Effects Of HPV16 E6/E7 Transcriptional Gene Stable Silencing And CyclinD1 Overexpressi-on Respective On Proliferation Apoptosis And Cyclical Factors Of Human Cervical Cancer SiHa Cells

Cervical cancer is one of the most common malignant in women worldwide. High-risk human papillomavirus(HPVs) are the necessary conditions for its development. HPV16 especially E6/E7 gene, is considered major causative agent for cervical cancer, plays an important role in the process of cervical epithelial magligant transformation. In study, we had built E6/E7 gene silencing stable cell line by small interfering RNA(siRNA). It had effection on proliferation apoptosis and related cyclical factors of human cervical cancer SiHa cells. Then, the cyclinD1 vector was designed, transfected into SiHa cells, whether influence on proliferation and cyclical factors for SiHa cells.E6/E7 shRNA plasmid was constrcucted and transfected into SiHa cells.Selecting by G418, we had built E6/E7 gene stable silencing cells named cm-16. Empty-plsmid as blank control named B3. E6/ E7 m RNA and protein expression were respectively detected by quantitative time-polymerase chain reaction(QT-PCR)and reverse transcription-polymerase chain reaction(RT-PCR)and Western blot. The results showed that E6/E7 gene silencing efficiency were 89% and 82%, respectively. Therefore, we determined that shRNA integrated into cellular genome DNA, inducing E6/E7 gene silence in transcription.Futhermore, cellular growth and proliferation were detected by MTT/ QT-PCR and immunohistochemistry(IHC).Cellular apoptosis were detected by TUNEL and flow cytometry. The proliferation activity of cm-16 cells was significantly lower than SiHa. However, the apoptosis was significantly higher in cm-16 than in SiHa cells.At the same time, related cyclical factors were detected by RT-PCR in SiHa/ cm-16 cells. The results displayed that cyclinD1/p21/CDK2/vimentin genes were increasing, p27/cyclin E/E-cadherin gene was reducing, other genes had no significant difference.We used QT-PCR and IHC detection expression of cyclinD1 in CIN and squamous cell carcinoma. The results revealed that expression of cyclinD1 is low in squamous cell carcinoma. mRNA and protein of cyclinD1 in SiHa/cm-16/Hcc94 cell lines were detected by RT-PCR and Western blot.The resulte showed that it was higher for cycinD1 expression in Hcc94 and cm-16 than SiHa cells. However, E7 gene was lower in Hcc94 and cm-16 than SiHa cells.We designed cyclinD1- pcDNA3.1 plasmid and transfected it into SiHa cells. Then, We had successful built cyclinD1 gene stable overexpression cell line named G-3 cells, empty plasmid transfected to SiHa cells and built cell line named C-2 cells. The related cyclical genes were detected RT-PCR in SiHa/G-3 cells. We found in G-3cells, p21/CDK2/ P27/ cyclinE/ vimentin were increasing, E-cadherin genes were reducing, E6/ E7 and other genes were no significant difference. cyclinD1 overexpression promoted prossibly epithelial mesenchymal transformation in G-3 cells. Then, cellular growth and proliferation were detected by MTT and RT-PCR. The results showed that cyclinD1 gene overexpression maybe promot proliferation in G-3 cells.Compared with gene silencing efficiency in domestric research, the E6/E7 was significantly decreased, which laid the foundation for molecular mechanism of cervcal cancer. Meanwhile, we discussed that cyclinD1 expression in CIN and squamous cell carcinoma. Then, we explored that the relationship between cyclinD1 gene and epithelial mesenchymal transformation, this the first time in related demestric research.