Involvement Of MiR-199a In Chemotherapy Resistance Of Ovarian Cancer Through Modulating Expression Of MTOR

BackroundOvarian cancer is the deadliest cancer of the female reproductive system.Ovarian carcinoma is often called the silent killer because it is characterised by few or late symptoms and has the highest mortality rate of all malignant gynaecological tumors.The standard initial management of ovarian cancer consists of surgical staging and administration of six cycles of intravenous chemotherapy with carboplatin and paclitaxel.However, 75% of patients present with advanced (stage III or IV) disease and, although more than 80% of these women benefit from first-line therapy, tumour recurrence occurs in almost all these patients at a median of 15 months from diagnosis. Second-line treatments can improve survival and quality of life but are not curative.The main limitation to a successful treatment is the development of resistance to combined chemotherapy, such as platinum-based drugs, cisplatin or carboplatin, coupled with paclitaxel. Cisplatin binds to double-stranded DNA and forms DNA adducts, interfering with DNA replication and RNA transcription ultimately triggering apoptosis. Paclitaxel is a mitotic inhibitor which binds and hyperstabilize microtubules, specifically binding to theβ-tubulin subunit, thereby suppressing microtubule dynamicity. Many molecular mechanisms implicated in the rise of resistance in cellular models of ovarian cancers have been characterized, emphasising its biological complexity: increased DNA repair activity and defective DNA damage response, increased antiapoptotic regulators activity, growth factor receptor deregulation and post-translational modification or altered expression ofβ-tubulin and other microtubule regulatory proteins. In the past few years the discovery of a new class of genomic regulators which are now universally recognized as central players in virtually any neoplasm development and progression, named microRNAs.MicroRNAs (miRNAs) are a growing class of small (～22 bp) endogenous non-coding RNAs which modulate gene expression mainly by base-pairing to the 3′-UTR of the target mRNA, and causing translational repression, mRNA cleavage, or estabilization miRNA-mediated posttranscriptional gene regulation is emerging as a critical regulator of many cellular processes, both physiologic and pathologic. Hundreds of miRNAs have been found in various organisms, suggesting their potential roles in all biological events. Aberrant levels of miRNA have been reported in a variety of human cancers. They have been shown to have both diagnostic and prognostic significance and to constitute a novel target for cancer treatment. The miRNA signature of drug resistance could be pursued to develop new strategies for targeted therapies in chemorefractive ovarian carcinoma patients. The therapeutic potential of miRNAs is still unexplored and miRNA-based cancer gene therapy offers the theoretical appeal of targeting multiple gene networks that are controlled by a single miRNA. Strategies of delivery"in patients"of miRNAs are currently in advanced development and are expected to enter clinical experimentation soon.ObjectivemiR-199a involved in the biological behavior of tumor cells and affected the growth of cancer cells by regulated target gene PAK4(p21-activated kinase 4)and mTOR (mammalian target of rapamicin);substantial researches have demonstrated that miR-199a could involve in the drug resistance of cancer cell through target protein.The study wants to determine whether miR-199a play an important role in cisplatin drug resistance of ovarian tumor cells.MethodsThe expression of miR-199a were analyzed by real-tmie PCR. OV2008 and C13* cells were transfected with the inhibitors or mimics of miR-199a or negative cotrol (NC) using Lipofectamine 2000 and treated by cisplatin;then apoptosis was analyzed by FACS. OV2008 and C13* cells were transfected with the inhibitors or mimics of miR-199a or negative cotrol (NC) using Lipofectamine 2000 and treated by different concentrations of cisplatin(DDP);CCK-8 determined ovarian cancer cell activity.ResultsReal-tmie PCR was used to determine the expression level of miR-199a in OV2008 and C13* cells.The expression level of miR-199a was significantly higher in OV2008 than C13* cells(P<0.05). When OV2008 and C13* cells transfected with the mimics or inhibitors of miR-199a and treated by DDP, apoptosis was analyzed by FACS shows that apoptosis of C13* cells was higher than control after transfected with the mimics of miR-199, OV2008 cells was lower than control after transfected with the inhibitors of miR-199. After transfection OV2008 cells with the inhibitor of miR-199a,and then treated with different concentrations of DDP,the cell cytotoxic effect of OV2008 cells for DDP were lower than control with increasing concentrations of DDP; the cell cytotoxic effect of C13* cells for cisplatin were higher than control after transfection the mimics of miR-199a.ConclusionsThe expression of miR-199a in OV2008 cells were significantly higher than C13* cells.After transfected with the mimics and inhibitors of miR-199a in OV2008 and C13* cells,the apopotosis ratio changed obviously. miR-199a may increase the sensitivity of ovarian tumor cells to DDP. ObjectiveTo research the effect of miR-199a on the sensitivity of OV2008 and C13* cells to DDP through transfected with the mimics and inhibitors of miR-199 and investigate its mechanism of action.MethodsWe applied real-tmie PCR and Western blot to analysis the expression of mTOR in OV2008 and C13* cells.After transfection OV2008 and C13* cells with the inhibitor of miR-199a with Lipofectamine 2000,we detected the change of expression level of mTOR in OV2008 and C13* cells by real-tmie PCR and Western blot.We construct luciferase reporter vectors of mTOR and then transfected them and the mimics of miR-199a into OV2008 cells by Lipofectamine 2000 to detect regulating change downstream target gene.ResultsReal-tmie PCR and Western blot were used to determine the expression level of mTOR in OV2008 and C13* cells.The expression level of mTOR was significantly higher in C13* than OV2008 cell(sP<0.05). After OV2008 and C13* cells transfected with the mimics and inhibitors of miR-199a ,real-tmie PCR and Western blot analyzed the results show that the mimics of miR-199a repress the expression of mTOR;and the inhibitors of miR-199a promote the expression of mTOR.After transfected the mimics of miR-199a and luciferase reporter vectors of mTOR into OV2008 cells , renilla luciferase reporter gene analysis indicated expression level of mTOR was negative regulated by the mimics of miR-199a.ConclusionsmiR-199a regulates the sensitivity of ovarian cancer cells to DDP by mTOR,and involved in the DDP resistance process of ovarian cancer.