J Biosci (2019) 44:126 Ó Indian Academy of Sciences DOI: 10.1007/s12038-019-9952-9 (0123456789().,-volV)(0123456789().,-volV) MiR-506-3p suppresses the proliferation of ovarian cancer cells by negatively regulating the expression of MTMR6 YUAN WANG,XIA LEI,CHENGYING GAO,YANXIA XUE,XIAOLIN LI,HAIYING WANG and YAN FENG* Department of Gynaecology, Yan’an University Affiliated Hospital, Yanan, Shaanxi Province, China *Corresponding author (Email, [email protected]) MS received 10 February 2019; accepted 26 July 2019; published online 22 October 2019 MicroRNAs have been reported to play a crucial role in ovarian cancer (OC) as the most lethal malignancy of the women. Here, we found miR-506-3p was significantly down-regulated in OC tissues compared with corresponding adjacent non- tumor tissues. Ectopic miR-506-3p expression inhibited OC cell growth and proliferation using MTT and colony formation assay. Additionally, flow cytometry analysis showed that the overexpression of miR-506-3p induced cell cycle G0/G1 phase arrest and cell apoptosis in OC cells. A luciferase reporter assay confirmed that the myotubularin-related protein 6 (MTMR6) was the target of miR-506-3p. The expression of MTMR6 was increased in OC tissues compared with adjacent tissues using immunohistochemistry. Elevated MTMR6 protein levels were confirmed in OC cells lines compared with immortalized fallopian tube epithelial cell line FTE187 using western blotting. In addition, knockdown of MTMR6 imitated the effects of miR-506-3p on cell proliferation, cell cycle progression and apoptosis in OC cells. Furthermore, rescue experiments using MTMR6 overexpression further verified that MTMR6 was a functional target of miR-506-3p. Our data indicate that miR-506-3p might serve as a tumor suppressor gene and propose a new regulatory mechanism of MTMR6 by miR-506-3p in OC. Keywords. Apoptosis; cell proliferation; miR-506-3p; MTMR6; ovarian cancer 1. Introduction binding predominantly to 30UTR of target mRNAs, micro- RNAs play a crucial role in fundamental cellular processes, In developed countries, ovarian cancer (OC) related mor- such as proliferation, differentiation, migration, apoptosis, tality currently account for more than any other female and tissue specific functions including angiogenesis, hor- genital tract tumors (McAlpine et al. 2014). Each year about mone production and innate immune response (Liz and 22,440 women will experience OC, and 14,080 deaths are Esteller 2016; Kim et al. 2017). The evident connection expected to occur in the United States (Siegel et al. 2017). between microRNA dysregulation and cancer was discov- OC typically presents with a few months of post-menopausal ered in 2002 (Acunzo et al. 2015). From then on, numerous bleeding, persistent pelvic, abdominal pain and distension information regarding the role of microRNAs in cancer (Brain et al. 2014). Despite the international standard of care tumorigenesis and drug resistance has been gained (Zhao for high-grade serous OC is surgery and platinum-based et al. 2015). MicroRNA-506-3p (miR-506-3p), a component cytotoxic chemotherapy, whereas the overall survival of of the X chromosome-linked cluster in the primate testis, these patients have not changed significantly for several was found to be involved in multiple physiological and decades (Kehoe et al. 2015). Recent epigenetic studies have pathological procedures in several human tumors (Huang challenged the traditional view on the pathomechanism of et al. 2017; Wu et al. 2018). The pleiotropic activities and OC, implying that microRNAs may be both cause and tumor-specific contributions of miR-506-3p to various can- consequences of aberrant epigenetic events in OC (Ramas- cer development and progression are achieved by binding to sone et al. 2018). its specific target genes (Wen et al. 2015; Guo et al. 2017). MicroRNAs are an evolutionarily conserved classical of Though miR-506-3p has recently been demonstrated to act short, regulatory non-coding RNAs (*22 nucleotides) that as tumor suppressor in non-small lung cancer (Guo et al. acts to negatively regulate transcripts expression of both 2017), retinoblastoma (Wu et al. 2018), cervical cancer coding and non-coding genes (Langhe 2015). Lately, more (Wen et al. 2015), and osteosarcoma (Jiashi et al. 2018). than 2588 mature human microRNAs have been discovered However, the role of miR-506-3p in OC remains to be and the list is growing (Strmsek and Kunej 2015). By elucidated. http://www.ias.ac.in/jbiosci 1 126 Page 2 of 11 Yuan Wang et al. The myotubularin (MTM)-related protein family in humans Table 1. Clinicopathological features of ovarian cancer patients consists of 14 members with specificity for phosphatidylinos- (n = 20) itol 3-phosphate (PtdIns3P) and PtdIns (3,5) (Clague and Lorenzo 2005). Members of this family encode a central pro- Clinical pathologic parameters Cases (n = 20) tein tyrosine phosphatase domain (PTP), pleckstrin homology/ Age GRAM (PH/G) and coiled-coil (CC) domains (Choudhury \50 8 et al. 2006). Several studies have been published in recent years C50 12 that the malignant biological behaviors are regulated by MTMs Tumor size (cm) \415 (Yoo et al. 2004). For instance, myotubularin-related protein 3 C45 (MTMR3) is responsible for miR-99a-mediated regulation of FIGO stage oral cancer cells proliferation, migration and invasion (Kuo I/II 14 et al. 2014). By using new software for cell tracking, Oppelt III/IV 6 et al. (2014) observed that MTMR3 deficiency caused severe Histological grade Low 9 reduction in velocity in three cancer cell lines. In colorectal Moderate 6 cancer, MTMR7 overexpression resulted in the inactivation of High 5 insulin-mediated AKT-ERK signaling and proliferation (Wei- dner et al. 2016). MTMR6 could negatively regulate Ca2?- FIGO federation of gynecology and obstetrics ? activated K channel KCa3.1 which implicate in the prolifer- ative activity of some cancer cells (Srivastava et al. 2005). A recent study revealed that MTMR6 has an unexpected role in cell line FTE187 cells were obtained from the American preventing cell death (Clague and Lorenzo 2005). Notably, Type Culture Collection (ATCC, Manassas, VA, USA). The MTMR6 was identified as miR-190b-regulated factor that OC cells were cultured in RPMI-1640 medium (Sigma, St. involved in the pathophysiologic mechanism of SIV infection Louis, MO, USA) with 10% fetal bovine serum (FBS, in the intestinal mucosa (Mohan et al. 2014). Sigma). FTE187 cells were kept in ovarian epithelial cell In the present study, the crosstalk between miR-506-3p medium (GIBCO, Carlsbad, CA, USA) with 10% FBS. All and MTMR6 as a cause for OC cancer cells proliferation, cell lines were maintained in a humidified atmosphere con- cell cycle progression and apoptosis, as well as the mecha- taining 5% CO2 at 37°C. nism underline this crosstalk were discovered. Our findings have the potential for broadening our understanding of epi- genetics of caner etiology. 2.3 Cell transfection The miR-506-3p mimics (miR-506-3p) and negative control 2. Materials and methods (miR-NC) were purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China). Two different small interfering 2.1 Clinical tissue samples RNAs for MTMR6 (si-MTMR6-1 and si-MTMR6-2) and si- NC were purchased from RiboBio Co., Ltd (Guangzhou, A total of 20 pairs of tumor tissues and adjacent non-tumor tissues China). The pcDNA3.1-MTMR6 (MTMR6) and pcDNA3.1 were collected from OC patients who underwent surgical resec- empty vector were synthesized by Guangzhou RiboBio Co., tion in the Department of Gynaecology, Yan’an University Ltd. (Guangzhou, China). For cell transfection, OVCAR3 Affiliated Hospital (Shanxi, China) between January 2016 and and SKOV-3 cells were seeded in six-well plates at a density December 2017. Before surgical resection, all patients were of 3 9 105 cells per well and transfected with miR-506-3p, confirmed not to receive chemotherapy or radiotherapy and miR-NC, si-MTMR6 or si-NC. In the rescue experiment, signed the written informed content. The basic clinicopatholog- OVCAR3 cells were co-transfected with miR-506-3p and ical characteristics of all enrolled patients were summarized in MTMR6 plasmids. All cell transfections were performed table 1. The stage for every patient was retrospectively assessed using LipofectamineTM 2000 Reagent (Invitrogen, Carlsbad, based on a modified International Federation of Gynecology and CA, USA) in accordance with the manufacturer’s Obstetrics (FIGO) staging system (Javadi et al. 2016). The col- instructions. lected tissues were immediately snap-frozen and stored in liquid nitrogen. The present study was approved by the Ethics Com- mittee of Yan’an University Affiliated Hospital (Shanxi, China). 2.4 Bioinformatics analysis To predict the potential target genes of miR-506-3p, several 2.2 Cell lines and culture condition software programs, including PicTar 5 (https://pictar.mdc- berlin.de/), TargetScan version 7.1 (http://www.targetscan. Human OC cell lines (ES-2, SKOV-3, HO-8910 and org) and miRBase release 22 (http://www.mirbase.org/) were OVCAR3) and the immortalized fallopian tube epithelial used. MiR-506-3p targets MTMR6 in ovarian cancer Page 3 of 11 126 2.5 Dual luciferase reporter assay Fisher Scientific), called as MTMR6 mutant type (MUT). Then, OVCAR3 and SKOV-3 cells were seeded in 24-well The 30-untranslated regions (30UTRs) of human MTMR6 plates at a density of 2 9 106 cells per well and co-trans- cDNA with the potential target sites for miR-506-3p were fected with 50 pmol of miR-506-3p or miR-NC and 20 ng of amplified and inserted into the psiCHECK2 plasmid (Pro- MTMR6 WT or MTMR6 MUT reporter plasmid using mega Corporation, Madison, WI, USA), named as MTMR6 LipofectamineTM 2000 (Invitrogen, USA). Luciferase wild type (WT). The corresponding site-directed mutagen- activities were quantified 48 h post-transfection using Dual- esis of miR-506-3p in the 30UTR of MTMR6 was performed Luciferase Reporter Assay System (Promega, USA) using Phusion Site-Directed Mutagenesis Kit (Thermo according to the manufacturer’s instructions.