HnRNP E2 Regulates Key Genes Of HPV16 And Its Role In Cervical Carcinogenesis

Objective:High-risk HPV?HR-HPV?infection is the primary cause for cervical carcinogenesis,and HPV16 is the key type of HR-HPV infection.At present,about 60.6% of cervical cancer is caused by HPV16 infection worldwide.HPV16 E2,E6,L1,and L2 are key genes for the cervical carcinogenesis,and they contain a variety of sites which can bind to RNA-binding proteins and protein complexes.hn RNP E2,which is regard as a special RNA-binding protein,is playing a vitally critical role in regulating gene transcription and post-transcriptional.hn RNP E2's KH domains provide a biological basis for specific binding to HPV16 genes.However,the role of hn RNP E2 in cervical carcinogenesis and the relationship between hn RNP E2 and HPV16 key genes are still inconclusive currently.In this study,population studies and in vitro cell experiments were used to explore roles of hn RNP E2 and HPV16 key genes in cervical carcinogenesis and their relationship,in order to provide a theoretical basis for the study of the etiology and pathogenesis of cervical cancer and new ideas for the prevention and treatment of cervical cancer.Methods:67 women with normal cervix?NC?,69 with low cervical intraepithelial neoplasia?CIN??and 68 with high cervical intraepithelial neoplasia?CIN?/??selected randomly from the cervical lesions cohort in Jiexiu County of Shanxi Province from June 2014 to September 2014 established by our research group,and 40 patients with cervical squamous cell carcinoma?SCC?treated at the Shanxi Provincial Cancer Hospital were diagnosed as subjects of this study in the same period.All datas of participates ageneral demographic characteristics,living habits,reproductive status,and past medical history were collected through the structured questionnaire designed by ourselves,and cervical tissues from operation and biopsy and exfoliative cells were collected.HPV16 infection was detected by flow-through hybridization and protein expression of hn RNP E2 and HPV16 E2,E6,L1,and L2 were detected by Western blot.At the same time,cervical cancer cell Si Ha?HPV16-positive?was selected as the study subject,and overexpression technology was used to regulate hn RNP E2 of Si Ha cell.The overexpression hn RNP E2 group was used as the experimental group,and the mock group and vector group were used as control.m RNA expression levels of each gene were measured by Real-time PCR.The database was established and analyzed by SPSS 21.0 software with ?²test,?² trend test,ANOVA and repeated measures data ANOVA.The Kruskal-Wallis H test was used for the data that did not obey normal distribution.The correlation between two variables was analyzed by Spearman rank correlation.All results were considered significant when P-value were less than 0.05.Results:1.Relationship between HPV16 infection and cervical carcinogenesis: infection rates of HPV 16 in NC,CIN?,CIN?/? and SCC were 10.4%,14.5%,41.2% and 67.5% respectively.The rate of HPV16 infection was increased gradually with the increasing severity of cervical lesions(?²_trend=46.627,P<0.001).HPV16 infection rates in CIN ?/?and SCC group were significantly higher than NC group.In additon,HPV 16 infection rate was positively associated with cervical lesions?CIN: a OR=3.23?0.61-17.07?;CIN ?/?: a OR=13.19?2.59-67.22?;SCC: a OR=49.45?7.87-310.61??,and it is the risk factor of cervical carcinogenesis.2.Relationship between HPV16 early genes and cervical carcinogenesis: expression levels of HPV16 E2 and E6 protein were significantly different in each group?HPV16 E2: H=19.870,P=0.001;HPV16 E6: H=16.201,P=0.001?.The expression level of HPV16 E2 protein in CIN?/? group was lower than NC group significantly,and the expression level of HPV16 E2 protein in SCC group was lower than CIN?/? and CIN?group.Protein expression level of HPV16 E6 in SCC group was higher than CIN?group significantly.The ratio of E2/E6 in SCC group was lower than NC and CIN?group significantly,but there was no significant difference between other groups?P>0.05?.3.Relationship between HPV16 late genes and cervical carcinogenesis: expression levels of HPV16 L1?H=12.011,P=0.007?and L2 protein?H=11.859,P=0.008?were significantly different in each group.Expression levels of HPV16 L1 and L2 in SCC group were lower than CIN?group significantly,but there was no significant difference between other groups?P>0.05?.4.Relationship between hn RNP E2 and cervical carcinogenesis: the expression level of hn RNP E2 was different significantly in each group?H=37.854;P<0.001?.The expression level of hn RNP E2 in SCC and CIN?/?group were lower than NC group,and SCC group was lower than CIN?group significantly,but there was no significant difference between other groups?P>0.05?.Low expression rates of hn RNP E2 was increased gradually with the increasing severity of cervical lesions(?²_trend=20.389,P<0.001).5.The correlation between hn RNP E2 and HPV16: hn RNP E2 was positively correlated with the ratio of HPV16 E2/E6?r=0.212,P<0.001?,HPV16 L1?r=0.278,P=0.022?and L2?r=0.446,P<0.001?.HPV16 E2 was negatively correlated with HPV16 E6?r=-0.295,P=0.025?,and positively correlated with the ratio of HPV16 E2/E6?r=0.572,P<0.001?,but there was no correlation between other factors?P>0.05?.6.Effects of hn RNP E2 on m RNA expression of HPV 16 key genes: m RNA expression levels of HPV16 E2,E6,L1 and L2 in overexpression hn RNP E2 group were higher than mock and vector group?P<0.05?.Conclusion:1.HPV16 infection could increases the risk of cervical carcinogenesis.Therefore,vaccination in uninfected individuals are important for early prevention of cervical carcinogenesis;Low expression of HPV16 E2,high expression of HPV16 E6,low expression of L1 and L2,and low E2/E6 ratio may increase the risk of CIN?/? and SCC and may be used as an auxiliary biomarker for CIN?/? and SCC for early screening of cervical cancer and have potential value in the prediction of carcinogenesis risk.2.The low expression of hn RNP E2 protein may increase the risk of CIN?/?and SCC,suggesting that expression level of hn RNP E2 may reflect the progression of cervical lesions,and the decrease of hn RNP E2 expression may serve as an auxiliary index of cervical carcinogenesis.3.There were positive correlation between hnRNP E2 and E2/E6 ratios,HPV16 L1 and HPV16 L2,suggesting that expression of hn RNP E2 and HPV16 E2,E6,L1,and L2 may affect each other during the development of cervical carcinogenesis.4.After upregulation of hn RNP E2,m RNA expression levels of HPV16 E2,E6,L1,and L2 in cervical cancer cells were decreased,suggesting that hn RNP E2 may act as a transcriptional repressor and inhibit transcription of HPV16 E2,E6,L1,and L2.