shh in The authors declare no conflicts of interest. limb development. Nature. 2005; 438:671-674. 15. Dahiya N, Morin PJ. MicroRNAs in ovarian carcinomas. references Endocr Relat Cancer. 2010; 17:F77-89. 16. Iorio MV, Visone R, Di Leva G, Donati V, Petrocca F, 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. Casalini P, Taccioli C, Volinia S, Liu CG, Alder H, et al. CA Cancer J Clin. 2013; 63:11-30. MicroRNA signatures in human ovarian cancer. Cancer 2. Mamane Y, Petroulakis E, Martineau Y, Sato TA, Larsson Res. 2007; 67:8699-8707. O, Rajasekhar VK, Sonenberg N. Epigenetic activation of 17. Laios A, O’Toole S, Flavin R, Martin C, Kelly L, Ring a subset of mRNAs by eIF4E explains its effects on cell M, Finn SP, Barrett C, Loda M, Gleeson N, D’Arcy T, proliferation. PLoS One. 2007; 2:e242. McGuinness E, Sheils O, et al. Potential role of miR-9 and 3. 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MicroRNAs 125a and 125b inhibit ovarian cancer cells through post-transcriptional inactivation of EIF4EBP1

Oncotarget , Volume 7 (8) – Dec 5, 2015

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Copyright: © 2016 Lee et al.
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1949-2553
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1949-2553
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10.18632/oncotarget.6474
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Abstract

www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 8 MicroRNAs 125a and 125b inhibit ovarian cancer cells through post-transcriptional inactivation of EIF4EBP1 1,* 1,* 1 Maria Lee , Eun Jae Kim and Myung Jae Jeon Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea These authors have contributed equally to this study Correspondence to: Maria Lee, email: marialee@snu.ac.kr Keywords: microRNA, microRNA 125a, microRNA 125b, epithelial ovarian cancer, EIF4EBP1 Received: June 04, 2015 Accepted: November 25, 2015 Published: December 05, 2015 AbstrAct The aim of the present study was to identify the specific miRNAs involved in regulation of EIF4EBP1 expression in ovarian cancer and to define their biological function. miRNA mimics and miRNA inhibitors were used in quantitative PCR, western blotting, and luciferase reporter assays to assess cell migration, invasiveness, and viability. miR-125a and miR-125b were downregulated in ovarian cancer tissue and cell lines relative to healthy controls. Increased expression of miR-125a and miR- 125b inhibited invasion and migration of SKOV3 and OVCAR-429 ovarian cancer cells and was associated with a decrease in EIF4EBP1 expression. The inverse relationship between miR-125a and miR-125b was corroborated by cotransfection of a luciferase reporter plasmid. Furthermore, miR-125a and miR-125b caused apoptosis and decreased cell viability and migration in an apparently EIF4EBP1-directed manner. Collectively, these results indicate that miR-125a and miR-125b are important posttranscriptional regulators of EIF4EBP1 expression, providing rationale for new therapeutic approaches to suppress tumour invasion and migration using miR-125a, miR-125b, or their mimics for the treatment of ovarian cancer. [9, 10]. Although miRNA genes represent approximately IntroductIon 1% of the predicted genes in the genome, approximately 30% of protein-encoding genes are regulated by at least Ovarian cancer is the fifth-leading cause of death one miRNA [11, 12]. miRNAs play key roles in diverse from gynaecological malignancies [1]. Although the pathways, including those involved in developmental molecular genetics of its initiation and progression are processes and cell growth, differentiation, and apoptosis not as well-described as other cancers, it is clear that [11, 13, 14]. In ovarian cancers, some miRNAs are translation initiation is deregulated during tumourigenesis positively associated with malignancy, including facets [2]. This deregulation could be attributed to eukaryotic such as tumour progression and chemotherapy resistance translation initiation factor 4E (EIF4E), an oncogene that [15-19]. However, the full regulatory landscape of regulates the translation of a specific subset of tumour- miRNAs in the pathogenesis of ovarian cancer has not promoting mRNAs [2, 3]. Like other oncogenes, such as been fully addressed. Akt and Ras, EIF4E induces senescence and acts as an Thus, we postulated that aberrantly-expressed intrinsic barrier to cancer [4, 5]. EIF4E-binding protein 1 miRNAs—whether over-expressed tumorigenic miRNAs (EIF4EBP1) plays a critical role in the control of protein or under-expressed protective miRNAs—contribute to the synthesis and cell growth and survival, thus promoting development of ovarian cancer by upregulating EIF4EBP1 tumourigenesis [6, 7]. Cells lacking EIF4EBP1s undergo expression. The aim of the present study was to identify p53-dependent senescence and are resistant to oncogenic the specific miRNAs involved in EIF4EBP1 expression in transformation [8]. However, it is unclear how EIF4EBP1 ovarian cancer cells and to define their functional effects. transcripts are regulated in ovarian cancer. MicroRNAs (miRNAs) are small, endogenous RNA molecules that play important regulatory roles by targeting mRNAs for cleavage or translational repression www.impactjournals.com/oncotarget 8726 Oncotarget EIF4EBP1 mRNA were identified for 15 miRNAs. Of results these, the two most notable were miR-125a and miR-125b, which were significantly downregulated in ovarian cancer relative to HOSE cells on microarray analysis. Alignment expression of mir-125a and mir-125b is of the 3′-UTR of EIF4EBP1 revealed that the putative significantly decreased in ovarian cancer tissue target sequences for miR-125a and miR-125b are highly and cell lines compared to normal ovarian tissue conserved across mammalian species. The downregulation of miR-125a and miR-125b We compared miRNA expression profiles in ovarian was also observed in ovarian cancer patients (Figure 1A, cancer cell lines and human ovarian surface epithelial 1B), accompanied by a significant increase in EIF4EBP1 (HOSE) cell lines using microarray analysis (data not mRNA expression (Figure 1C). The role of miR-125a shown). In an effort to identify specific miRNAs that and miR-125b as an inhibitor of EIF4EBP1 was further might regulate EIF4EBP1, we used the biocomputational suggested by a significant, inverse correlation between prediction algorithms of three different programs the expression levels of miR-125a and miR-125b, and (miRanda, TargetScan, and PicTar). This approach is EIF4EBP1 mRNA (Pearson correlation coefficient = -0.73 known to provide a good balance of sensitivity and and -0.83, respectively; p < 0.01; Figure 1D). specificity [20]. Potential regulatory relationships with We next examined the relationship between www.impactjournals.com/oncotarget 8727 Oncotarget Figure 1: miRNA expression in ovarian cancer tissue and normal ovarian epithelial tissue. A. qPCR analyses of two miRNAs in normal ovarian tissue and cancer tissue. B. Expression of miR-125a and miR-125b was significantly decreased in women with ovarian cancer (n = 20) compared to controls (n = 7). C. 4EBP1 mRNA expression was significantly increased in ovarian cancer patients compared with controls. D. An inverse correlation was observed between the expression levels of miR-125a or miR-125b and EIF4EBP1 mRNA. Quantitative data representing the mean ± standard deviation (SD) are presented in the bar graph. *P < 0.01 compared with control expression levels. www.impactjournals.com/oncotarget 8728 Oncotarget EIF4EBP1 expression and outcome. We searched high- EIF4EBP1 is a direct target of miR-125a and grade serous epithelial ovarian carcinoma ( HGS EOC) mir-125b cases in The Cancer Genome Atlas (TCGA) for cases with EIF4EBP1 alterations using cBioPortal [21]. Overall, 316 To assess whether miR-125a and miR-125b directly ovarian cancers with genome-wide gene expression data alter EIF4EBP1 expression, we transfected SKOV3 cells were available. In keeping with our in vitro and in vivo with luciferase expression plasmids containing the full- results, we found that EIF4EBP1 mRNA expression was length wild-type 3′-UTR of the EIF4EBP1 transcript significantly higher in ovarian cancer tissue than in normal or the 3’-UTR in which the miR-125a and miR-125b ovarian surface epithelium (P < 0.001). Furthermore, binding sites had been deleted. Co-transfection of the patients whose tumours exhibited EIF4EBP1 expression plasmid containing the wild-type UTR plus a miR-125a alteration had significantly poorer disease-free survival or miR-125b mimic, but not a negative control, resulted (Figure 2A; P = 0.042) and overall survival (Figure 2B; in a significant decrease in relative luciferase activity P < 0.001). (Figure 3C). In contrast, co-transfection of the plasmid with the mutated UTR plus either mimic completely Both miR-125a and miR-125b inhibit EIF4EBP1 abolished miR-125a and miR-125b-mediated repression, mRNA and protein levels demonstrating the specificity of these miRNAs for EIF4EBP1 suppression. Because miR-125a and miR-125b mimics reduced We performed a series of functional studies to EIF4EBP1 3’-UTR-driven luciferase activity, inhibitors determine the roles of miR-125a and miR-125b in the of these two miRNAs were assessed to determine if they regulation of EIF4EBP1. First, using specific miR mimics, exerted the opposite effect. Co-transfection with the we investigated whether overexpression of miR-125a or miRNA mimics plus a miR-125a or miR-125b inhibitor miR-125b was sufficient to reduce EIF4EBP1 levels in significantly increased the luciferase activity relative to SKOV3 and OVCAR-429 ovarian cancer cells. The miR- controls (Figure 3C). Taken together, these results show 125a and miR-125b mimics repressed EIF4EBP1 mRNA that both miR-125a and miR-125b can directly influence and protein levels in both cancer cell lines (Figure 3A). EIF4EBP1 through specific binding to its 3′-UTR. Next, cultured SKOV3 cells were transfected with a miR- Furthermore, co-treatment with miR-125a or miR-125b 125a inhibitor, a miR-125b inhibitor, or a negative control. plus their inhibitors enhanced EIF4EBP1 mRNA and Treatment with an inhibitor of miR-125a or miR-125b protein levels (Figure 3D). Taken together, these results enhanced EIF4EBP1 mRNA and protein levels (Figure show that both mi miR-125a and miR-125b can directly 3B). influence EIF4EBP1 through specific binding to its 3’- UTR. Figure 2: Kaplan-Meier plots for epithelial ovarian cancer patients stratified according to EIF4EBP1 expression. Patients whose tumours showed EIF4EBP1 alteration had significantly worse disease-free survival A. (P = 0.042) and overall survival B. (P = < 0.001). www.impactjournals.com/oncotarget 8729 Oncotarget www.impactjournals.com/oncotarget 8730 Oncotarget Figure 3: Effects of overexpression and inhibition of miR-125a and miR-125b and co-transfection of miR-125a or miR-125b and miR-125a or miR-125b inhibitors on 4EBP1 expression. qRT-PCR analysis of miR-125 and miR-125b levels in SKOV3 and OVCAR-429 cells transfected with specific miRNA mimic (A., left) or miRNA inhibitor (B., left). Transfection with miR- 125a or miR-125b mimic decreased 4EBP1 mRNA and protein levels (A., right). Conversely, transfection with miR-125a or -125b inhibitor resulted in an increase in 4EBP1 levels (B., right). miR-125a or -125b overexpression significantly decreased the relative luciferase activity of the wild-type 3′-UTR, but not of the mutant 3′-UTR in which the miRNA-binding sites were deleted (C., left). Conversely, treatment of miR-125a or -125b inhibitors significantly increased the relative luciferase activity (C., right). Co-transfection of miR-125a or miR- 125b and miR-125a or miR-125b inhibitors in SKOV3 and OVCAR-429 cells enhanced 4EBP1 protein levels D.. Each experiment was conducted in triplicate. Quantitative data representing the mean ± SD are presented in the bar graph. *P < 0.01 compared with negative controls. silencing by RNA interference on invasion in SKOV3 and miR-125a and miR-125b mimics decrease cell OVCAR-429 cells. Diminished invasion abilities were invasion and migration in EOC cells present in siRNA-treated SKOV3 and OVCAR-429 cells (Figure 4D). An invasion assay was performed to evaluate To assess whether EIF4EBP1 plays a role in whether EIF4EBP1 regulation by miR-125a and miR- migration inhibited by miR-125a and miR-125b in EOC 125b prevents invasion in EOC cells. To confirm the cells, we conducted a wound-healing assay to assess possible role of miR-125a and miR-125b in cell invasion, cell motility. Treatment with miR-125a and miR-125b we evaluated the invasive potential of SKOV3 and mimics resulted in decreased motility of SKOV3 and OVCAR-429 ovarian cancer cells treated with mimics OVCAR-429 cells compared to control treatment. In of these miRNAs. Indeed, treatment with miR-125a and contrast, miR-125a and miR-125b inhibitor treatment miR-125b mimics decreased invasion relative to control increased cell motility of these cells (Figure 4E, 4F). treatment (Figure 4A, 4C). In contrast, miR-125a and Similarly, EIF4EBP1-knockdown inhibited cell motility in miR-125b inhibitor treatment increased invasion by these SKOV3 and OVCAR-429 cells (Figure 4G). E-cadherin cells (Figure 4B, 4C). expression was decreased in miR-125a- and miR-125b- We next assessed the impact of EIF4EBP1- www.impactjournals.com/oncotarget 8731 Oncotarget www.impactjournals.com/oncotarget 8732 Oncotarget www.impactjournals.com/oncotarget 8733 Oncotarget www.impactjournals.com/oncotarget 8734 Oncotarget Figure 4: Invasion and wound healing assays in miR-125a, miR-125b-treated, and knockdown of EIF4EBP1 in SKOV3 and OVCAR-429 ovarian cancer cells. Treatment with miR-125a and miR-125b mimics resulted in decreased invasion compared to controls A., C.. In contrast, treatment with miR-125a and miR-125b inhibitors increased invasion by SKOV3 and OVCAR-429 cells B.,C.. In EIF4EBP1 siRNA-treated cells, cell invasion was decreased compared to controls D.. Treatment with miR-125a and miR-125b mimics resulted in decreased motility of SKOV3 and OVCAR-429 cells compared to control cells E., F.. In contrast, treatment with miR-125a and miR-125b inhibitor increased cell motility E., F.. In EIF4EBP1 siRNA-treated cells, cell motility was decreased compared to controls G.. E-cadherin expression was decreased in miR-125a- and miR-125b-treated EOC cells and siR-EIF4EBP1 cells compared to controls H.. *P < 0.01 compared with negative controls. mimic-treated EOC cells and in siR-EIF4EBP1-treated miR-125a or miR-125b inhibits ovarian cancer SKOV3 and OVCAR-429 cells relative to control cells cell proliferation by repressing EIF4EBP1 (Figure 4H). Taken together, these results demonstrate expression that miR-125a- and miR-125b-mediated suppression of EIF4EBP1 plays an important role in cell invasion and In addition to their role in reducing invasive and mobility in ovarian cancer. migratory abilities of ovarian cancer cells, we assessed the role of miR-125a and miR-125b in cellular proliferation in www.impactjournals.com/oncotarget 8735 Oncotarget Figure 5: Cell proliferation after treatment with miR-125a or miR-125b, their inhibitors, or knockdown of EIF4EBP1. miR-125a or miR-125b overexpression significantly decreased ovarian cancer cell proliferation 48 to 72 h after transfection. Treatment with miR-125a or miR-125b inhibitor increased viability and proliferation of SKOV3 and OVCAR-429 cells A.,B.. In EIF4EBP1 siRNA-treated cells, cell proliferation was decreased compared to controls C. TUNEL assay revealed that overexpression of miR-125a or miR-125b and knockdown of EIF4EBP1 increased apoptosis in these cells, as indicated by the intense dark brown staining of the majority of SKOV3 and OVCAR-429 cells compared to control cells. Annexin V/PI-based flow cytometric analysis was conducted to check apoptotic cell death by miR-125a, miR-125b, or knockdown of EIF4EBP1 in ovarian cancer cells 24 h after treatment D.. *P < 0.01 compared with negative controls. www.impactjournals.com/oncotarget 8736 Oncotarget SKOV3 and OVCAR-429 cells. miR-125a and miR-125b by miRNA-mediated downregulation of EIF4EBP1 overexpression significantly decreased proliferation of expression was caused by apoptotic cell death, we ovarian cancer cells within 48 to 72 h of treatment (Figure measured the levels of apoptosis in SKOV3 and 5A, 5B), as did the knockdown of EIF4EBP1 (Figure OVCAR-429 cells stably transfected with miR-125a or 5C).Conversely, treatment with a miR-125a or miR-125b miR-125b, and in control cells. TUNEL assay revealed inhibitor increased proliferation (Figure 5A, 5B). that overexpression of miR-125a or miR-125b increased the rate of apoptosis relative to control cells (Figure To explore the possibility that suppression of cell growth 5D). Cellular apoptosis was also increased in EIF4EBP- Figure 6: Effects of overexpression and inhibition of miR-125a or miR-125b and EIF4EBP1 knockdown on expression of 4EBP1, VEGF, and mTOR. VEGF expression was downregulated in miR-125a- or miR-125b-overexpressing and EIF4EBP1 knockdown SKOV3 and OVCAR-429 cells. However, no significant difference in mTOR expression was observed between miR-125a- or miR-125b-overexpressing cells and cells in which miR-125a or miR-125b was inhibited. In knockdown of EIF4EBP1 cells, no significant difference in mTOR expression was observed relative to controls. www.impactjournals.com/oncotarget 8737 Oncotarget knockdown cells relative to the siRNA-control group another member of the miR-125a family, the isoform (Figure 5D). miR-125a-3p, is downregulated in non-small cell lung cancer cells; its expression is negatively correlated with pathological stage and metastasis [30]. miR-125a-3p is miR-125a and miR-125b regulates the expression also suspected to regulate genes encoding chemokine of EIF4EBP1 and VEGF ligand 4 (CCL4) and IGF2. Both promote tumour cell migration, implying a role for miR-125a-3p in To explore the molecular mechanism underlying tumour suppression [30-32]. In the current study, miR- miR-125a or miR-125b inhibition of cell viability and 125b was also downregulated in ovarian cancer tissue. proliferation, we examined the expression levels of mTOR Overexpression of miR-125b inhibits tumour-induced and VEGF, as well as EIF4EBP1, by western blotting angiogenesis associated with HER2 and HER3 expression (Figure 6). Interestingly, VEGF expression was markedly in ovarian cancer cells [33]. On the other hand, poly-A downregulated in miR-125a or miR-125b-overexpressing binding protein-1 (PAPB-1), known to promote translation SKOV3 and OVCAR-429 cells. However, no significant of capped mRNAs, may also be targeted by miR-125b difference in mTOR expression was observed between [34]. A previous study reported that loss of miR-125b either the cells overexpressing miR-125a or miR-125b, or is associated with increased EIF4EBP1 protein levels in the cells treated with miR-125a or miR-125b inhibitor and breast cancers [35]. their respective control cells. These results were consistent EIF4EBP1 was significantly elevated in ovarian with those of EIF4EBP1-knockdown cells (Figure 6). cancer cells relative to control cells. EIF4EBP1 mRNA levels were also elevated. Our results indicated that dIscussIon EIF4EBP1 was a direct target of miR-125a and miR- 125b, and that reduction of miR-125a and miR-125b In this study, we elucidated the post-transcriptional levels using miR-125a and miR-125b inhibitors partially inhibition of EIF4EBP1-mediated pathways in ovarian restored EIF4EBP1 expression, as well as invasiveness cancer cells by the miRNAs miR-125a and miR-125b. We and migratory capabilities in SKOV3 and OVCAR429 showed a direct interaction between miR-125a, miR-125b, cells. EIF4EBP1 siRNA decreased cell proliferation and and EIF4EBP1; overexpression of miR-125a and miR- invasion in SKOV3 and OVCAR429 cells, indicating that 125b was associated with suppression of luciferase activity these abilities were EIF4EBP1-dependent. under control of the EIF4EBP1 3’-UTR. Furthermore, In case studies, EIF4EBP1 alteration was strongly we demonstrated that overexpression of miR-125a and related to survival in ovarian cancer patients. In keeping miR-125b caused significant downregulation of both with our in vitro findings, we found that EIF4EBP1 EIF4EBP1 mRNA and protein. expression was significantly elevated in ovarian cancer Translation factors functionally interact with tissue and cell lines and that protein levels were strongly oncogenes and underlie most human cancers. The associated with prognosis of patients with ovarian cancer. increased or reduced expression of these factors is Moreover, miR-125a and miR-125b downregulates VEGF associated with the development of specific cancers and and EIF4EBP1 levels, suggesting that suppression of these crucially affects disease progression. eIF4E mediates tumorigenic targets may be the mechanism by which these phenotypic changes by selectively enhancing translation of miRNAs suppress tumour growth. a limited pool of mRNAs that code for proteins involved In conclusion, we found that miR-125a and miR- in malignancy. As such, enhanced eIF4E can contribute to 125b were frequently downregulated in ovarian cancer every aspect of malignant progression [4]. EIF4EBP1, a cells. Ectopic expression of miR-125a and miR-125b protein that can inhibit the translation initiation of capped inhibited ovarian cancer cell proliferation, migration, mRNAs, is a downstream target of the AKT/mTOR and invasion in vitro. Further experiments revealed that pathway, and when EIF4EBP1 is phosphorylated, its EIF4EBP1 was a direct and functional target of miR-125a inhibitory impacts decrease, allowing increased translation and miR-125b in ovarian cancer cells. Our functional initiation [6]. Indeed, in a previous study at our institution, analysis of these two miRNAs, therefore, suggests that an elevated level of p-eIF4EBP1 was associated with miR-125a and miR-125b may represent both diagnostic advanced stage, higher histologic grade, shorter disease- markers and therapeutic targets in the treatment of ovarian free survival rate, and chemoresistance [22].The increase cancer patients. in eIF4EBP1 mRNA is somewhat surprising because such an increase would inhibit translation of capped mRNAs that can promote tumour progression. miR-125a is a tumour-suppressing miRNA downregulated in several cancers [23-27]. In the present study, we found that miR-125a was downregulated in ovarian cancer tissue relative to normal tissue. Similarly, www.impactjournals.com/oncotarget 8738 Oncotarget Biosystems, Carlsbad, CA, USA), and the two siRNAs MATERIAlS AND METHODS to each targeted gene were (EIF4EBP1 #1 sense: 5’CGAACCCUUCCUUCCGAAUtt-3’, antisense 5’AUUCGGAAGGAAGGGUUCGtt-3’; EIF4EBP1 #2 Tissue collection sense: 5’GAUCAUCUAUGACCGGAAAtt-3’, antisense 5’UUUCCGGUCAUAGAUGAUCct-3’). Cells were All experiments were performed with the approval plated to 40-60% confluency and transfected with 10 of the review board for human research of Seoul National nM EIF4EBP1-siRNA using Lipofectamin 3000 reagent University Hospital. Samples were collected between July (Invitrogen, Carlsbad, CA, USA) according to the 2012 and July 2014 from 20 ovarian cancer patients who manufacturer’s instructions. Knockdown efficiency was underwent surgery and a control group of seven patients con firmed by RT-qPCR. The transfected cells were used with benign gynaecologic disease. Informed consent was for various assays 48-72 h after transfection to allow for obtained from all of the participating women. Ovarian the effective knockdown of EIF4EBP1. tissue samples 1 × 1 cm in size were collected at the time of surgery. The samples were immediately snap-frozen in Quantitative real-time PCR analysis (qPCR) liquid nitrogen and kept at -80°C until RNA extraction was performed. Total RNA was extracted from cell lines and tissue samples using the mirVana miRNA Isolation Cell culture Kit (Ambion, Austin, TX, USA). cDNA was generated using the GoScript Reverse Transcription system SKOV3 and OVCAR429 ovarian cancer cell lines (Promega, Madison, WI, USA) according to the were purchased from Korean Cell Line Bank (KCLB, manufacturer’s protocol. PCR amplification was carried Seoul, Korea) and six types of ovarian cancer cell lines out with the following primers: EIF4EBP1, forward (SNU840, OVCAR3, TOV112D, YDOV-151, YDOV-161 5′-ATGTCCGGG GGCAGCAGCTGCAGCCAG-3′ and and YDOV-139) were provided by Korea Gynecologic reverse 5’-ACAGGUGAGGUUCUUGGGAACU-3′; and Cancer Bank through Bio & Medical Technology GAPDH, forward 5′-GTCGGAGTCAACGGATTTGG-3′ Development Program of the MSIP, Korea. Cell lines and reverse 5′-AAAAGCAGCCC TGGTGACC-3′. were maintained in Dulbecco’s modified Eagle medium Reaction mixtures were prepared using the SYBR (DMEM; Sigma-Aldrich, St Louis, MO, USA) at 37°C in Premix Ex Taq (TaKaRa, Tokyo, Japan) according to the an atmosphere of 5% CO . Culture medium was replaced manufacturer’s protocol. Each 20-μL reaction mixture with fresh medium every 2-3 days. Cells were used contained10 μL 2× SYBR Premix Ex Taq, 0.4 μL 50× between passages 5 and 10. ROX Reference Dye, 4 μL 10 μM forward and reverse primer mixture, 3.6 μL nuclease-free water, and 2 μL Transfection of miRNAs cDNA. PCR was carried out under conditions of 95°C for 5 sec followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 34 sec, and extension at 95°C SKOV3 and OVCAR429 cells were plated at a 5 for 15 sec, 60°C for 1 min, and 95°C for 15 sec. Reverse density of 5 × 10 cells per 100-mm culture dish, cultured transcription and miRNA quantification were carried in DMEM containing 10% foetal bovine serum (FBS) out using TaqMan miRNA Assays (Applied Biosystems, without antibiotic or antimycotic, and incubated at 37°C Carlsbad, CA, USA). PCR amplification was conducted in a humidified atmosphere of 5% CO until the cells using the TaqMan Universal PCR Master Mix according reached 40-50% confluence. Cells were then transfected to the manufacturer’s protocol. The samples were with synthetic precursor miRNA, miR-125a, or miR-125b analysed using the 7500 real-time PCR system (Applied at a final concentration of 100 nM using Lipofectamine Biosystems). All PCRs were performed in triplicate, 3000 (Invitrogen, Carlsbad, CA, USA). Forty-eight hours and the specificity of each reaction was determined after transfection, cell lysates were collected for RNA by melting curve analysis at the dissociation stage. For or protein isolation. Expression levels based on reporter relative quantification, the qPCR data were analysed using Cy3 fluorescence revealed high transfection efficiencies, -ΔΔCt the 2 method, where β-actin and U6B were used as exceeding 50% in some experiments. internal controls for EIF4EBP1 and miRNAs, respectively. Knockdown of EIF4EBP1 Western blot analysis Small interfering RNAs (siRNA) were used to Cells were lysed in 200 µl RIPA buffer (150 mM inhibit endogenous EIF4EBP1 in ovarian cancer cell sodium chloride, 1% NP 40, 0.5% sodium deoxycholate, lines. Scrambled siRNA (universal nega tive control 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl [pH siRNA), EIF4EBP1-siRNA was purchased (Applied www.impactjournals.com/oncotarget 8739 Oncotarget 8.0], 100 mM PMSF) and centrifuged at 14,000 g for 10 (Promega). Firefly luciferase activity was normalized to min at 4°C. The supernatant was mixed with denaturing Renilla luciferase expression level for each sample. Each sample buffer (1:1) and boiled for 5 min at 94°C. Equal experiment was conducted in triplicate. amounts of protein (50 µg) were loaded and separated by 10% SDS-polyacrylamide gel electrophoresis Invasion and wound healing assays and blotted onto nitrocellulose membranes (BioRad, Hercules, CA, USA). The membranes were blocked in Invasion by tumour cells was analysed using Cell Tris-buffered saline with Tween 20 (TBST) containing Invasion Assay Kit (8-μm pore size; Chemicon, Billerica, 5% non-fat dry milk for 1 h at 4°C and incubated with MA, USA) according to the manufacturer’s protocol. anti-4E-BP1 (1:2000), anti-phospho-4E-BP1 (1:1000), After transfection, cells were suspended in serum-free anti-mTOR (1:2000), anti-E-cadherin (1:1000) (all from medium and plated at a density of 5 × 10 cells per well Cell Signalling Technologies, Danvers, MA, USA), or in the upper chamber. The lower chamber was filled with anti-VEGF (1:2000; Santa Cruz Biotechnology, Santa culture medium supple mented with 10% FBS as the Cruz, CA, USA) antibodies overnight at 4°C. Anti-β-actin chemoattractant. After 48 h, the non-invading cells on the antibody (1:5000; Sigma-Aldrich) was used as a control. upper side of the membrane were gently removed with a Membranes were washed in TBST and incubated with cotton swab. The cells that had invaded the lower surface horseradish peroxidase-conjugated secondary antibodies of the membrane were stained, air-dried, photographed, (Jackson Immunoresearch, West Grove, PA, USA) for 1 and counted under a light microscope. For quantification, h at room temperature and washed again in TBST. The the stained cells were dissolved with 10% acetic acid, signal was detected using an enhanced chemiluminescence and absorbance was measured at 560 nm. The assay was kit (Thermo Scientific, Rockford, IL, USA), and intensity performed in triplicate. was quantified using ImageJ software. The wound healing assay was performed with a Cytoselect 24-Well Cell Invasion Assay Kit (Cell Biolabs, luciferase reporter assay San Diego, CA, USA) according to the manufacturer’s protocol. Transfected cells were added to either side of To validate the 4EBP1 3′-untranslated region (3′- the open end at the top of the insert. When the cells had UTR) as a target of miR-125a and miR-125b, in vitro formed a monolayer, the insert was removed to gener ate a assays used the miTarget miRNA 3′-UTR target clones consistent 0.9-mm gap wound in the mid dle. To determine (HmiT004676-MT01; Genecopoeia, Rockville, MD, migration distance, the size of the wound was measured at USA). These miRNA target clones consisted of the each time point. At multiple time points, cells were fixed pEZX-MT01 vector containing the coding sequences and stained with methylene blue and photographed. of both firefly and Renilla luciferase; the full 3′-UTR of the EIF4EBP1 transcript (GenBank accession number: Cell viability assay NM_004095) was inserted downstream of the firefly luciferase sequence. TargetScan (www.targetscan.org) An equal number of cells (1 × 10 ) transfected with predicted that the miR-125a and miR-125b binding sites miR-125a and miR-125b were seeded in 96-well plates are located at nucleotides 142 to 148. For mutagenesis and incubated for 48 h. The number of viable cells was assays, these two miRNA-binding sites within the 3′- determined using a Cell Counting Kit (CCK; Dojindo, UTR of the 4EBP1 transcript were deleted. After heat- Kumamoto, Japan). CCK reagents were added to cultures shock transformation in competent Escherichia coli and incubated for 2 h; measurement of absorbance of each cells (One Shot TOP 10 competent cells; Invitrogen), well at 540 nm with a micro-ELISA reader (Molecular the plasmids were amplified in Luria-Bertani medium Devices; Sunnyvale, CA, USA) was then performed. supplemented with 50 μg/ml kanamycin (Bio Basic, Markham, ON, Canada). Plasmid DNA was prepared on TUNEl assay columns (NucleoBond PC 500; Macherey-Nagel, Düren, Germany), and the identities of the amplified plasmids were confirmed by capillary sequencing (ABI 3730XL, An equal number of cells (5 × 10 ) transfected Applied Biosystems) using the sequencing primers with miR-125a and miR-125b were seeded in a Lab- 5′-CUCACUCAGGGCACCUGC-3′ (forward) and Tek II Chamber Slide w/Cover RS Glass Slide Sterile 5′-UUCAAUCCCAGAGUCCCU-3′ (reverse). SKOV3 and incubated for 48 h. The TUNEL assay was and OVCAR429 cells were plated at a density of 1 × 10 performed using the ApopTag Kit S7100 (Millipore, cells per well in 96-well plates. A total of 100 ng plasmid Billerica, MA, USA) according to the manufacturer’s DNA was co-transfected with miRNA mimic, miRNA instructions. Colour development was carried out inhibitors, or negative controls, as described above. using a 3,3’-diaminobenzidine solution, and sections Luciferase assays were performed 48 h after transfection were counterstained with methyl green. Negative using the Dual-Luciferase Reporter Assay System www.impactjournals.com/oncotarget 8740 Oncotarget control sections, processed in the absence of terminal 5. Wendel HG, De Stanchina E, Fridman JS, Malina A, Ray S, deoxynucleotidyl transferase, showed no staining. For Kogan S, Cordon-Cardo C, Pelletier J, Lowe SW. 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Published: Dec 5, 2015

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