Characteristics And Function Prediction Of Vaginal Microbiota In Women With Cervical Cytology Abnormalities Under HPV16 Integration

Objective:Cervical cancer is one of the malignant tumors that endanger women’s health.HPV16 is the most important oncogenic type,and integration of viral DNA into the host genome is the key step in carcinogenesis.However,the factors affecting HPV16 integration are still unclear.With the development of high-throughput sequencing technology,human’s understanding of microorganisms has been further improved.The relationship between vaginal microbiota and HPV16 integration status has become a research hotspot.Based on our previous study,women with abnormal cervical cytology were selected as the high-risk group of cervical cancer,and metagenomic sequencing technology was used to fully reveal the characteristics and functions of vaginal microbiota under HPV16 integration.Random forest algorithm was used to construct a vaginal microbiota model to predict HPV16 infection and integration status,evaluate and screen potential biomarkers,and combine bioinformatics and machine learning methods to reveal the relationship between HPV16 integration status and vaginal microbiota in a deeper way.In order to provide a new theoretical basis for the prevention and control of HPV16 infection and its related cervical lesions from the perspective of vaginal microorganisms.Method:A total of 510 women with abnormal cervical cytology by Thinprep Cytologic Test(TCT),who came from the gynecological clinic of Second Hospital of Shanxi Medical University during January to June 2018,were enrolled in this study.The cervical exfoliated cells were detected by Flow-through Hybridization technology to determine human papillomavirus(HPV)infection typing.DNA loads of HPV16 E2 and E6 and m RNA expression levels of HPV16 E2,E6 and E7 were detected by RT-PCR.The protein expression levels of E2,E6 and E7 of HPV16 were detected by Western-blot.Those who used antibiotics within 2 weeks,had sex within 3 days,and had vaginal irrigation within 48 hours before the collection of vaginal secretions were further excluded.Finally,59 HPV16 positive women and 73 HPV16 negative women were selected for metagenome sequencing of vaginal microbiota.R Vegan software was used to analyze the diversity of vaginal microbiota,LEf Se software was used to analyze the characteristic bacteria,and Kyoto Encyclopedia of genes and genomes(KEGG)database was used to analyze the function of vaginal microbiota.The R Random Forest software package was used to construct the prediction model of vaginal microbiota for HPV16 infection and integration status,and the area under the receiver operating characteristic(ROC)curve(AUC)was used to evaluate the model.SPSS 26.0 and R4.1.0 software were used for t test and Mann-Whitney U test,and the test level wasα=0.05.R4.1.0 and Graph Pad 9.0 software were used to draw statistical maps.Result:1.Characteristics of vaginal microbiota in women with abnormal cervical cytology: The top five vaginal microbiota of women with abnormal cervical cy tology were Gardnerella＿vaginalis(40.91%),Lactobacillus＿crispatus(23.59%),Lactobacillus＿iners(15.11%)and Staphylococcus＿aureus(3.52%)and Streptomy ces＿sp.＿ICC4(1.50%).The functions of vaginal microbiota were mainly Protein families: genetic information processing,Carbohydrate metabolism,Translation,and Nucleotide metabolism.2.Characteristics and functions of vaginal microbiota under HPV16 infecti on: The diversity of vaginal microbiota was increased under HPV16 infection(P<0.05).Under HPV16 infection,the proportion of Lactobacillus＿iners and La ctobacillus＿crispatus decreased,and the proportion of Gardnerella＿vaginalis incr eased.The vaginal characteristics of HPV16 infection were Prevotella＿intermedi a,Streptococcus＿anginosus,Peptoniphilus＿harei and Mageeibacillus＿indolicus.A t the L2 level,the functions of vaginal microbiota in the state of HPV16 infec tion are Metabolism of cofactors and vitamins,Glycan biosynthesis and metabo lism,Aging,and Environmental adaptation and Cancer: overview predominated.At L3 level,Chaperones and folding catalysts,Transfer RNA biogenesis,Thiam ine metabolism,and Protein play important roles in the vaginal microbiota duri ng HPV16 infection kinases and Aminoacyl-t RNA biosynthesis were predomina nt.3.The relationship between m RNA and protein expression of HPV16 E2,E6 and E7 and vaginal microbiome: The diversity of vaginal microbiome was increased in groups with low E2 expression and high E6 and E7 expression.The proportion of Lactobacillus＿iners and Lactobacillus＿crispatus in E2 m RNA and protein high expression group was higher than that in low expression group,and the proportion of E6 and E7 m RNA and protein high expression group was lower than that in low expression group.4.Characteristics and functions of vaginal microbiota under HPV16 integration:According to the ratio of HPV16 E2/E6 DNA load,the subjects were divided into HPV16 episomal status and mixed/integrated status.The diversity of vaginal microbiota increased in HPV16 mixed/integrated status(P<0.05).In the episomal status,Lactobacillus＿iners and Lactobacillus＿crispatus were predominant.In the episomal status,Fructose and mannose metabolism and Glycerolipid metabolism were relatively high.Phenylalanine,tyrosine and tryptophan biosynthesis,DNA repair and recombination proteins,Porphyrin metabolism,Secretion system,Oxidative phosphorylation,Chaperones and folding catalysts and Thiamine metabolism accounted for low proportion.5.Evaluation of predictive value of vaginal microbiota for HPV16 infectio n and integration status: Random forest algorithm was used to construct a pred iction model of vaginal microbiota for HPV16 infection and integration status.The subjects were randomly divided into training set and test set at a ratio of7:3.The importance of microorganisms was ranked according to Mean Decrease Gini coefficient.The ROC curve was drawn to evaluate the prediction efficienc y.Peptoniphilus＿harei,Lactobacillus＿crispatus and Prevotella＿intermedia were t he main biomarkers for predicting HPV16 infection(AUC=0.667).Lactobacillus＿iners,Enterococcus＿faecium,Neisseria＿gonorrhoeae,Ureaplasma＿parvum and Prevotella＿intermedia were the main strains to predict HPV16 integration status Biomarkers(AUC=0.733).Conclusions:1.In women with HPV16 infection,low E2 expression and high E6 and E7 gene expression,the diversity of vaginal microbiota increased,Lactobacillus crispatus and other probiotics decreased,and pathogenic anerobacteria were the vaginal characteristic bacteria.It is suggested that HPV16 infection,low expression of E2 gene and high expression of E6 and E7 gene are closely related to the changes of vaginal microbiota.The function of vaginal microbiota in the state of HPV16 infection is mainly carcinogenic and cancer-promoting,which provides a theoretical basis for the study of carcinogenic mechanism of HPV16 infection.2.The diversity of vaginal microbiota is increased,Lactobacillus crispatus and Lactobacillus iners are decreased,and the function of vaginal microbiota is mainly carcinogenic and carcinogenic.Vaginal microbiota may be used as an indicator to evaluate the integration status of HPV16 virus and provide intervention ideas and theoretical basis for inhibiting the integration of the virus into the host genome.3.The vaginal microbiota model based on metagenomic sequencing technology combined with random forest algorithm to predict HPV16 integration status has good performance,and the selected biomarkers have a high degree of fit with the bioinformatics analysis results,suggesting that the combination of bioinformatics and machine learning methods should be strengthened in the future.To provide deeper and more accurate research techniques and methods for vaginal microbiome research.