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Downregulation of Microrna-1274A Induces Cell Apoptosis Through Regulation of BMPR1B in Clear Cell Renal Cell Carcinoma

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Downregulation of Microrna-1274A Induces Cell Apoptosis Through Regulation of BMPR1B in Clear Cell Renal Cell Carcinoma ONCOLOGY REPORTS 39: 173-181, 2018 Downregulation of microRNA-1274a induces cell apoptosis through regulation of BMPR1B in clear cell renal cell carcinoma HIROFUMI YOSHINO*, TOMOKAZU YONEZawa*, Masaya YONEMORI, KAZUtaKA MIyamoto, TAKASHI SAKAGUCHI, SatorU SUGIta, YOUICHI OSAKO, SHUICHI Tatarano, MasayUKI NAKagawa and HIDEKI ENOKIDA Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan Received May 24, 2017; Accepted October 12, 2017 DOI: 10.3892/or.2017.6098 Abstract. Our previous studies of the microRNA (miRNA) advanced-stage RCC patients is extremely poor (5-10%) due to expression signature in clear cell renal cell carcinoma (ccRCC) recurrence or distant metastasis (2). At diagnosis, nearly 30% indicated that miRNA-1274a (miR-1274a) was significantly of RCC patients present with metastasis (3). Current treat- upregulated in clinical specimens, suggesting that miR-1274a ments for RCC include molecular-targeted therapeutics such may act as an oncogenic miRNA in ccRCC. The aim of this study as anti-angiogenic multi-tyrosine kinase inhibitors or mTOR was to investigate the functional roles of miR-1274a and identify inhibitors, which are being widely used for patients with downstream tumor-suppressive targets regulated by miR-1274a metastatic or recurrent RCC. However, these types of therapies in ccRCC cells. Functional studies of miR-1274a were carried are not expected to have curative effects as they only slightly out by anti-miRNA to investigate cell proliferation and apoptosis extend progression-free survival (4). Therefore, to improve the using the A498, ACHN and Caki1 ccRCC cell lines. Suppression outcome of patients with RCC, it is necessary to fully elucidate of miR-1274a significantly inhibited cancer cell proliferation the molecular mechanisms underlying the development and and induced apoptosis in the ccRCC cells. Gene expression data progression of RCC and the associated oncogenic pathways. combined with in silico analysis and luciferase reporter assays The discovery of non-coding RNAs (ncRNAs) in the demonstrated that bone morphogenetic protein receptor type 1B human genome was an important conceptual breakthrough, (BMPR1B) was directly regulated by miR-1274a. Moreover, and the understanding of ncRNAs is important for progress in TCGA database as well as immunohistochemistry demonstrated cancer research. MicroRNAs (miRNAs) are endogenous small low expression of BMPR1B in ccRCC clinical specimens ncRNA molecules (19-22 bases in length) that regulate protein- compared to that in normal kidney tissues. We conclude that loss coding gene expression by repressing translation or cleaving of oncogenic miR-1274a reduced cancer cell proliferation and RNA transcripts in a sequence-specific manner (5). miRNAs induced apoptosis in ccRCC through targeting BMPR1B. Our are predicted to regulate more than 60% of the protein-coding data revealing molecular pathways and a target gene regulated genes in the human genome (6). Accumulating evidence by oncogenic miR-1274a provide new insight into the potential suggests that miRNAs are aberrantly expressed in many mechanisms of ccRCC oncogenesis. human cancers and play significant roles in human oncogenesis and metastasis (7). Therefore, identifying aberrantly expressed Introduction miRNAs is an important first step to elucidate the details of miRNA-mediated oncogenic pathways in cancer cells. Renal cell carcinoma (RCC) is a disease in which cells in Previously, our miRNA expression signature of ccRCC the tubules of the kidney undergo oncogenic transformation. revealed that microRNA-1274a (miR-1274a) was significantly The most common subtype (approximately 80% of cases) of upregulated in cancer tissues, suggesting that this miRNA RCC is clear cell RCC (ccRCC) (1). The 5-year survival rate of functions as an oncogenic miRNA (8). Therefore, the aim of the present study was to investigate the functional significance of miR-1274a and to identify the molecular targets and pathways mediated by miR-1274a in ccRCC cells. Our data demonstrated Correspondence to: Dr Hideki Enokida, Department of Urology, that downregulation of miR-1274a inhibited cancer cell prolif- Graduate School of Medical and Dental Sciences, Kagoshima eration and induced apoptosis. Moreover, gene expression data University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan and in silico database analysis showed that bone morphogenetic E-mail: [email protected] protein receptor type 1B (BMPR1B) gene was identified as a candidate target of miR-1274a. The discovery of genes and * Contributed equally pathways mediated by oncogenic miR-1274a provides important insights into the potential mechanisms of ccRCC oncogenesis Key words: microRNA, miR-1274a, renal cell carcinoma, apoptosis, upregulated miRNA and suggests novel therapeutic strategies for the treatment of ccRCC. 174 YoshIno et al: MOLECULAR TARGETS REGULATED BY ONCOGENIC miR-1274a Materials and methods Table I. Clinicopathological characteristics of the ccRCC patients. Clinical specimens and cell culture. The ccRCC and adja- cent normal tissues were collected from 40 ccRCC patients Characteristics Data immediately after undergoing radical nephrectomies at Kagoshima University Hospital between 2003 and 2013. The Total number of cases 40 clinicopathological information of the patients is shown in Mean age ± SD (years) 64.4±13.5 Table I. The specimens were staged according to the American Sex, n (%) Joint Committee on Cancer-Union Internationale Contre le Male 14 (35.0) Cancer (UICC) tumor-node-metastasis (TNM) classification Female 26 (65.0) and histologically graded (9). This study was approved by the Bioethics Committee of Kagoshima University; prior written Stage, n (%) informed consent and approval were obtained from all patients. pT1a 18 (45.0) We used human ccRCC cell lines, 786O, A498, ACHN and pT1b 13 (32.5) Caki1, obtained from the American Type Culture Collection pT2 2 (5.0) (ATCC; Manassas, VA, USA). These cell lines were incubated pT3a 4 (10.0) in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, pT3b 3 (7.5) MA, USA) supplemented with 10% fetal bovine serum (FBS) pT4 0 (0.0) and maintained in humidified incubators (5% CO ) at 37˚C. 2 Grade, n (%) G1 8 (20.0) Tissue collection and RNA extraction. Tissues were immersed in RNAlater (Thermo Fisher Scientific) and stored at -20˚C until G2 30 (75.0) RNA extraction was conducted. Total RNA, including miRNA, G3 0 (0.0) was extracted using the mirVana™ miRNA isolation kit (Thermo Unknown 2 (5.0) Fisher Scientific) following the manufacturer's protocol. Infiltration, n (%) α 27 (67.5) Quantitative real-time reverse transcriptase-polymerase β 13 (32.5) chain reaction (qRT-PCR). Stem-loop RT-PCR (TaqMan γ 0 (0.0) MicroRNA Assays; product ID: 2883 for miR-1274a; Applied Biosystems, Foster City, CA, USA) was used to quantify Venous invasion, n (%) miRNAs according to the manufacturer's protocol for PCR v (-) 29 (72.5) conditions. We used human RNU48 (product ID: 001006; v (+) 11 (27.5) Applied Biosystems) as internal control. For investigating the genes, we applied SYBR-Green quantitative PCR-based array approach, and the following primers were used: BMRP1B forward primer, 5'-CTTTTGCGAAGTGCAGGAAAAT-3' the Cell Proliferation Kit II (Roche Molecular Biochemicals, and reverse primer, 5'-TGTTGACTGAGTCTTCTGGAC Mannheim, Germany) according to the manufacturer's AA-3'; GUSB forward primer, 5'-CGTCCCACCTAGAAT CT instructions. Cell apoptosis assays were carried out using GCT-3' and reverse primer, 5'-TTGCTCACAAAGGTCAC ccRCC cell lines transfected with the transfection reagent, AGG-3'. qRT-PCR was performed with 500 ng of total RNA anti-miR-control and anti-miR-1274a, in 6-well tissue culture using the Power SYBR-Green Master Mix (cat. no. 4367659; plates. Cells were harvested 72 h after transfection by trypsin- Applied Biosystems) on the 7300 Real-Time PCR System ization and washed in cold phosphate-buffered saline (PBS). (Applied Biosystems). The experimental procedures were Double staining with FITC-Annexin V and propidium iodine conducted according to the protocol recommended by the (PI) was carried out using the FITC Annexin V apoptosis manufacturer. The specificity of amplification was monitored detection kit (BD Biosciences, Bedford, MA, USA) according using the dissociation curve of the amplified product.BMPR1B to the manufacturer's recommendations and analyzed within data values were normalized to GUSB. The ∆∆Ct method was 1 h by flow cytometry (FACScan; BD Biosciences). Cells employed to calculate the fold-change of expression level rela- were discriminated into viable cells, dead cells, early and late tive to the internal controls. apoptotic cells by CellQuest software (BD Biosciences), and then the percentages of total apoptotic cells from each samples miRNA inhibitor transfection. ccRCC cell lines were trans- were compared. Experiments were conducted in triplicate. fected with Lipofectamine RNAiMAX transfection reagent and Opti-MEM (Thermo Fisher Scientific) with 10-60 nM Putative target gene analysis of miR-1274a. To investigate miRNA inhibitor (hsa-miR-1274a; product ID: AM13432, cat. the expression status of candidate miR-1274a target genes in no. AM17000) and negative control #1 anti-miRNA (cat. no. ccRCC clinical specimens, we examined Oligo Microarray AM17010; Thermo Fisher Scientific). human 44K (Agilent Technologies, Santa Clara, CA, USA) expression profiling of ACHN with anti-miR-control and Cell proliferation and apoptosis assays
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