Study On The Effect Of Different Polymorphic Sites Of HPV16 Virus E6-295T/G And E7-647A/G On The Expression Of STAT1 And IRF9

Object: To investigate the effect of HPV16 E6 295T/G and E7 647A/G polymorphisms on the expression of immune-related molecules STAT1 and IRF9,and to lay a foundation for the prevention and treatment of cervical cancer.Methods:（1）DNA was extracted from 165 cervical cell samples infected with HPV16,and the E6 and E7 genes were amplified by PCR assay and sequenced by Sanger sequencing.The sequencing results were compared with the standard European sequences provided on NCBI,and the phylogenetic tree was constructed.（2）Four types of cells,Ha Ca T,293 T,C33A and Si Ha,were infected with HPV16 E6-295T/E7 647 A overexpression unmutated group,E6-295T/E7-647 G,E6-295G/E7-647 A and E6-295G/E7-647 G overexpression mutated group lentiviral vectors,respectively.（3）q RT-PCR was used to detect the m RNA expression of HPV16 E6 and E7,and Western Blot was used to detect the effects of different polymorphism sites of E6 and E7 on the expression of STAT1 and IRF9 molecules.（STAT1 and IRF9 are important complexes of the IFNκ signaling pathway and can influence the expression of natural immune molecules）（4）The data obtained were analyzed statistically and scientifically using IBM SPSS Statistics 26,and differences were considered statistically significant at P < 0.05.Results:（1）DNA was extracted from cervical cell samples,the HPV16 E6 and E7 genes were sequenced to obtain information on the variant loci of E6 and E7 genes,and statistical analysis was performed,and it was found that the E6-295T/G variant in cervical cancer cells was not significantly different between Uyghurs and Han Chinese（P>0.05）.The variation of E7-647A/G sites in cervical cancer cells of Han ethnic group was significantly higher than that of Uygur ethnic group,and the difference was statistically significant（P<0.05）.After constructing phylogenetic trees for E6 and E7,142 samples were found to belong to European strains（NCBI: NC₀01526.4）,the remaining 23 were Asian strains（NCBI: AF534061）,and no American or other strains were seen.（2）Lentiviral overexpression vectors for E6 and E7 polymorphic sites were constructed and infected with four cell lines,and the expression levels of signal transducer and activator of transcription 1（STAT1）and interferon regulatory factors 9（IRF9）were detected by Western Blot.showed that overexpression of E6-295T/E7-647 A,E6-295T/E7-647 G,E6-295G/E7-647 A and E6-295G/E7-647 G polymorphic lentiviral vectors,respectively,in Ha Ca T cells resulted in STAT1 expression levels were significantly lower than those of the control group,with statistically significant differences（P<0.05）.And the E6-295G/E7-647 A and E6-295G/E7-647 G variant groups were significantly lower than the E6-295T/E7-647 G variant group,and the E6-295T/E7-647 G variant group was significantly lower than the E6-295T/E7-647 A unvaried group,and the difference was statistically significant（P<0.05）.Overexpression of E6-295G/E7-647 A and E6-295G/E7-647 G polymorphic locus lentiviral vectors in293 T cells resulted in significantly lower STAT1 expression levels than controls,with statistically significant differences（P<0.05）.Overexpression of these four polymorphic loci lentiviral vectors in C33 A cells,respectively,resulted in significantly higher STAT1 expression levels than in the control group,with statistically significant differences（P<0.05）,and E6-295G/E7-647 G was significantly higher than E6-295G/E7-647A;E6-295G/E7-647 A was significantly higher than E6-295T/E7-647 G and E6-295T/E7-647 A groups,and the differences were statistically significant（P<0.05）.Overexpression of these four polymorphic loci lentiviral vectors in Si Ha cells respectively could result in significantly higher STAT1 expression levels in the E6-295T/E7-647 A,E6-295G/E7-647 A and E6-295G/E7-647 G groups than in the control group,and the highest STAT1 expression levels in the E6-295G/E7-647 A group were significantly higher than in the other groups.The differences were statistically significant（P<0.05）.Overexpression of these four polymorphic loci lentiviral vectors in 293 T cells,respectively,resulted in significantly lower IRF9 expression levels than the control group,and IRF9 expression in the E6-295G/E7-647 G group was significantly lower than that in the E6-295G/E7-647A;IRF9 expression in the E6-295G/E7-647 A group was significantly lower than that in the E6-295T/E7-647 A and E6-295T/E7-647 G groups,and the differences were statistically significant（P<0.05）.After these four different polymorphic lentiviral vectors infected Ha Ca T cells separately,IRF9 expression was significantly lower than that of the control group,and E6-295G/E7-647 A was significantly lower than that of the E6-295G/E7-647 G group;E6-295G/E7-647 G group was significantly lower than that of the E6-295T/E7-647 A and E6-295T/E7-647 G groups,and the difference was statistically significant（P<0.05）.Overexpression of these four polymorphic lentiviral vectors in C33 A cells respectively resulted in significantly higher IRF9 expression levels than the control group,and the IRF9 expression levels in the E6-295G/E7-647 A group were significantly higher than those in the E6-295G/E7-647 G and E6-295T/E7-647 G groups;the E6-295G/E7-647 G and E6-295 T /E7-647 G groups were significantly higher than E6-295T/E7-647 A,and the differences were statistically significant（P<0.05）.After these four different polymorphic lentiviral vectors infected Si Ha cells separately,IRF9 expression was significantly higher than that of the control group,and E6-295G/E7-647 G was significantly higher than that of the E6-295G/E7-647 A and E6-295T/E7-647 G groups;the E6-295G/E7-647 A and E6-295T/E7-647 G groups were significantly higher than the E6-295T/E7-647 A unvaried group,and the difference was statistically significant（P<0.05）.Conclusion:（1）The cervical cell samples infected with HPV16 E6 and E7 types in this study were mainly European strains,and the E7-647 G locus variant occurred mainly in Han Chinese cervical cancer.（2）When HPV16 E6-295T/G and E7-647A/G lentiviral vectors with different polymorphic loci were infected with 293 T and Ha Ca T non-cancer cells,they caused a significant decrease in the cellular STAT1 and IRF9 protein expression levels,and the E6-295 G variant decreased to the strongest extent.Infection of C33 A and Si Ha cervical cancer cells significantly increased STAT1 and IRF9 protein expression levels,and the E6-295 G variant was the most strongly elevated.