MicroRNA93Via Targeting RAB11FIP1Promotes Oncogenesis Of Cervical Cancer

Cervical carcinoma is one of the most common malignant diseases among women. Carcinogenesis of the cervix is thought to be a multistep process involving virus infection, tumor suppressor gene activation or proto-oncogene inactivation, immunity suppression, and others. Although HPV vaccination has been carried out in various countries, it prevents only about70%occurrences of the diseases and has no therapeutic effect on already existed cervical cancer or precursors. The identification of molecular regulators associated with initiation or progression would contribute to prevention and treatment of cervical cancer. The finding of microRNAs (miRNAs) provides a new approach for exploring new molecules and mechanisms in oncogenesis and progression in cervical cancer.MiRNAs are a group of conserved noncoding RNAs that can bind to the3’ untranslated region (UTR) of target mRNA and regulate stability and translation of mRNAs, resulting in either inhibition of translation or degradation of the target mRNA. Components of the miRNA machinery and miRNAs themselves are involved in many cellular processes that are altered in cancer, such as differentiation, proliferation and apoptosis. It has been verified that miRNAs show differential expression levels in cancer and are able to affect cellular transformation, carcinogenesis and metastasis, acting as oncogenes or tumor suppressors.In a previous study, we screened the different expression of miRNAs between cervical normal and cancer tissues through miRNA array and found that miR-93was remarkably up-regulated in cervical cancer tissues compared with normal tissues (11), which imply that miR-93may participate in the pathogenesis or development of cervical cancer. In the present study, we examined miR-93expression in cervical cancer tissues and investigated the function of miR-93in proliferation, migration, and invasion of cervical cancer cells. We found that MiR-93might act as an oncogenic miRNA and serve as a potential therapeutic target in cervical cancer. Part Ⅰ The expression signature of miR-93and its values in high grade CIN and cervical cancerObjectiveTo investigate altered expression of miR-93in cervical cancer tissues and identify the predictive value of miR-93in the progression of cervical cancer.MethodsUsing stem-loop RT and Real time PCR to detect the expressions of miR-93in168cervical cancer tissue samples,60CIN2or3tissue samples and48cervical normal tissue samples. Collect the clinicopathological parameters of these patients with cervical cancer. The correlation between the miR-93levels and the prognostic factors were analyzed.Results1. miR-93is up-regulated in squamous cervical cancer tissue samples and CIN2or3tissue samples, comparing to normal tissue.2. The expression of miR-93was not correlated with FIGO stage, pelvic lympho nodes metastasis, deeper stroma invasion, vaginal extension and lymphovascualr involvements.Conclusions1. MiR-93is up-regulated in squamous cervical cancer tissue samples and there is no significant difference of relative expression of miR-93among different clinicopathologic characteristics2. In high grade CIN, MiR-93is also up-regulated, while no difference is found between squamous cervical cancer tissue samples and CIN sample. The over-expression of miR-93as an early event plays a role in promoting oncogenesis of cervical cancer. Part Ⅱ The impact of miR-93to cervical cancer cell biology behaviorsObjectiveTo investigate the impact of miR-93to cervical cancer cell biology behaviors.MethodsSiha and CaSki cells were transfected with miR-93inhibitor. MTT method was used to the changes of cellular proliferation. Flow cytometry technique was used to detect the cell apoptosis and cell cycle distribution induced by miR-93. Transwell assay were used to detect the alterations of invasion.Results1. Low expression of miR-93in Siha and CaSki inhibited cell proliferation, and suppresses apoptosis.2. Low expression of miR-93can not effected the cell cycle and the cell invasion.ConclusionsThe down-regulation of MiR-93expression promots proliferation and suppresses apoptosis in cervical carcinoma cells, while there isno affection to the cell cycle and the cell invasion. These indicated that miR-93plays a role in promoting oncogenesis of cervical cancer. Part III MiR-93targeted control RAB11FIP1protein expression in cervical cancer cellsObjectiveIdentify the target gene of miR-93and explore the molecular mechanisms of miR-93in cervical cancer oncogenesis.MethodsImmunohistochemical analysis was performed on human cervical normal, CIN2or3, and cervical cancer tissue. Siha and CaSki cells were transfected with miR-93inhibitor or RAB11FIP1Ientiviral. MTT method and flow cytometry technique were used to detect the changes of cellular proliferation and cell apoptosis induced by miR-93. Examine the expressions of RAB11FIP1by real time RT PCR and western blot. Luciferase activity reporter assays were used to identify RAB11FIP1as the direct target of miR-93.Results1) RAB11FIP1protein expression is decreased in CIN and cervical cancer tissues. A significantly negative correlation between miR-93and RAB11FIP1expression was identified in CIN and cervical tissues.2) MiR-93negatively regulates RAB11FIP1protein expression3) The relative luciferase activity of pmirGC214-UTR containing miR-93binding sites was significantly suppressed by cotransfected with miR-93but not negative control. Also, the relative luciferase activity was not altered when cotransfection was done with miR-93and pmirGC214-mUTR containing a mutant binding site or empty pmirGC214.4) Overexpression of RAB11FIP1inhibits proliferation and promotes apoptosis in cervical cancer cells Conclusions1. RAB11FIP1is one of the direct target genes of miR-93. The important role of miR-93in cervical cancer is at least partially mediated by targeting RAB11FIP1.2. Overexpression of RAB11FIP1inhibits proliferation and promotes apoptosis in cervical cancer cells. The expression of RAB11FIP1protein in high grade CIN and cervical caicinoma is down regulation. These all indicate that RAB11FIP1protein can inhibit oncogenesis of cervical cancer in early phase.