Study On CFLIP Expression And CFLIP-Targeted Gene Silencing With ShRNA In Cervical Cancer

Apoptosis plays a considerable role in embryo growth, cellular homeostasis and balance of immune system. It also opens a new territory for neoplasia research. Neoplasia results from cells proliferation out of control along with failure of apoptosis. The apoptotic mechanism is triggered by intracellular as well as extracellular stimuli. The central event in the intracellular apoptotic parth way is the proteolytic activation of cystein aspartyl-specific proteases-8 (caspase-8), also named FLICE (FADD-like interleukin-1-beta converting enzyme). cFLIP (cellular FLICE-like inhibitory protein), inhitor of caspase-8, was first described in the study of melanoma in 1997 by Irmler, et al. FLIP is an intracellular survival factor structurally similar to caspase-8, and modulates cell surface receptor-mediated cell death. Overexpression of cFLIP is crucial to the development of tumors. cFLIP-targeted silencing can promote cell apoptosis and enhance the sensitivity to chemotherapy in gastric cancer, colon cancer and rhabdomyosarcoma et al. Cervical cancer is the second commonest cancer and the second major cause of cancer-related mortality in women worldwide. Although the detailed molecular mechanism remains to be elucidated, studies have now confirmed that it was the consequence of synergism of multiple factor, such as infection of virus especially for the human papilloma virus, mutation of certain genes and immune escape. There is little research on the function of cFLIP in the carcinogenesis of cervix and its relation with HPV. RNA interference (RNAi), a new technique, can be used to silence certain genes to study the expression, regulation and function of the genes. It is one of the useful tools in the study of tumor and its therapy. This study was to elucidate these questions. OBJECTIVE:A. To investigate the expression and significance of cFLIP and HPV16E7 in cervical cancer tissueB. To silence cFLIP in Hela cells by small hairpin RNA (shRNA) and relief the inhibition of caspase-8 by cFLIPC. To explore the potential of cFLIP-targeted gene silencing in inhibiting cellular growth, raising cellular apoptosis and enhancing the sensitivity to radiochemotherapy in cervical cancerMETHODS:A. The expression of cFLIP and HPV16E7 were detected with immunohistochemistry in 100 samples of cervical tissues involving 40 invasive cervical cancers, 40 cervical intraepithelial neoplasias (CIN) and 20 normal cervixes and their clinical significance were analyzed.B. Amplification of cFLIPS and cFLIPL genes were examined with RT-PCR, and the expression of cFLIPS and cFLIPL proteins were detected with Weston Blot in Hela, Siha, C33a and Caski. And Hela was choosed to be studied in the following steps.C. A shRNA plasmid targeted cFLIP was constructed to silence cFLIP expression and transfected to Hela cells with lipo 2000. RT-PCR and Weston Blot were applied to determine the silencing effect.D. Along with the down-regulated cFLIP, the change of caspase-8 protein expression were examined with Weston Blot. The cellular proliferation and apoptosis after transfetion were examined with MTT assay, flow cytometry (FCM) and confocal imaging system.E. Hela cells were treated with cisplatin(DDP), irinotecan(CPT-11) and Go60 after transfection and their sensitivities to radiochemotherapy with the down-regulated cFLIP were examined with MTT assay and FCM. RESULTS:A. The positive rates of cFLIP and HPV16E7 expression in cervical cancer, CIN and normal cervix were lower by turns (P<0.05). There were no significant difference of cFLIP expression between cervical cancer and CINⅡ-Ⅲ(P>0.05). The positive rates of cFLIP in cervical cancer and CINⅡ-Ⅲwere markedly higher than those in CINⅠ(P<0.05). The expression level of cFLIP in cervical cancer has no association with pathological classification, histological grade, FIGO stage and lymph node involvement. cFLIP expression in HPV-16 E7 positive CINⅡ～Ⅲwas markedly greater than that of HPV-16 E7 negative CINⅡ～Ⅲ(P<0.05). There were no significant difference of cFLIP expression between HPV-16 E7 (+) and HPV-16 E7 (-) CINⅠ, and so were cervical cancer tissues (P>0.05).B. cFLIPS and cFLIPL were expressed in Hela, Siha, C33a and Caski cells both on mRNA and protein levels.C. The shRNA plasmid targeted cFLIP gene succuccefully and specifically silenced cFLIPS and cFLIPL and regulated caspase-8 protein expression.D. Down-regulating cFLIP gene in Hela cells have significance effects on cellular proliferation, apoptosis and sensitivity to radiochemotherapy (P<0.05).CONCLUSIONS:A. Overexpression of cFLIP and HPV16E7 may be involved in the devolpment and progression of cervical cancer. cFLIP may interact whith HPV-16 E7 during the transformation of CIN into cervical cancer.B. The over expression of cFLIP may act as a novel molecular marker for diagnostics and a specific target for therapeutics in human cervical cancer. cFLIP-targeted RNAi might be useful to control this disease.