Regulation Of HPV E2 On The Expression Of Genes Related To Proliferation And Differentiation Of Cervical Cancer Siha Cells

Cervical cancer is one of the most common malignant tumors in women.The incidence rate is second only to breast cancer,ranking second in female malignant tumors.China is a high incidence area of cervical cancer.A large number of studies have found that human papillomavirus infection is the main cause of cervical cancer development.The high-risk HPV virus continues to infect cervical basal cells,and the HPV genome integrates into the host chromosome,resulting in high expression of HPV E6 and E7,and decreased or absent expression of HPV E2.However,the molecular mechanism of abnormal expression of this HPV cancer-related gene is still unclear.This study selected cervical squamous cell carcinoma,cervical lesions of different lesions,and SiHa and Caski cell lines of cervical squamous cell carcinoma as subjects,using QT-PCR,Western Blot,gene transfection and in vitro animal experiments.The relationship between the expression of HPV E2 and E6,E7 and the genes related to proliferation and differentiation of cervical cancer cells were discussed.First,105 cases of cervical squamous cell carcinoma(SCC)and cervical intraepithelial neoplasia(CIN)were collected,including 35 cases of SCC and 70 cases of CIN(26 cases of CIN I and 23 cases of CIN II,CIN III(21 cases),RT-PCR was used to detect the expression levels of E2,E6 and E7 mRNA in cervical squamous cell carcinoma and precancerous lesions.The results showed that the expression levels of E6 and E7 mRNA increased with the increase of CIN level,and the expression level of CIN III was the highest.At the same time,E6 and E7 mRNA in cervical squamous cell carcinoma also showed high expression but CIN.There was no significant difference in III;the expression level of E2 gene decreased with the increase of CIN level,and the expression level was the lowest and the expression level was the lowest in CIN III and cervical squamous cell carcinoma.The HPV16 E2 specific primer was designed,the E2 gene was amplified by RT-PCR,and the E2 gene overexpression plasmid pcDNA3.1-HPV16E2 was constructed.The HPV16 E2 gene overexpression plasmid and the control empty plasmid were transiently transfected into SiHa cells by liposome method.Real-time quantitative PCR and Western blotting were used to detect the expression of E2,E6 and E7 genes and the expression of Ki67,p16,CyclinD1 and caspase14 mRNA and protein in SiHa cells.The E2 gene-specific siRNA interference plasmid was designed and synthesized.The interference plasmid and the empty plasmid vector were transfected into Caski cells by liposome.The E2 gene silencing efficiency was detected by real-time fluorescent quantitative PCR and Western blotting.,E6,E7 gene and proliferation cycle differentiation related genes Ki67,p16,CyclinD1,caspase14 mRNA and protein expression levels.The results showed that the expression levels of E6 and E7 in SiHa cells overexpressing E2 gene were significantly decreased at mRNA and protein levels,and the expression levels of CyclinD1 and caspase14 were increased,and the expression levels of p16 and Ki67 were significantly decreased.In the silent E2 Caski cells,the expression levels of E6 and E7 genes were significantly increased at mRNA and protein levels,and the expression levels of CyclinD1 and caspase14 were decreased,and the expression levels of p16 and Ki67 were significantly increased.In order to further study the effect of E2 gene on the proliferation and differentiation of cervical squamous cell carcinoma,the HPV16 E2 gene overexpression plasmid was stably transfected into SiHa cells,and screened and identified by G418 to obtain E2 gene stably overexpressing cell line SiHa-E2.The empty-loaded SiHa cell tumor suspension and the E2 gene-stable overexpressed SiHa cell tumor suspension were injected into the right axilla of female BALB/c nude mice,and the nude mice were inoculated with tumor cells,and the growth of nude mice was observed at the same time every week.Tumor growth at the diet and inoculation site,vernier caliper measured and recorded the size of the transplanted tumor(length a,width b),calculated tumor volume V=ab~2/2,plotted tumor growth curve of nude mice,and sacrificed tumors of nude mice at 5th week Organize the photo to weigh.The results showed that the animal model was successfully constructed,and the tumor formation rate was 100%.After inoculating E2 stably overexpressing SiHa cells and empty plasmid vector SiHa cells,the tumors showed continuous growth and the growth rate of subcutaneous xenografts in the experimental group was significantly slower than that in the control group.The tumor mass was significantly reduced compared with the control group.This study preliminarily explored the molecular mechanism of HPV16E2 gene affecting the proliferation and differentiation of cervical squamous cell carcinoma.The study showed that E2 gene inhibits the proliferation of cervical squamous cell carcinoma SiHa cells by regulating the expression levels of E6 and E7 genes,and promotes cell differentiation;E2 gene expression is missing.One of the molecular features of cervical squamous cell carcinoma and high-grade intraepithelial neoplasia provides a new possible target for the prevention and treatment of cervical squamous cell carcinoma.