| ObjectivesHigh-risk human papillomavirus(hrHPV)infection is an important pathogenic factor of cervical lesions.Recent studies have shown that telomere length and the distribution of related gene polymorphisms are significantly correlated with the incidence of inflammation and tumor.In this study,by detecting the variation of the relative telomere length(RTL)of the cervical epithelial cells of women infected with hrhPV and the mutation of the related gene single nucleotide polymorphism(SNP)sites,we explored the susceptibility factors of female hrhPV,and provided more theoretical basis for the pathogenesis and clinical diagnosis of cervical lesions..Methods1.In this study,a cross-sectional study design was adopted.Women who underwent cervical cancer screening at the Second Hospital of Shandong University between June 2019 and August 2020 were included in the study,and women with reproductive system infectious diseases such as condyloma acuminatum,cervical intraepithelial neoplasia,cervical cancer and other systemic tumors were excluded.Exfoliated cervix cells were collected and stored in SurePathTM cell preservation solution,while thin-layer liquid-based cytology and hrHPV DNA testing were performed.2.We extracted DNA and HPV DNA from exhumed cervical cells and performed HPV typing detection by using Roche Cobas4800 detection system.3.In our experiment,quantitative real time polymerase chain reaction(qRT-PCR)was used to determine the RTL of cervical epithelial cells.4.We used nucleic acid microarray technology to detect the distribution of 19 SNP sites of telomere related genes in cervical epithelial cells,including TERT(rs2736108,rs10069690,rs2736098,rs2736100,rs2853669,rs2853676,rs2853677,rs7726159,rs2242652,rs13167280),OBFC7(rs12415148),TERT-CLPTM1(rs401681),TERC(rs2293607,rs75316749,rs 12696304),TP53(rs 1042522),POT1(rs 116895242),TNF-α(rs1800629),MYNN(rs 10936599).5.We analyzed the differences of RTL in cervical epithelial cells between different groups by Student T-test.We used Pearson’s test to analyze the correlation between RTL of cervical epithelial cells and age and hrHPV infection.The evaluation of distribution differences in alleles of 19 SNP sites between different groups were done using χ2 test.Unconditional logistic regression analysis was used to investigate the association between telomere related gene polymorphisms and hRHPV infection.At the same time,we also analyzed the relationship between telomere related gene polymorphisms and the RTL of cervical epithelial cells through variance analysis.Results1.We divided the subjects into three groups according to the results of high risk HPV DNA testing:control group,hrHPV infection group,and HPV16/18 infection group.In the control group,508 patients,aged 19-83 years(40±14),had negative HPV DNA test.In the hrHPV infection group,826 patients with 14 high-risk types of HPV DNA were positive(including HPV16/18),aged 17-83 years(38±11).In the HPV16/18 infection group,only 305 patients(37±11 years old)were positive for HPV16/18.In the hrHPV infection group and HPV 16/18 infection group,the age of women was mainly 26-35 years,followed by 36-45 years,and>55 years was the lowest.2.The concentration of DNA extracted from cervical epithelial cells by Roche cobas 4800 detection system was above 100ng/ul,and OD260/280 was between 1.5-1.6,which could be used for subsequent experiments.3.In the control group,the average RTL of cervical epithelial cells was significantly negatively correlated with age(R2=0.09,P<0.0001),and the RTL of women aged 15-25,26-35,36-45,46-55 and>55 was gradually shortened.RTL in hrHPV(including HPV16/18)infected women aged 15-25,>55,26-35,36-45 and 46-55 decreased in turn.There was no significant correlation between RTL and age in hrHPV infection group(P=0.127).Compared with the control group,the RTL of hrHPV(including HPV16/18)infected women aged 15-25 years was significantly shorter,and the RTL of>aged 55 years was longer.4.The relationship between cervical epithelial RTL and hrHPV infection:the mean and standard deviation of RTL were 1.31±0.60 in the control group,1.19±0.48 in the hrHPV infection group,and 1.16±0.47 in the HPV16/18 infection group.The RTL of hrHPV infection group and HPV16/18 infection group was significantly shorter than that of control group(P<0.001).Although the RTL of the HPV16/18 infection group was shorter than that of the hrHPV infection group,it was not statistically significant(P=0.523).5.Associations between mutations in TERT rs2736108 and hrHPV susceptibility:allele T and genotype TT frequencies at rs2736108 were significantly increased in the hrHPV infection group,which was associated with the risk of hrHPV infection(TT vs.CC:OR=1.547,95%CI=1.021-2.343,P=0.039;T vs.C:OR=1.255,P5%CI=1.036-1.519,P=0.02).Differences in the distribution of allele T and genotype TT at this locus were both associated with susceptibility to HPV16/18 infection(TT vs.CC:OR=1.803,95%CI=1.079-2.963,P=0.019;T vs.C:OR=1.323,95%Ci=1.041-1.682,P=0.022).6.Correlation between OBFC1 rs12415148 mutation and hrHPV susceptibility:The allele C of rs12415148 increased in hrHPV infection group and was associated with hrHPV susceptibility(T vs.C:OR=0.695,95%Ci=0.490-0.985,P=0.040).In the HPV16/18 infection group,the difference in the distribution of this locus was not associated with the risk of HPV16/18 infection(T vs.C:OR=0.668,95%Ci=0.438--1.020,p=0.060).7.The frequency of TERT rs13167280 genotype AG was increased in the hrHPV infection group,and the genotype AG was associated with the risk of hrHPV infection(AG vs AA:OR=1.742,95%CI=1.012-2.998,P=0.044).The distribution of genotypes and alleles in the HPV16/18 infection group was not significantly associated with the risk of HPV16/18 infection(P>0.05).8.The differences in genotype and allele frequency of the remaining 16 loci were not significantly correlated with the susceptibility of hrHPV and HPV16/18(P>0.05).9.The RTL of different genotypes at rs2736108:There was no significant difference in RTL of CC,CT and TT genotypes in the control group(P>0.05),while the RTL of CC was shorter than that of CT in the hrHPV infected group(P=0.038),but there was no statistically significant difference in RTL of TT between the other two genotypes(P>0.05).Otherwise there was no significant difference in RTL of CC,CT and TT genotypes in the HPV16/18 infection group(P>0.05).10.The relative telomere length of different genotypes at rs13167280:RTL of AG in the control group was significantly longer than that of AA and GG(P=0.039 and 0.05),and no statistically significant difference was found between AA and GG(P>0.05).The RTL of AG in hrHPV infection group was longer than that of AA and GG,and it was statistically significant(P 0.004,0.01,respectively),but there was no statistically significant difference between AA and GG(P>0.05).In the HPV16/18 infection group,the RTL of AG was significantly longer than that of GG(P=0.028),but there was no statistically significant difference among other genotypes(P>0 0.05).11.Because the CC genotype frequency of OBFC1 rs12415148 is too low,we cannot analyze the RLT of different genotypes of this locus,which requires us to further expand the sample size to analyze the relationship between the variation of this locus and telomere length.Conclusions1.In this study,it was found that the age of women infected with hrHPV in Jinan,Shandong Provinc was mainly 26-35 years old.The telomere length of cervical epithelial cells in women without hrHPV infection gradually shortened with the increase of age,indicating that the telomere length of cervical epithelial cells was significantly correlated with age.However,the telomere length of the cervical epithelial cells of women infected with hrHPV did not change with age,suggesting that hrHPV could affect telomere homeostasis.The RTL of cervical epithelial cells infected with hrHPV,especially in women infected with HPV16/18,was significantly shorter than that in women not infected with hrHPV,indicating that telomere homeostasis had been impaired in patients infected with hrHPV,and the RTL of cervical epithelial cells is expected to be a marker for early diagnosis of cervical lesions.2.Telomere related gene mutation has been proved to be the risk factor of many kinds of tumor,our study is the first to investigate telomere related gene polymorphism and the risk of hrHPV infections in the relationship.We found that the TERT rs2736108 TT genotype and allele T,TERT rs13167280 AG genotype,OBFC1 rs12415148 allele C were associated with a significant risk hrHPV infections,suggesting they are hrHPV susceptible factors;In addition,the TT genotype and allele T of TERT rs2736108 were associated with HPV16/18 susceptibility,but allele C of TERT rs13167280 and AG genotype OBFC1 rs12415148 were not significantly associated with HPV16/18 susceptibility,and they may be susceptibility factors of other types of hrHPV.The RTL of TERT rs13167280 AG genotype was significantly longer,which may be due to the mutation of this locus promoting telomere elongation,or the bias caused by sampling error.In women infected with hrHPV,the relative telomere length of TERT rs2736108 CT genotype was longer than that of CC genotype,while no significant differences were found in RTL of all genotypes at this site in women not infected with hrHPV and in women infected with HPV16/18,which may be due to differences caused by other types of hrHPV infection or due to sampling error.This study provides a new theoretical basis for the early diagnosis of hrHPV infection and the determination of whether further clinical treatment is needed,and has important clinical application value. |
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