The Roles And Mechanisms Of JunD In The Progression Of Cervical Cancer

Cervical cancer is the fourth most commonly diagnosed cancer and the fourth leading cause of cancer death in females worldwide. Although the occurrence and mortality rates of cervical cancer are reduced with a large-scale application of screening and considerable progression of surgery, radiotherapy and chemotherapy, the prognosis of patients with advanced cervical cancer is still not satisfactory. Finding effective treatment for middle and advanced cervical cancer is significant for improving the prognosis of patients with advanced cervical cancer. Infection with high-risk human papillomavirus (HR-HPV) is a necessary factor in the development of cervical cancer. The virus oncogenes expand their own malignant effects by affecting the host celluar genes. It’s a main emphasis for looking for potential therapeutic targets in clinical research by exploring the related molecules affected by the virus in host cells.JunD protein encoded by the JUND gene is the latest member of the Jun family. It’s different from the other members of the family by its expression and biological functions. JunD mediate cell proliferation, apoptosis, differentiation and other functions by activations or inhibitions of a variety target genes. In tumor cells, the dysfunction of JunD expression, abnormal regulation of JunD and the interaction with other factors such as cyclinDl, MEN1, p21, MMP-7 involved in processes of turnor formation, metastasis, angiogenesis and so on.In order to illustrate the function and mechnisms of JunD in the progression of cervical cancer, we firstly explored the effects of JunD on cell biological behavior in cervical cancer by using cell function experients in present study, then searched for target gene by bioinformatics methods, verified and confirmed the target gene by gene functional experiments. Finally, we examed the expression of JunD and target gene in cervical tissues, verified the correlation with each other and compareed the correlation between the expression level of JunD and target genes with the pathological parameters of cervical cancer. This aim of study is to investigate the potential therapeutic targets of cervical cancer and to provide evidence for the treatment of advanced cervical cancer.Part ⅠThe biological function of JunD in cervical cancer cellsObjectiveTo investigate the biological control of JunD on cell proliferation, apoptosis, invasion, migration and other biological behaviors of cervical cancer cells.MethodsmRNA and protein levels of JunD in HPV positive cervical cancer cell lines and HPV negative cell lines were detected by real-time RT PCR and western blot. Cervical carcinoma cell line SiHa and CaSki were transfected with small interfering RNA targeting JunD or negative control to down-regulate JunD expression. HPV negative cell line U2OS were transfected with JunD plasmid or the empty vector to increase JunD expression. Cell proliferation experiments were carried out by CCK-8 assay. Cell apoptosis analysis were conducted by flow cytometry technique.Cell migration and invasion were detected by transwell assays.Results1. Comparing to HPV negative cell lines, the expression of JunD in HPV positive cervical cancer cell lines is higher.2. Knock down of JunD in SiHa and CaSki by siRNA inhibited cell proliferation, cell migration and invasion, promoted apoptosis.3. Up regulation of JunD in HPV negative cell line U2OS enhacned cell proliferation, migration and invasion surpressed cell apoptosis.Conclusion:1. In cervical cancer cells, JunD promotes cell proliferation, avoids apoptosis and exhances cell migration and invasion. It suggests that JunD may play a cancer -promoting role in the progression of cervical cancer.Part ⅡBioinformatics analysis of JunD in cervical cancer and verification of JunD target genes.ObjectiveTo identify the target gene of JunD and to reveal the mechanism of JunD in cervical cancer progression.MethodsFirstly, screen genes which were coexpressed with JunD in cervical cancer by coexpression analysis supported by Oncomine database included the data sets of clinical samples and cell lines. Secondly, search for genes which were interacted with JunD in BioGRID database. Finally, Combined the coexpression genes and protein interacted genes of JunD, find out the candidate genes. mRNA levels of candidate genes were detected by real-time RT-PCR in HPV positive cervical cancer cell lines and HPV negative cell lines. The expression of JunD was regulated by small interfering RNA transfection. Examine the expression of SIRT1 by real-time RT-PCR and western blot. The relationship between JunD and target gene SIRT1 was detected by immunoprecipitation assay.Results1. Candidate genes co expressed and protein interacted wih JunD in cervical cancer were predicted by combinding with Oncomine database and BioGRID database.2. SIRT1 was predicted after verifing candidate genes in HPV positive cervical cancer cell lines and HPV negative cells.3. JunD can bind to SIRT1 and regulate the expression of SIRT1.Conclusion1. Oncomine database is an effective tool for screening functional related genes.2. The expression of SIRT1 is regulated by JunD at post transcriptional levels, and it is the direct target gene of JunD.Part ⅢThe effects of SIRT1 on biological behaviors in cervical cancerObjectiveTo verify the biological effects of target gene SIRT1 on cell proliferation, apoptosis, invasion and migration in cervical cancer.MethodsSiHa cells were transfected with siRNA targeting SIRT1 or negative control to down-regulate SIRT1 expression. CCK-8 method and flow cytometry technique were used to detect the changes of cellular proliferation and apoptosis induced by siR-SIRT1. Transwell assay were used to detect the alterations of migration and invasion. Examine the expressions of SIRT1 by real time RT PCR and western blot.Results1. Down regulation of SIRT1 in SiHa by siRNA surpressed cell proliferation, cell migration and invasion, promoted apoptosis.Conclusion1. High expression of SIRT1 promotes the proliferation, invasion and migration of cervical cancer cells, and inhibits apoptosis. It suggests that SIRT1 plays a similar role as JunD in carcinogenesis in cervical cancer progression.Part IVThe expression and significance of JunD and SIRT1 in cervical cancerObjectiveTo investigate altered expression of JunD and SIRT1 in cervical related tissues and identify the predictive value of JunD and SIRT1 in the progression of cervical cancer.MethodsThe expressions of JunD and SIRT1 were examed by immunohistochemical staining in 138 cases of primary cervical epithelial carcinoma,77 cases of high level of cervical lesions and 59 cases of cervical normal tissues. Collect the clinicopathological parameters of these patients with cervical cancer. The correlation between the JunD and SIRT1 levels and the prognostic factors were analyzed.Result1. The expression of JunD and SIRT1 in cervical squamous cell carcinoma was higher than that in high-grade lesion group and normal epithelial tissue group.2. JunD high expression was significantly correlated with FIGO stage, differentiation, stromal invasion and vascular invasion.3. SIRT1 high expression was significantly correlated with FIGO stage, differentiation and vascular invasion.4. The high expression of JunD was significantly with the high expression of SIRT1.Conclusion:1. The expression of JunD are related with SIRIT1 significantly in cervical cancer, and Their higher expression in cervical cancer tissues are associated with adverse.