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Dysregulation of Micro-143-3P and BALBP1 Contributes to the Pathogenesis of the Development of Ovarian Carcinoma

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Dysregulation of Micro-143-3P and BALBP1 Contributes to the Pathogenesis of the Development of Ovarian Carcinoma ONCOLOGY REPORTS 36: 3605-3610, 2016 Dysregulation of micro-143-3p and BALBP1 contributes to the pathogenesis of the development of ovarian carcinoma Hongyan ZHANG1 and WANBIN LI2 1Department of Gynecology, Affiliated Hospital of Jining Medical University, Jining, Shandong 272029; 2Jining Medical University, Jining, Shandong 272113, P.R. China Received February 28, 2016; Accepted April 9, 2016 DOI: 10.3892/or.2016.5148 Abstract. The objective of the present study was to identify the inhibitor. These findings provide support that downregula- association between mir-143-3p and RalA-binding protein 1 tion of the miR-143-3p is associated with a decreased risk of (RALBP1), and their roles in regulating the development of ovarian cancer. ovarian cancer. Overexpression of RALBP1 induced apoptosis of the ovarian cancer cells, and developed ovarian cancer. Introduction In silico analysis and luciferase assay were used to identify whether RALBP1 was the target of mir-143-3p. Subsequently, Ovarian cancer is the most life-threatening malignancy in real-time PCR and western blotting were used to determine reproductive tract and fourth leading cause of cancer-related the expression level of mir-143-3p, RALBP1 mRNA and deaths in women. Majority of ovarian cancers stem from protein in different groups, furthermore, MTT assay and flow epithelium, though there are also stromal and germ cell cytometry were used to detect the viability and apoptosis of tumors (1,2). Generally, ovarian tumors are silent at early stage cells in different treatment groups. We identified RALBP1 and remain unrecognized in most cases (>80%) until ovarian as a target gene of miR-143-3p using computational analysis, carcinoma has metastasized to other areas out of the ovaries. and the luciferase activity of cells transfected with wild-type Most recently, 21,550 American new cases of ovarian cancer RALBP1 and RALBP1 siRNA were much lower than the and 14,600 deaths from the cancer in 2009 were reported by scramble control, however, the luciferase activity of cells the American Cancer Society (www.cancer.org). There is little transfected with mutant RALBP1 was similar with scramble improvement in 5-year survival rates over the past 10 years, control. The real-time PCR and western blot results suggested and long-term survival is significantly unsatisfactory and that the miR-143-3p level was markedly lower in participants among the worst of all the anatomic sites of cancer. with ovarian cancer compared with normal control, while the Ral paralogs bind to an array of effectors such as ZONAB, expression of RALBP1 mRNA and protein were evidently phospholipase D1 (PLD1), filamin, exocyst components overexpressed in participants with ovarian cancer compared (Exo84 and Sec5) and RalBP1 (RLIP) upon activation, leading with normal control. Furthermore, the RALBP1 mRNA and to various functions associated with Ral (3). Earlier findings protein level in cells transfected with miR-143-3p mimics showed that RalBP1, the best characterized effector of Ral, was and RALBP1 siRNA were downregulated, while notably correlated with Ral-mediated tumorigenesis (4). Additionally, upregulated subsequent to transfection with miR-143-3p the human bladder cancer tissues had higher expression of inhibitor, when compared with scramble control. Additionally, RalBP1 than normal tissues, which was associated with Ral the viability of cells were inhibited following transfec- expression (5). The subcutaneous xenograft tumor growth in tion with miR-143-3p mimics and RALBP1 siRNA, while cancer cell lines could be reduced by deletion of RalBP1 by notably promoted subsequent to transfection with miR-143-3p suppression of its function by antibody or by antisense reduc- inhibitor. Apoptosis of cells were promoted following trans- tion (6). In RalBP1, there is association between the exocyst fection with miR-143-3p mimics and RALBP1 siRNA, while complex component Sec5 and transformation mediated by notably inhibited subsequent to transfection with miR-143-3p Ral (4). There was also correlation between PLD1 and cell transformation by oncogenes such as Ras (7). MicroRNAs are non-protein-coding RNAs that play post-transcriptional regulator of genes. microRNAs have Correspondence to: Dr Wanbin Li, Jining Medical University, been shown to serve a role in almost all cellular processes 16 Taibai Lake Lotus Road, Jining, Shandong 272113, P.R. China in animals and plants first discovered as critical regulators E-mail: [email protected] of developmental timing in Caenorhabditis elegans (8,9). MicroRNAs have ~22 nucleotides, derived from large major Key words: micro-143-3p, BALBP1, ovarian carcinoma, tumori- transcripts which generate imperfect structures of stem loop. genesis, apoptosis, proliferation MicroRNAs bind to target mRNAs and suppress transition or enhance transcript degradation, thereby regulating the expres- sion of genes (10). 3606 ZHANG and li: miR-143-3P AnD BALBP1 Dysregulation Notably, a great deal of microRNAs serve as crucial System (Bio-Rad, Hercules, CA, USA) was used to carry out regulator of gene expression and are generally present in the reactions. The internal control was U6. Bio-Rad CFX the non-protein-encoding regions (11). More than 60% of Manager quantitative PCR software (Bio-Rad) was used to protein-coding transcripts are regulated by small non-coding examine the change in expression of miR-143-3p and RALBP1 RNAs known as microRNAs (12). Each microRNA regulates mRNA, and the 2-ΔΔCt method was used to calculate the change a range of target genes at the post-transcriptional level. They in expression of RALBP1 gene and miR-143-3p. Three inde- are associated with a variety of disorders and serve as tumor pendent tests were performed. inhibitors and oncogenes to affect tumorigenesis (13). For instance, microRNAs have been associated with ovarian tumor Cell culture and transfection. Dulbecco's modified Eagle's development and progression (14). It is reported that germline medium (DMEM) (Cellgro Mediatech, Manassas, VA, USA) variations in processing genes and microRNAs known as containing 1% non-essential amino acids (Sigma-Aldrich, messenger RNA transcripts of their target genes play a role St. Louis, Mo, USA), 2 mM L-glutamine, 50 µg/ml strepto- in tumor progression as well as the risk of development of mycin sulfate, 50 U/ml penicillin and 10% fetal bovine serum cancers, such as ovarian cancer (15). (FBS) (Invitrogen) was used to maintain the A2780 cells in It has been previously shown that miR-143-3p is differ- an atmosphere of 5% CO2/95% air at 37˚C. When cells were entially expressed in the A2780 cells collected from ovarian cultured to 80% confluency, transfection was performed carcinoma, and dysregulation of RalA-binding protein 1 using Lipofectamine 2000 (Invitrogen) according to the (RALBP1) has also been reported to be involved in the manufacturer's protocol. A2780 cells were analyzed 48 h after molecular mechanism of apoptosis of A2780 cells (16,17). By transfection. searching the online miRNA database, we found that RALBP1 is a virtual target of miR-143-3p. In the present study, we Cell proliferation assay. A2780 cell proliferation was validated RALBP1 as a target of miR-143-3p and verified the measured using the Alamar Blue assay according to the involvement of miR-143-3p and RALBP1 in the development quantitative metabolic conversion of non-fluorescent resazurin of ovarian carcinoma. and blue to fluorescent resorufin and pink by living A2780 cells. An Alamar Blue (Invitrogen) stock solution was added Materials and methods to the wells equal to 10% of the total incubation volume for 2-6 h. Synergy HT Multi-Mode Microplate Reader (Bio-Tek Subjects. The present study consisted of 35 patients treated at instruments, Winooski, VT, USA) was used to determine the Department of Gynecology, Affiliated Hospital of Jining the reduction of resazurin in the cultures according to the Medical Unversity (Jining, China) between november 2013 absorbance at 590 and 530 nm wavelengths. Each test was and January 2015. All data processing and sample collection performed at least three times. were approved by the Ethics Committee of Affiliated Hospital of Jining Medical Unversity. Participants or their first-degree Luciferase assay. The PCR fragment including the binding relatives had already signed the informed consents before start site was used to make the luciferase constructs. The of the experiments after carefully explained all potential risk 3' untranslated region (3'UTR) of RALBP1 with the target factors. Cancerous tissue samples and adjacent non-cancerous site of miR-143-3p was inserted into the XhoI and NotI sites control were collected from each participant. All of the of the psiCHECK-2 (Promega, Madison, Wi, USA) following specimens were obtained from surgical resection. None of the the manufacturer's recommendations. Mutations were intro- patients was treated with any neoadjuvant treatment, such as duced using site-directed mutagenesis (Stratagene, La Jolla, radiotherapy and chemotherapy, before surgical resection and CA, USA). For the reporter assays, Lipofectamine 2000 the followed tumor resection to collect clinical data. was used to co-transfect A2780 cells with negative control (NC mimics), mutant miR-143-3p or wild-type miR-143-3p RNA isolation and real-time PCR. TRIzol reagent (Invitrogen, mimics (SunBio Corporation, Shanghai, China) and reporter Carlsbad, CA, USA) was used to extract the total RnA from plasmids (500 ng). Promega Luciferase Assay System was A2780 cells and tissue
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