The Role Of Human Papillomavirus Infection And Its Synergistic Factors In The Progression Of Cervical Intraepithelial Neoplasia

Cervical cancer is the fourth most common cancer in women worldwide and presents a high mortality rate,especially in developing countries.There are an estimated 604,127 new cases of cervical cancer and 341,831 deaths globally in 2020.Nearly 90%of cervical cancer deaths occurred in developing countries.In recent years,with the popularization of cervical cancer screening and the widespread application of HPV vaccines,the incidence of cervical cancer in developed countries has dropped significantly,while the incidence of cervical cancer in China is on the rise.It is estimated that 98,900 new cases and 30,500 deaths occurred in 2015 in China.Shanxi Province is a high-risk rural area for cervical cancer in China.According to literature reports,the incidence of cervical cancer in Yangcheng County,Shanxi Province from 2003 to 2007 was 78.23/100,000,and the mortality rate of cervical cancer in 2005 was 25.07/100,000,which are much higher than the average incidence and mortality rate in China.Cervical intraepithelial neoplasia（CIN）,as a precancerous lesion of the cervix,provides a good opportunity to block the occurrence and progression of cervical cancer.Due to the unclear understanding of CIN’s outcome rules and related factors,there is a lack of more refined clinical diagnosis and treatment plans.Therefore,finding the relevant factors affecting the progression of CIN is a problem that researchers are very concerned about,but there is still a lack of large-scale population-based prospective cohort studies.Persistent infection with high-risk human papilloma virus（HR-HPV）is the primary factor leading to CIN progression to cervical cancer.There are 13-14 most common HR-HPV genotypes associated with cervical cancer and CIN.However,the risk of progression to cervical cancer and CIN varies substantially among individual carcinogenic genotypes.Can existing screening strategies be optimized by combining different HR-HPV genotypes?HR-HPV is not the only factor in cervical cancer and CIN.So what factors are its synergistic factors?Whether there are special geographical factors for such a high incidence in Shanxi Province deserves further study.Biogeographic evidence shows that the high incidence area of cervical cancer in Shanxi Province is located in the Taihang Mountains,which is consistent with the high incidence area of neonatal neural tube defects caused by folate deficiency.Folate deficiency may be a special geographical factor in the high incidence area of cervical cancer in our province.However,it is not clear whether folate deficiency is associated with the risk of CIN progression and infection with HR-HPV.Recent studies have shown that circular RNAs（circRNAs）play an important role in tumorigenesis and development.Exploring the expression differences of circRNAs in cervical carcinogenesis is expected to discover some novel biomarkers of CIN progression.This study is planned to be carried out in the following parts:1)To establish a study cohort of CIN in Shanxi and evaluate its baseline characteristics;2)Combined with follow-up data,to evaluate the effect of HPV genotyping for the triage of women with atypical squamous cells of undetermined significance（ASC-US）;3)To analyze the relationship of red blood cell（RBC）and serum folate concentrations with cervical intraepithelial neoplasia and high-risk HPV infection;4)Based on the Biobanks established by Shanxi CIN cohort study,we screened and verified the differentially expressed circRNAs during cervical carcinogenesis.The purpose of this study is to discover several key influencing factors of CIN outcomes,optimize screening strategies,and provide an important theoretical basis for subsequent in-depth study.Part 1 Prospective cohort of cervical intraepithelial neoplasia in Shanxi Province:participants enrollment and comparison of baseline characteristics.Objective:The primary objective of this study was to establish a study cohort of CIN in Shanxi and evaluate its baseline characteristics.Methods:The Shanxi CIN cohort study is a prospective cohort study,based on a large-scale combined community-and hospital-based cervical cancer screening program.From June 2014,a baseline survey was carried out on a total of 40,000 women from the two community sites of Yangqu and Jiexiu in Shanxi Province and 1,200 women from the Second Hospital of Shanxi Medical University and Shanxi Provincial Cancer Hospital,including epidemiological questionnaires,gynecological examinations,and experimental index detection（including liquid-based cytology,colposcopy,histopathological examination and HPV genotyping,etc.）and collection of biological specimens.The database was established by Epidata3.0 software.Results:The detection rate of CIN was 2.00%（95%CI:1.82-2.09）among 40,000 women in the community field,and the detection rate of CC was 48/100,000.The mean age of 3504 women in the Shanxi CIN follow-up cohort was 47.0±9.5 years old.The top 5 genotypes of HPV infection rate were HPV16（19.1%）,51（3.4%）,33（3.3%）,58（3.3%）and 18（3.0%）.The median of serum folate concentration 16.1（12.6-20.6）nmol/L and RBC folate concentration 325.9（228.1-445.6）ng/ml in the CIN2+group were lower than those in the<CIN2 group.In the CIN2+group,the strong positive expression rates of P16（33.8%）,Ki67（39.7%）,and folate receptor（FR α）（39.2%）were significantly higher than those in the<CIN2 group.Conclusions:By establishing the Shanxi CIN cohort,the prevalence rates of CIN and CC in the Shanxi population were clarified,and the distribution characteristics of HPV genotypes in the cohort population were found.Based on the differences in baseline characteristics between different subgroups,a research-sharing platform was established to conduct follow-up research on biological indicators affecting CIN progression.Part 2 Efficient Combination of Human Papilloma virus Genotyping for the Triage of Women with Atypical Squamous Cells of Undetermined SignificanceObjective:In this prospective,population-based study,we evaluated the utility of high-risk human papilloma virus（HR-HPV）genotyping for triaging women with atypical squamous cells of undetermined significance（ASC-US）in the Chinese rural area.Methods:A total of 40,000 women were recruited from rural areas of Shanxi Province,China,between June 2014 and December 2014.Women with Pap results of ASC-US underwent HPV genotyping,colposcopy and histopathological examination.For those with normal cervixes or cervical intraepithelial neoplasia（CIN）1 on the initial evaluation,a 2-year follow-up study was performed.Results:The reporting rate of ASC-US was 5.76%（2,304/40,000）in the study population.The detection rates of CIN 2 or above（CIN2+）and CIN 3 or above（CIN3+）in women with ASC-US were 7.28%and 1.75%,respectively.HPV 16（39.53%）,HPV 58（17.83%）,and HPV 52（15.50%）were the three most prevalent HR-HPV genotypes among all women with ASC-US cytology.The five most common HR-HPV genotypes in CIN3+lesions were HPV16,HPV58,HPV33,HPV31 and HPV18.Compared with the 15 HR-HPV testing,genotyping for a combination of HPV16/18/31/33/58 increased specificity significantly with virtually no loss of sensitivity for detecting CIN2+ and CIN3+lesions,as well as significantly reduced colposcopy referral rate（23.15%vs 33.70%,P<0.01）.In addition,in the 2-year follow-up period,women with infection of HPV16,18,31,33 or 58 genotypes were the most likely population（92%,23/25）to develop CIN2 lesions.Conclusion:Our results demonstrate that genotyping for a combination of HPV16/18/31/33/58 provides a more efficient and cost-effective model to risk-stratify women with ASCUS in the Chinese rural population.Part 3 Associations of RBC and serum folate concentrations with cervical intraepithelial neoplasia and high-risk HPV infectionObjective:To evaluate the associations of RBC and serum folate concentrations with prevalence of CIN grades and HR-HPV infection,their interactions with prevalence of CIN grades,and RBC folate with the risk of CIN1 progressing to CIN2.Methods:Using data from the Shanxi CIN cohort of 2304 female Chinese adults,we used logistic-regression model to estimate ORs and prevalence ratios（PRs）of RBC and serum folate concentrations with prevalence of CIN grades and HR-HPV infection.Categoric and spline analyses were used to evaluate the dose-response relations.We estimated the association of RBC folate with risk of CIN1 progressing to CIN2 in the nested case-control cohort.Results:An inverse association was observed between increased RBC folate concentration and the odds of all CIN grades（Q1 versus Q4:OR:2.28;95%CI:1.77,2.93,P-trend<0.001）.Significant interaction of RBC folate and HR-HPV infection was observed for prevalence of CIN2 or above（P-interaction<0.01）.No associations of RBC and serum folate with PRs of HR-HPV in each CIN grade.Over a median follow-up of 21.0 months,RBC folate was associated with increased risk of CIN1 progressing to CIN2.（Q1 versus Q4:OR=3.86;95%CI:1.01-14.76）.Conclusion:Our study indicates that RBC folate is associated with prevalence of CIN grades and CIN1 progression in female Chinese adults.Maintenance of normal folate status is important for reducing the risk of CIN and its progression in women with or without HR-HPV infection.Part 4 Comprehensive analysis of circRNA expression profiles during cervical carcinogenesisObjective:To explore some potential biomarkers of CIN progression,we comprehensively analyzed the differentially expression of circRNAs during cervical carcinogenesis and preliminarily explored their effects on the biological behavior of cervical cancer cells.Methods:The differential expression profiles of circRNAs and mRNAs in the process of cervical carcinogenesis were screened by Affymetrix gene chip technology.Differentially expressions of circRNAs were analyzed and selected by bioinformatics analysis,including GO,KEGG,STEM and STRING,TargetScan,and miRanda databases.qRT-PCR was used to further verify the analysis results.The expression of Hsa_circ₀003954 was knocked down by siRNA,and its effect on the cell proliferation and cell cycle of cervical cancer cells was detected through the CCK-8 assay and flow cytometer.Results:Short time-series expression miner（STEM）was utilized to characterize the timecourse expression patterns of circRNAs and mRNAs from NC to HSIL and CSCC.We observed a significant enrichment of cancer-related signaling pathways in the DEMs over time,such as DNA replication,cell cycle,and DNA repair-related GO functions,based on the DEMs obtained by STEM.CDK1,KIF11,CCNB2,PCNA and CCNB1were found to be hub genes by a PPI network.qRT-PCR analysis of CSCC,HSIL and NC samples was performed to validate the top 5 circRNAs.The expression patterns of hsa_circ₀001955 and hsa_circ₀003954 were both continuously upregulated in NC to HSIL and CSCC by qRT-PCR（P<0.01）.Subsequently,a competing endogenous RNA（ceRNA）network analysis and real-time PCR validation indicated that hsa_circ₀001955/hsa-miR-6719-3p/CDK1,hsa_circ₀001955/hsamiR-1277-5p/NEDD4L and hsa_circ₀003954/hsa-miR-15a-3p/SYCP2 were highly correlated with cervical carcinogenesis.Silencing of hsa_circ₀003954 inhibited SiHa cell proliferation and perturb the cell cycle in vitro.Conclusion:This study established the expression profile of circRNAs in the process of cervical carcinogenesis,and found that hsa_circ₀001955 and hsa_circ₀003954 were continuously up-regulated,which are expected to become new biomarkers in the process of cervical carcinogenesis,and provide theoretical support for further research on the molecules of circRNAs in the progression of CIN.