Relationship Between FTO And HPV16/18 In High-grade Squamous Intraepithelial Lesion And Risk Prediction

Objective:Cervical cancer ranks fourth among female malignant tumors.The occurrence of cervical cancer is the result of the interaction of virus,environment and body.The main cause is long-term and persistent HR-HPV infection,among which HPV16 and18 are the most common carcinogenic types.SIL is a precancerous lesion of cervical cancer,which can be divided into ASCUS,LSIL and HSIL according to cytology.Among them,LSIL has a high natural regression rate,and clinical follow-up observation is often given priority to,while a large proportion of HSIL progressed to cervical cancer.It can be seen that finding the factors affecting the progression from low-grade to high-grade lesions can provide an opportunity for early prevention of cervical cancer.m6A modification is the most common form of post-transcriptional modification of eukaryotic m RNA.FTO is the first m6A demethylation regulator to show demethylase activity on m RNA,which can promote the occurrence and development of many kinds of malignant tumors,but its expression in cervical cancer has not been consistent.On the basis of obtaining the related factors and HPV infection,we detected the expression level of FTO in different levels of SIL.The relationship between FTO,HPV16/18 infection and HSIL was discussed by path analysis,and the predictive value of related factors to HSIL was evaluated by nomogram,ROC and DCA,which provides a theoretical basis for revealing the research mechanism of FTO regulating HSIL.Methods:From June 2021 to June 2022,64 cases of ASCUS,74 cases of LSIL and 66cases of HSIL patients diagnosed by TCT in the Gynecology Clinic of Jiexiu Maternal and Child Health Hospital in Shanxi Province were selected as the study subjects.General demographic factors and related factors of SIL were collected,and cervical exfoliated cells were collected.HPV infection was detected by diversion hybridization and FTO m RNA expression was detected by Rt-q PCR.SPSS 25.0,Amos 24.0,R 4.2.2 and Graphpad prism 8.0 packages were used for statistical analysis and graphing respectively.Quantitative data were used for Wilcoxon rank sum test and Kruskal-Wallis H test,and qualitative data were used forX² test and trendX² test.Univariate and multivariate analyzes were performed with multivariate Logistic regression analysis,and the correlation strength index between the research variables and SIL used the OR value and 95%CI.Amos 24.0 was used to conduct pathway analysis on the relationship between FTO,HPV16/18 infection and HSIL.The fitting effect evaluation index was goodness of fit index GFI,GFI for adjusting degrees of freedom was AGFI,root mean square residual RMR,comparative fitting index CFI,normal fit index NFI,value-added fit index IFI.Nomogram,ROC and DCA were used to evaluate the predictive value of related factors on HSIL.The inspection levelα=0.05.Results:1.Single factor analysis of demographic characteristics and related factors of cervical lesions:The average age of 204 patients with ASCUS was 44.50±13.00 years old,LSIL was 44.78±11.28 years old,and HSIL was 49.68±11.69 years old.There were significant differences in age（X²=8.929,p=0.012）.There were no significant differences in marital status,education level and monthly family income（p>0.05）.Passive smoking（X²=6.363,p=0.042）,alcohol consumption（X²=12.872,p=0.001）,frequency of vaginal washing（X²=21.239,p<0.001）,menstrual cycle（X²=6.902,p=0.032）,menstrual period（X²=6.244,p=0.044）,menopause（X²=7.904,p=0.019）,postmenopausal bleeding（X²=6.503,p=0.039）,age of first sexual life（X²=7.008,p=0.030）and number of sexual partners（X²=6.565,p=0.038）were significantly different among the different pathological groups.Combining ASCUS and LSIL as the low-grade and below lesion group（≤LSIL）,the results of univariate analysis showed that age（X²=8.317,p=0.004）,passive smoking（X²=4.796,p=0.029）,alcohol consumption（X²=14.671,p<0.001）,frequency of vaginal washing（X²=20.807,p<0.001）,menstrual period（X²=6.220,p=0.013）,menopause（X²=7.881,p=0.005）,postmenopausal bleeding（X²=6.485,p=0.011）,age at first sex（X²=6.573,p=0.010）,and number of sexual partners（X²=4.530,p=0.033）were significantly different between≤LSIL and HSIL.2.Univariate analysis of the relationship between HPV infection and cervical lesions:HPV infection rate（X²=17.445,p<0.001）,HR-HPV infection rate（X²=15.724,p<0.001）and HPV16/18 infection rate（X²=11.469,p=0.003）were significantly different among multiple groups.HPV infection rate,HR-HPV infection rate and HPV16/18 infection rate increased with the severity of cervical lesions（p<0.05）.The relationship between HPV16/18 infection and HSIL showed that HPV infection rate（X²=8.452,p=0.004）,HR-HPV infection rate（X²=8.405,p=0.004）and HPV16/18infection rate（X²=11.419,p=0.001）showed statistically significant difference between≤LSIL and HSIL groups.3.Univariate analysis of the relationship between FTO and cervical lesions:There was a statistically significant difference in the expression of FTO m RNA among the three groups（H=35.171,p<0.001）.Further comparison between the groups showed that there was a statistically significant difference in the expression of m RNA between the ASCUS and LSIL groups and the HSIL group（p<0.001）.Using the median of FTO m RNA expression in ASCUS group(2^-△△Ct=8.14)as the threshold,FTO was divided into high expression(2^-△△Ct>8.14)and low expression(2^-△△Ct≤8.14)levels.It was found that the level of FTO m RNA expression showed an upward trend with the progress of SIL(X_trend²=31.462,p<0.001).The relationship between FTO and HSIL showed that there was a statistically significant difference in the expression of FTO m RNA between≤LSIL and HSIL groups（Z=5.732,p<0.001）,and the expression level of FTO（X²=31.705,p<0.001）was significantly different between≤LSIL and HSIL groups.4.Multivariate Logistic regression analysis of HSIL related factors:Factors that showed statistical differences in single factor analysis were included in multivariate unconditional logistic regression for analysis,and≤LSIL was used as the control group for analysis using stepwise regression method.The results showed that high expression of FTO（OR=1.023,95%CI:1.010-1.036）,menopause（OR=2.734,95%CI:1.335-5.601）,age of first sexual intercourse<20 years old（OR=3.361,95%CI:1.143-9.882）,HPV16/18 infection（OR=2.326,95%CI:1.156-4.678）,and frequency of vaginal washing<3 days/week（OR=4.334,95%CI:1.600-11.742）were risk factors for HSIL,and menstrual period of 3-7 days（OR=0.330,95%CI:0.138-0.786）were protective factors for HSIL.5.Pathway analysis of FTO expression and HPV16/18 infection in HSIL:Through pathway analysis,it was known that FTO has a direct effect on HSIL（path coefficient=0.37）,and it may also have an indirect effect on HSIL through HPV16/18infection（path coefficient=0.03）,with the direct effect being more significant.The fitting effect of the model was evaluated,and the results showed that the fitting indicators GFI,CFI,NFI,and IFI were all greater than 0.900,with an RMR of 0.003,indicating a good fit of the model.6.Analysis of the predictive value of relevant factors on the risk of HSIL occurrence:Nomogram results showed that high level of FTO,menopause,age of first sexual life<20 years old,menstrual period<3 days or>7 days,HPV16/18infection,frequency of genital cleaning<3 days/week,the risk of HSIL is higher.The internal validation results of Bootstrap 1000 repeated sampling showed that the predicted value was basically consistent with the measured value,suggesting that the nomogram prediction model was accurate.Taking FTO in the nomogram,menopause,age of first sexual life,menstrual period,HPV16/18 infection,and genital cleaning frequency as predictors,the ROC curve was drawn,and the area under the ROC curve（Area under curve,AUC）was calculated to be 0.804,suggesting that the constructed nomogram has good predictive ability to predict the risk of HSIL.The potential value of the nomogram is further evaluated using DCA.The DCA results showed that the DCA curve was on the right side of the gray slanted line（All）,indicating that the obtained prediction model had more net benefit than the"complete intervention"and"no intervention"strategies.When the high risk threshold is about 0.1-0.9,it has certain clinical significance When the net benefit is greater than 0.If the personal threshold probability is>0.1（that is,if the patient’s HSIL risk is greater than 0.1,we will choose to intervene in HSIL）,and the high-risk threshold is between 0.1-0.9,the higher the value smaller,the higher the net benefit.Intervention（such as enhanced follow-up or treatment）in≤LSIL population with related risk factors is helpful to prevent more patients from progressing to HSIL.Conclusion:1.Among the influencing factors related to HSIL,high expression of FTO,menopause,age of first sexual life<20 years old,HPV16/18 infection,and vaginal washing frequency<3 days/week are risk factors for HSIL,and menstrual period of 3-7 days is protective factor for HSIL.It suggests that developing good hygiene habits,not starting sexual life early,and regular HPV screening may reduce the risk of HSIL.When evaluating the risk of HSIL,multiple influencing factors should be considered comprehensively,and high-risk groups should be found as early as possible,to achieve early detection and early treatment of HSIL.2.FTO can have a direct effect on HSIL,and may also have an indirect effect on HSIL through HPV16/18 infection,suggesting that HPV16/18 infection may be affected by the level of FTO,and the etiology of HSIL can be analyzed from two aspects:direct effect and indirect effect.3.The nomogram model predicts the risk of HSIL,and the model is well calibrated.DCA is used to evaluate the predictive value of the nomogram.The results show that the predictive nomogram model can achieve higher than the"complete intervention"and"no intervention"strategies,indicating that intervention（such as enhanced follow-up or treatment）in≤LSIL population with related risk factors is helpful to prevent more patients from progressing to HSIL.This prediction model has a good prediction effect.In the future,the predictive model can be used to further explore the influencing factors of HSIL,and provide ideas for whether to implement clinical intervention.