The Roles And Mechanisms Of MiR-375Targeting Sp1in The Progression Of Cervical Cancer

Cervical cancer (CC) is one of the most common gynecologic malignancies in women worldwide. With over99.7%of cervical lesions containing HPV DNA sequences, high-risk human papillomavirus (HR-HPV) has been considered as the necessary factors in the etiology of most cervical cancers, and HPVs16and18are the majority types responsible for the70%cervical cancer. Dates show that HR-HPV E6and E7are the primary transforming viral proteins, who can inactivate the tumor suppressors, P53and Rb respectively, and contribute to cancer progression through a variety of pathways, including promotion of proliferation, inhibition of apoptosis and lost control of cell cycles.miRNAs are a group of conserved noncoding small RNAs which can bind to the untranslated region (UTR) of target mRNA and regulate the stability and translation of mRNAs, resulting in either inhibition of translation or degradation of the target mRNA. miRNAs are important factors involved in a wide array of biological processes including proliferation, apoptosis, metabolism, cellular differentiation, carcinogenesis and development of cancer, and also play key roles in viral persistent infection and clearance.MiR-375is a functional miRNA play important roles in both biological and pathogenic processes. In our previous study, we screened the different expressions of miRNAs between cervical normal and cancer tissues by microarray and found that miR-375was remarkably down-regulated in cervical cancer tissues, compared with normal tissues. In the present study, we examined miR-375expression in sufficient series of well-defined clinical cervical tissues and analyzed the correlations between miR-375expression and poor prognostic clinicopathologic parameters. We further investigated the biological functions of miR-375in migration, and invasion in cervical cancer cells and identified the target gene for miR-375through gain-of-function experiments. Moreover, we explored the relationship between miR-375and HPV16E6/E7expression. This aim of the study is to investigate the roles and mechanisms of miR-375in the progression of cervical cancer, and to identify a new predictor for prognosis or target for therapy. ObjectiveTo investigate altered expression of miR-375in cervical cancer tissues and identify the predictive value of miR-375in the progression of cervical cancer.MethodsUsing stem-loop RT and Real time PCR to detect the expressions of miR-375in170cervical cancer tissue samples and68cervical normal tissue samples. Collect the clinicopathological parameters of these patients with cervical cancer. The correlation between the miR-375levels and the prognostic factors were analyzed.Results1. miR-375is down-regulated in squamous cervical cancer tissue samples comparing to normal tissue.2. The expressions of miR-375was correlated with FIGO stage, pelvic lympho nodes metastasis, deeper stroma invasion, vaginal extension and lymphovascualr involvements.Conclusions1. miR-375is down-regulated in squamous cervical cancer tissue samples.2. Low level of miR-375is significantly correlated with poor prognosis factors, suggesting miR-375might be a candidate of prognostic predictor. ObjectiveTo investigate the biological functions of miR-375in cervical cancer cells and identify the target gene of miR-375and to explore the molecular mechanisms of miR-375in cervical cancer progression.MethodsSiha and CaSki cells were transfected with miR-375mimic or siRNA targeting Spl. MTT method and flow cytometry technique were used to detect the changes of cellular proliferation and cell cycle distribution induced by miR-375. Wound healing test and transwell assay were used to detect the alterations of migration and invasion. Examine the expressions of Spl by real time RT PCR and western blot. Luciferase activity reporter assays were used to identify Spl as the direct target of miR-375.Results1) Over expression of miR-375in Siha and CaSki inhibited cell proliferation, migration, invasion and blocked G1to S transition.2) Over expression of miR-375reduced the expression of Spl both at mRNA and protein level. There was a negative correlation between the expression of miR-375and the mRNA level of Spl in the cervical cancer tissues.3) The relative luciferase activity of pmirGLO-UTR containing miR-375binding sites was significantly suppressed by cotransfected with miR-375but not negative control. Also, the relative luciferase activity was not altered when cotransfection was done with miR-375and pmirGLO-mUTR containing a mutant binding site or empty pmirGLO.4) Knock down of Sp1in Siha and CaSki by siRNA inhibited cell proliferation, migration, invasion and block G1to S transition.Conclusions1Over expression of miR-375in cervical cancer cells inhibits the cell proliferation, migration, invasion and blocks cell cycle G1transition to S, resulting in G1arrest. It implies miR-375may participate the progression of cervical cancer.2Spl is one of the direct target genes of miR-375. The important role of miR-375in cervical cancer is at least partially mediated by targeting Spl. ObjectiveTo analyze the relationships between miR-375and HPV16E6/E7expression, and further explore the possible mechanism through which miR-375regulate the expression of HPV16E6/E7.MethodsEmploying Stem-loop RT PCR to detect miR-375expressions in normal tissue samples with HR-HPV infection or without, and in cervical cancer cells with different E6/E7levels induced by plasmid expressing E6/E7or knock down of E6/E7by siRNA. Detected the expressions of E6/E7and the downstream molecules with real time RT PCR and western blot.Results1. There was no significant difference between normal tissue samples with or without HR-HPV infection.2. Neither overexpression of HPV16E6/E7by transfection with HPV-16E6/E7expression vector into C33A cells nor down-regulated expression of HPV16E6/E7using siRNAs in SiHa cells significantly altered miR-375transcripts.3. The expressions of miR-375in cervical tissue samples with simple HPV16infection were negative correlated with the mRNA level of HPV16E6or E7.4. Over expression of miR-375in Siha cells suppressed the expression of E6and E7, leading to increase the expression of P53and pRB protein, and decrease the expression of MCM7protein. 5. Over expression of miR-375in HCT-116did not change the expression of P53and pRB protein.Conclusions1. In cervical cancer cells, over expression of miR-375suppresses the expression of E6/E7, leading to influence the expression of downstream molecules mediated by E6/E7.2. HR-HPV infection or E6/E7protein don't change the expression of miR-375.