The Role Of TLR9 And High-risk HPV16 Oncogene Expression In Cervical Lesions And Their Relationship

Objective:Cervical cancer is the fourth largest malignant tumor in women.Persistent high-risk human papillomavirus（HR-HPV）infection is the main cause of cervical cancer and precancerous lesions.Among which HPV16 is the most common carcinogenic type.Studies have shown that TLR9 exists on the endosomal membrane of cervical epithelial cells,and can specifically bind to the unmethylated Cp G motifs of HPV16 E6 and E7genes,and participate in the occurrence and development of cervical cancer and other tumors closely related to HPV infection.Based on cell studies,it has been found that HPV16 E6 and E7 oncoproteins can inhibit the transcription process of TLR9,suggesting that TLR9 is closely related to high-risk HPV16 infection and the expression of oncogenes.However,the relationship between the two in the occurrence and development of cervical lesions is still unclear.In this study,women with normal cervix,cervical precancerous lesions and cervical cancer patients were selected as the research objects to form the natural gradient of the occurrence and development of cervical lesions.Based on the clear relationship between the expression of TLR9 or HPV16oncogenes and cervical lesions,the relationship between TLR9 and HPV16 infection or the expression of oncogenes in the development of cervical lesions was explored,which provides scientific basis for the etiology of cervical cancer and open up new ideas for the formulation of effective strategies and measures to prevent malignant progression of cervical lesions in HPV-infected women.Methods:From April 2017 to August 2018,124 women with normal cervical（NC）,168women with cervical intraepithelial neoplasia（CIN）and 49 women with squamous cell carcinoma of the cervix（SCC）were collected according to strict inclusion and exclusion criteria in the department of gynaecology of the Second Hospital of Shanxi Medical University.A unified structured questionnaire was used to collect general demographic characteristics,living habits,hygiene habits,menstrual and marital history and other factors that may be related to cervical lesions.The cervical exfoliated cells of the patients were collected,and the HPV infection and typing were detected by using hybridization technology.The Cervical biopsy specimens were collected of the patients were collected.The TLR9 mRNA levels were detected by using real-time fluorescent quantitative PCR,and the protein levels of TLR9 and HPV16 E2,E6,E7,L1,L2 were detected by Western-blotting.SPSS 22.0 and Graph Pad Prism 8.0.2 software were used for analysis of variance,χ²test,Fisher’s exact test,pearson correlation,spearman rank correlation,multiple logistics regression and other statistical analysis.The inspection level isα=0.05（two-sided）.Results:1.Analysis of general demographic characteristics and related factors of cervical lesions:The results of multiple logistics regression model analysis showed that compared with the NC group,age≤35 years old（OR=4.073（1.329,12.483））and passive smoking（OR=1.874（1.133,3.100））were the risk factors for CIN.Passive smoking（OR=3.469（1.453,8.287））,divorce,widow and separation（OR=4.698（1.316,16.776））were risk factors for SCC.2.The relationship between HR-HPV infection or HPV16 oncoprotein expression and cervical lesions:With the progress of cervical lesions,the infection rates of HR-HPV(χ²_trend=55.549,P<0.001)and HPV16(χ²_trend=80.718,P<0.001)both showed an upward trend.Compared with the NC group,the high expression of E2 protein could reduce the risk of CIN（a OR=0.255（0.066,0.980））and SCC（a OR=0.026（0.004,0.187））;high expression of E6 and E7 proteins could increase the risk of CIN（a OR=4.299（1.224,15.093）;a OR=6.634（1.853,23.746））and SCC（a OR=11.815（1.844,75.698）;a OR=6.538（1.394,30.664））.High expression of L1（a OR=0.076（0.016,0.368））or L2（a OR=0.061（0.011,0.344））protein and high E2/E6 ratio（a OR=0.016（0.001,0.251））could reduce the risk of SCC.No similar relationships between high expression of L1 or L2 protein,high E2/E6 ratio and CIN were observed.3.The relationship between TLR9 expression and cervical lesions:The expression of TLR9 mRNA（F=15.943,P<0.001）and protein（F=22.808,P<0.001）was significantly different among different cervical lesions.The expression level of TLR9protein in SCC group was higher than that in NC group,while the expression level of TLR9 mRNA was lower than that in NC group,with statistical significance.With the aggravation of cervical lesions,the expression of TLR9 protein increased gradually(χ²_trend=9.661,P=0.002),and the high expression of TLR9 protein increased the risk of SCC（OR=3.912（1.341,11.409））.No similar relationship between TLR9 mRNA and cervical lesions was observed.4.The relationship between TLR9 and HPV16 infection or oncogene expression:In HPV16-positive women,the expression of TLR9 mRNA was lower than that of HPV16-negative women,while the expression of TLR9 protein was higher than that of HPV16-negative women（P<0.05）,a similar relationship was observed in HR-HPV-positive women.Spearman rank correlation analysis showed that TLR9 protein was positively correlated with HPV16 E2,E6,L1,and L2 proteins（r_s=0.250,P=0.004;r_s=0.174,P=0.05;r_s=0.257,P=0.003;r_s=0.253,P=0.004）.There was no significant correlation between TLR9 mRNA and HPV16 oncoprotein.5.Relationship between TLR9 and HPV16 infection or oncogene expression in the progression of cervical lesions:In NC and CIN groups,TLR9 protein expression in HPV16 positive women was higher than that in HPV16 negative women,while in SCC group,TLR9 protein expression in HPV16 positive women was lower than that in HPV16 negative women（P<0.05）.TLR9 mRNA expression in HPV16 positive women was lower than that in HPV16 negative women in the CIN group（P=0.001）,no similar relationship was found in NC and SCC groups.Spearman rank correlation analysis showed that TLR9 was positively correlated with E2,E6,E7,L1 and L2 proteins of HPV16 in CIN women（r_s=0.241,P=0.04;r_s=0.231,P=0.05;r_s=0.271,P=0.02;r_s=0.280,P=0.016;r_s=0.368,P=0.001）,A similar association was found in the SCC group.There was no significant correlation between TLR9 mRNA and HPV16 oncoprotein in NC,CIN and SCC groups.Conclusion:1.Women younger than 35 years of age had an increased risk of cervical intraepithelial neoplasia,women who were divorced,widowed or separated had a greater risk of cervical cancer,and passive smoking increased the risk of cervical cancer and its precancerous lesions.2.HR-HPV and HPV16 infection may increase the risk of cervical intraepithelial neoplasia and cervical cancer.Especially from the perspective of oncogenes,high expression of HPV16 E6 and E7 oncoproteins could significantly increase the risk of cervical cancer and its precancerous lesions.which once again confirmed the important role of HPV16 oncogene in cervical cancer.With the progression of the malignant degree of cervical lesions,the E2/E6 ratio,L1 and L2 protein decreased,which verified the landmark role of E2/E6 ratio,L1 and L2 protein on the integration of HPV and host DNA.3.High expression of TLR9 protein can increase the risk of cervical lesions,and TLR9 protein elevation may be an early warning marker of cervical lesions.In particular,TLR9 protein expression was significantly higher in HPV16-positive precancerous women than in HPV16-negative women,and was positively correlated with the expression of HPV16 oncoprotein.It is suggested that the high expression of TLR9protein is related to the HPV16 infection and the increase of oncoprotein.Therefore,TLR9 detection should be promoted in women to provide some basis for the application of TLR9 intervention agents in clinical practice.