Oncology Research, Vol. 21, pp. 83–91 Printed in the USA. All rights reserved. Copyright Ó 2014 Cognizant Comm. Corp.

0965-0407/14 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096504013X13775486749218 E-ISSN 1555-3906 www.cognizantcommunication.com

MiR-138 Induces Renal Carcinoma Cell Senescence by Targeting EZH2 and Is Downregulated in Human Clear Cell Renal Cell Carcinoma Jiaqian Liang,*1 Yajing Zhang,†1 Guosong Jiang,* Zhouqiang Liu,* Wei Xiang,* Xuanyu Chen,* Zhaohui Chen,* and Jun Zhao* *Department of Urology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China †Department of Nuclear Medicine, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China

MiR-138 has been shown to be downregulated in various cancers, including head and neck squamous cell carcinoma (HNSCC) and clear cell renal carcinoma (ccRCC). In the present study, we aimed to reveal the mechanism of miR-138 induction of senescence in renal carcinoma cells and identify its specific target genes. We used qRT-PCR to analyze miR-138 expression levels in renal carcinoma cell lines and ccRCC samples. The activity of b-galactosidase was measured for functional analysis after miR-138 mimic transfection. To identify the targets of miR-138, we used three types of target prediction software to determine three candidate target genes. Furthermore, a 3¢UTR luciferase assay was performed. Western blotting was used to detect the protein expression levels of candidate target genes. Additionally, knockdown of EZH2 by its siRNA was performed. The expression of miR-138 was downregulated in RCC cells lines and in tumor samples compared with their controls. Transfection of miR-138 mimic induced SN-12 cell senescence, decreased the protein expression of EZH2, and increased the protein expression of P16. Furthermore, miR-138 decreased the 3¢UTR luciferase activity of EZH2. The knockdown of EZH2 by siRNA induced SN-12 cell senescence, decreased the protein expression level of EZH2, and increased the protein expression of P16. MiR-138 is a tumor-suppressor miRNA in ccRCC that induces SN-12 cell senescence by downregulating EZH2 expression and upregulating P16 expression. Key words: MiR-138; EZH2; P16; Renal carcinoma; Senescence

INTRODUCTION Renal cell carcinoma (RCC) is the most common type of kidney cancer, accounting for approximately 2% of total adult malignancies. Clear cell renal cell carcinoma (ccRCC) is a subtype of RCC and histologically comprises 80% of RCC cases (1). Radical or partial nephrec­ tomy has thus far remained the best treatment strategy. Unfortunately, there is no effective treatment to cure met­ astatic cancer, which is resistant to radiation therapy and chemotherapy and has low response rates and severe toxicity in patients. Thus, there is an urgent need to identify new therapeutic targets for metastatic RCC. Cellular senescence is a state of growth arrest in which cells may maintain their metabolic activity but usually undergo a change in their gene expression profiles. Cellular senescence can be activated by normal cells in response to multiple cellular stresses, including oncogene

activation, oxidative damage, and replicative senescence (2–5). Although a variety of cell cycle regulators participate in the progression of replicative senescence, the most commonly used marker of cell senescence is a senescence-associated b-galactosidase (SA-b-gal) (6). Cellular senescence not only dominates the natural aging of normal cells but also controls a process of malignant phenomena transformation, thereby protecting the organism against tumor development (3). MicroRNAs (miRNAs) are a type of short, noncoding single-stranded RNA sequences, approximately 22–25 nucleotides in length. Mature miRNAs negatively regu­ late  gene expression by binding to the 3¢ untranslated regions (UTRs) of target messenger RNAs (mRNAs), exerting their function as oncogenes or tumor-suppressor genes during tumor development and progression (7,8). Recent evidence has shown that dysfunctional miRNAs

These authors provided equal contribution to this work and should be considered as co-first authors. Address correspondence to Jun Zhao, Department of Urology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, PR China. Tel: +86-27-85351624, +86-13807165235; E-mail: [email protected] or Zhaohui Chen, Department of Urology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, PR China. Tel: +86-27-85351625, +86-18627956868; E-mail: [email protected]

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83 University Libraries Delivered by Ingenta to: Purdue IP: 185.106.104.177 On: Wed, 16 Nov 2016 12:44:24 Article(s) and/or figure(s) cannot be used for resale. Please use proper citation format when citing this article including the DOI,

84 liang ET AL.

can regulate senescence by targeting downstream genes (9). Here we found that overexpression of miR-138 could induce senescence in SN-12 cells. Thus, we sought to address the mechanism of the miR-138 induction of SN-12 cell senescence. The genes EZH2, SIRT1, and hTERT, which are potential targets of miR-138, were selected as candidate downstream target genes. MATERIALS AND METHODS Cell Culture Human renal carcinoma cell lines SN-12 and 786-O were kindly provided by Dr. Xiaoping Zhang (Urology Department, Union Hospital, Wuhan, China). The HK-2 cells, obtained from Dr. Xiaoping Zhang, were derived from normal kidneys and used as control cells. SN-12, 786-O, and HK-2 cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and 1% penicillin/ streptomycin (Invitrogen). Small Interfering RNA and miRNA Mimic Transfection The transfections were carried out using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. In brief, prior to transfection, the SN-12 cells were replated onto six-well plates at a density of 2.5 × 105 cells/well and incubated overnight. For small interfering RNA (siRNA)-mediated­ gene knockdown, 50 nmol/L of EZH2 (GGT GAT CAC AGG ATA GGT ATT) or negative control siRNA (Gene­ Pharma, Shanghai, China) was used. For overexpression of miR-138, the cells were transfected with 50 nmol/L of miR-138 mimic (AGC UGG UGU UGU GAA UCA GGC CG) or a negative control (RiboBio Co. Ltd, Guangzhou, Guangdong, China). After 48–72 h of transfection, the cells were harvested for further analysis. Clinical Samples and RNA Extraction In total, 32 pairs of ccRCC tissue specimens, along with their patient-matched normal kidney tissues, were obtained from patients who underwent nephrectomy for RCC after approval by the Research Ethics Board of Union Hospital. Each specimen was then examined by a pathologist at the institution and histologically classified. Eleven of the specimens were classified as high-grade RCC (Fuhrman grade 3 or 4), and 21 of the specimens were classified as lowgrade RCC (Fuhrman grade 1 or 2). The specimens were collected in liquid nitrogen and stored at −80°C. The tissues were cryopulverized using a glass homogenizer. Total RNA was isolated from the tissues using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The concentration and quality of the RNA from each patient tissue were measured by an Amersham Bioscience Spectrophotometer and verified by gel electrophoresis.

Quantitative Real-Time PCR The synthesis of cDNA and the quantitative real-time PCR (qRT-PCR) analysis of gene expression were carried out with Perfect Real Time assay kits (Takara Bio, Dalian, China) according to the manufacturer’s protocol. Briefly, total RNA was extracted using TRIzol Reagent (Invitrogen), according to the manufacturer’s instructions, from the clinical samples and the HK-2, 786-O, and SN-12 cell lines and was used to synthesize cDNA with a gene-specific primer or oligo(DT). RNU6B(U6) and GAPDH were used as the housekeeping genes for detecting the expression levels of miR-138 and EZH2, respectively. The primers used to detect EZH2 and GAPDH are listed below, and bulge-loop miRNA qRTPCR primer sets (one reverse transcription primer and a pair of quantitative PCR primers for each set) specific for miR-138 and U6 were designed by RiboBio. The reverse transcriptase (RT) reactions contained 0.5 mg total RNA, 1 ml oligo(DT), or miR-138/U6 reverse transcription primer (RiboBio) and dNTP mixture with PrimeScript™ RT reagent Kit (Takara Bio, Dalian, China), according to the manufacturer’s protocol. The 10-ml reaction volume was incubated at 42°C for 15 min, then at 85°C for 15 s, and then held at 4°C. The cDNA product was used directly for the following qRT-PCR analysis. The 10-ml qRT-PCR reactions consisted of 1 ml of RT product, 5 ml of SYBR® Premix Ex Taq™ (Takara Bio), and 0.2  mM of each primer for miR-138 (RiboBio) or EZH2. The reactions were incubated at 95°C for 30 s and underwent 40 cycles of PCR at 95°C for 10 s and then at 60°C for 35 s. The reactions were performed in Applied Biosystems 7300 Real-Time PCR System. Finally, the mRNA and miRNA expression levels were determined using the 2−DCt method. The miRNA primers of miR-138 and U6 of real-time PCR were Bulge-Loop™ miRNA qRT-PCR (RiboBio). The primers used were human EZH2 forward primer: 5¢-CCC TGA CCT CTG TCT TAC TTG TGG A, reverse primer: 5¢-ACG TCA GAT GGT GCC AGC AAT A; human GAPDH forward primer: 5¢-GAA GGT GAA GGT CGG AGT C, reverse primer: 5¢-GAA GAT GGT GAT GGG ATT TC. Dual Luciferase Reporter Assay A region in the 3¢UTR of EZH2 containing the predicted binding site of miR-138 was amplified from genomic DNA (forward primer: GAT GCC CTT AAG TAT GTG GGT A, reverse primer: TGT TTG CTT TCT CAA CTG GCT A) and cloned into the Xba1 site of the pGL3 control vector (Promega, Madison, WI, USA), termed pGL3-EZH2-3¢UTR. The plasmid that carried the mutated sequence in the complementary sites for the seed region of miR-138, termed pGL3-EZH2-3¢UTRmut, was generated based on the pGL3-EZH2-3¢UTR plasmid using a MutanBEST Kit (Takara Bio), a forward

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MiR-138 INDUCES RENAL CARCINOMA CELL SENESCENCE

mutagenic miR-138 primer: ATT GCC TTC TGT CGT CCA CAG TGT TTT GTA CCT AGC CTT TTG C and reverse mutagenic miR-138 primer: GCA AAA GGT CTA GGT ACA AAA CAC TGT GGA CGA CAG AAG GCA AT were used to synthesize for the PCR experiment. The constructs were then verified by sequencing. The pRL-TK vector (Promega) was cotransfected as an internal control for normalization of the transfection efficiency. Each vector was cotransfected into SN-12 cells with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, SN-12 cells were seeded onto 24-well plates (25,000 cells/well) and incubated overnight. The following morning, the cells were transfected with 100 ng of either wild-type or mutated 3¢UTR vector and with 4 ng of pRL-TK in the presence of miR-138 or miR-138 control. Reporter assays were performed 48 h posttransfection using the Dual-Luciferase Assay System (Promega). Senescence Assay b-Galactosidase activity was analyzed using a senescence detection kit (Beyotime, Shanghai, China). Briefly, 2 × 105 cells/well in six-well plates were fixed in fixative solution for 15 min and washed three times in phosphatebuffered saline (PBS). A staining solution containing X-gal was added to the cells, which were then incubated for 24 h at 37°C. Blue-stained cells were counted in four separate areas.

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Western Analysis At 72 h after transfection, the cells were washed with ice-cold PBS and added to RIPA lysis and extraction buffer (Beyotime, China) containing Protease Inhibitor Cocktail I (Roche, Mannheim, Germany). The cells were collected with a cell lifter and rotated for 30 min at 4°C, followed by centrifugation at 12,000 rpm for 10 min at 4°C. The total protein was analyzed by 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) mem­ branes. The membranes were blocked with 5% nonfat­ milk for 1 h at room temperature and were incubated with anti-EZH2(1:1,000), anti-SIRT1(1:1,000), anti-GAPDH (1:2,000) antibodies (Proteintech™, Wuhan, China #218001-AP, #13161-1-AP, #10494-1-AP), anti-hTERT (1:500) (Epitomics Inc., CA, USA #1531-1), and anti-P16 (1:500) (Anbo, USA, #C0285) at 4°C overnight. On the second day, the membranes were followed up with anti-mouse or anti-rabbit IgG HRP-conjugated secondary antibodies (ProteintechÔ) for 1 h at room temperature and were visualized with BeyoECL Plus (Beyotime). A chemiluminescent signal was detected using BioSpectrum 610 (UVP Bioimaging System, Upland, CA, USA). Statistical Analysis The data were shown as the mean ± SEM. SPSS 12.0 statistical software (SPSS Inc.) was applied for statistical analysis. A two-tailed Student’s t test was used for comparisons, with p ＜ 0.05 considered to be significant.

Figure 1.  MiR-138 was downregulated in RCC cell lines and primary tumor samples. qRT-PCR was performed to assess the expression level of miR-138. The miR-138 expression was normalized to U6. (A) The miR-138 was detected in three types of cells, and HK-2 was used as a normal control cell. (B) MiR-138 expression was detected in 32 pairs of ccRCC samples and normal control tissue samples and found to be downregulated in 81% of ccRCC samples. Student’s t test: no asterisk, not significantly different (p > 0.05); **p