HPV16E6/E7Promoter-targeting SiRNA Induces Owth Inhibition Of Cervical Cancer Cell Line SiHa In Vitro And In Vivo

Cervical carcinoma is the second most common malignancy in women worldwide. Of all the malignant tumors, the incidence of cervical cancer has the most specific factor. The simultaneous expression of human papillomavirus (HPV) E6and E7oncogenes is pivotal for malignant transformation and maintenance of malignant phenotypes. A number of epidemiologic studies reveal that HPV16is most prevalent and accounts for more than50%of all genotypes in cervical cancers. RNA interference has recently been recognized as a powerful and promising tool to inhibit specific gene expression through endonucleasic cleavaging and degradating of homologous mRNA. Until recent research, siRNA can effectively knock down target genes through siRNA-induced transcriptional gene silencing (TGS), and posttranscriptional gene silencing (PTGS), but more on PTGS. However, like other virus, HPV also has ability of variation, which indicates new pathway research is necessary. It has been known that the expression of high-risk HPV16E6/E7is controlled by a unique and common promoter (P97) that is located immediately up-stream of the E6gene. Thus, it is likely that targeting for P97via TGS can result in abolition of both HPV16E6and E7activity. We previously demonstrated that siRNA targeting P97effectively knocked down HPV16E6/E7, suggesting that a promoter-targeting siRNA can induce gene silencing of an extraneous viral gene such as HPV, but it was an in vitro study.In vitro study, HPV16-transformed SiHa cells were used as a model system; Realtime RT-PCR, Western Blotting, MTT assay, Annexin V apoptosis assay and flow cytometry were applied to examine the effects of two category of siRNA. In vivo, The aim was to verify the inhibitory effects of HPV16promoter-targeting siRNA on tumor growth by injecting siRNA into BALB/c mice inoculated by SiHa cells and by transfecting siRNA into SiHa cells in vitro followed by animal inoculation, to estimate the value of E6/E7specific siRNA-induced transcriptional gene silencing as a potential therapeutic strategy for cervical cancer. Part I. HPV16E6/E7promoter-targeting siRNA induces growth inhibition of cervical cancer cell line SiHa in vitroObjective:To verify whether HPV16E6/E7can be reduced by promoter-targeting siRNA in SiHa cells.Methods:The siRNA targeting the promoter of HPV16E6/E7was transfected into cervical cancer cell line SiHa. Then the E6and E7expression, cellular proliferation and apoptosis was observed.Results:In vitro, whether with promoter-targeting siRNA or E7-targeting siRNA, E6/E7mRNA expression were simultaneously reduced. In protein expression, As an alternative P53protein was up to199%and577%, P16protein was induced by49.5%and44.7%. Proliferation were obviously inhibited by29.29%and26.19%, while apoptosis was remarkably increased by161.54%and124.62%.Conclusions:The promoter-targeting siRNA could effectively and simultaneously knocks down both extraneous viral genes E6and E7, and consequently inhibited the proliferation and induced the apoptosis in SiHa cells. Part â…¡. HPV16E6/E7promoter-targeting siRNA induces growth inhibition in transpalnted tumor of cervical cancerObjective:To confirm the growth inhibition in transpalnted tumor of cervical cancer with HPV16E6/E7promoter-targeting siRNA.Methods:In vivo, we designed two methods, injecting siRNA into BALB/c mice inoculated by SiHa cells and by transfecting siRNA into SiHa cells in vitro followed by animal inoculation, Tumor volume and growth curves were assessed. Furthermore, cellular proliferation and apoptosis of inoculated tumors were determined by immunohistochemistry staining and TUNEL assay.Results:1. In vivo, Tumor growth in mice using in vitro HPV16promoter-targeting siRNA transfection was decreased by average89.7%at day80compared to scramble control, while E7-targeting siRNA was reduced by average64%. The tumor weight in control group was was4.59-fold heavier than promoter group, while E7transcript scramble group was2.6-fold heavier.2. With siRNA intratumoral injection in vivo, the tumor volume and tumor weight were decreased compared to the control group. The tumor volume in mice using HPV16promoter-targeting siRNA was reduced by29.8%and E7-targeting siRNA by42.8%compared to scramble control. The tumor weight was induced by1.65-fold and1.45-fold similarly.3. Pathologic characteristics of tumor tissues showed little significant difference in proliferation by ki-67staining among all groups, but TUNEL assay presented a remarkably increased apoptotic cells in promoter-targeting siRNA group compared to related scramble group. Conclusions:1. RNAi targeting the promoter of HPV16E6/E7acts effectively in vivo to inhibit the growth in transplanted tumor of cervical cancer.2. Tumor retardation of HPV16E6/E7promoter-targeting siRNA is mainly through induction of cellular apoptosis.