Association Of Maternal Expressed Gene 3 （MEG3） Methylation Levels With Ankylosing Spondylitis

Objective:Ankylosing spondylitis（AS）is a chronic immune-mediated inflammatory disease characterized by chronic inflammation and new bone growth,often accompanied by joint osteophyte formation and joint stiffness,resulting in limited mobility of the patient.At present,the pathogenesis of AS remains unclear.Maternally expressed gene 3（MEG3）is both a maternal lnc RNA and a tumor suppressor gene located on chromosome 14q32.A variety of studies have shown that abnormally expressed MEG3 is not only related to cancer,but also involved in the occurrence and development of autoimmune diseases.However,the mechanism leading to the change of MEG3 gene expression level remains unclear.Therefore,this study aims to compare MEG3 gene methylation level and expression level between AS patients and healthy controls from the perspective of epigenetics,and analyze the relationship between MEG3 gene and genetic susceptibility to AS,so AS to provide clues for finding new therapeutic targets for AS.Methods:In this study,10ml blood samples and epidemiological data were collected from patients admitted to the Department of Rheumatology and Immunology of the First Affiliated Hospital of Anhui Medical University and the Second Affiliated Hospital of Anhui Medical University from June to September 2021,as well as 10ml blood samples from the control group in the Physical Examination Department of the First Affiliated Hospital and the Second Affiliated Hospital of Anhui Medical University.A total of 83 cases in the case group and 83 cases in the control group were included.General demographic data,epidemiological data and clinical characteristics data were collected by using the AS epidemiological questionnaire,and the differences of MEG3gene methylation levels were analyzed and the expression levels were compared.Methylation detection:DNA kit was used to extract DNA from the collected blood samples,and methylation level was detected by Methyl Target method.Firstly,peripheral blood samples from 77 AS patients and 65 healthy controls were selected for PBMC extraction.Quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）was used for detection.Kolmogorov-smirnov was used to test the normality of the data.The quantitative data conforming to the normal distribution were represented by mean±standard deviation,and the quantitative data conforming to the skewness distribution were represented by median and Interquartile range（IQR）.T test and Mann-Whitney U test were used to compare the mean values of the quantitative data of normal distribution and skewness distribution respectively.The Kruskal Wallis Test was used to find relationships between categorical variables.Spearman rank correlation coefficient test was used to explore the correlation between MEG3 gene and clinical features.Receiver operating characteristic curve（ROC）and area under curve（AUC）were used to evaluate the predictive ability or feasibility of MEG3 gene AS a biomarker of AS.All statistical analyses were performed using SPSS 25.0 software（SPSS Inc.,Chicago,IL,USA）and graphing was performed using Graph Pad Prism version 9.03（Graph Pad Software,La Jolla,CA,USA）.A P double-tailed value≤0.05 is considered significant.Results:The study was divided into two parts:（1）a total of 83 AS patients（63 males and 20 females）and 83 healthy individuals（63 males and 20 females）were included in the methylation level detection phase.The ages of healthy controls and AS patients were33.00（28.00,41.00）and 33.00（29.00,42.00）years,respectively.There were no significant differences in age（Z=0.151,P=0.881）and gender（X~2=0.002,P=0.965）between the two groups.（2）A total of 77 AS patients and 65 healthy controls were included in the expression level detection phase.There were no significant differences in age（Z=-0.096,P=0.923）and gender（x~2=0.001,P=0.993）between the two groups.Among the 42 Cp G loci of the 3 Cp G islands of MEG3 gene,the methylation levels of35 loci were statistically different between AS patients and healthy controls,indicating that the METHYLation levels of MEG3 gene in AS patients were significantly changed.In addition,the methylation levels of the three Cp G regions of the MEG3 gene promoter also showed significant differences between AS patients and healthy controls,and the methylation levels of AS patients were all higher than those of healthy controls.Stratified analysis by gender showed that there was no statistical difference in the methylation level of MEG3 promoter between AS patients and healthy controls in both male and female stratified analysis.Grouping according to drug use,it was found that there was no statistical difference in MEG3 gene methylation level among AS patients in the non-medication group,the biologics group and the non-biologics group.According to the STATUS of HLA-B27,all subjects were divided into healthy control group,HLA-B27 positive group and HLA-B27 negative group,and it was found that there were significant differences in the methylation level of MEG3 gene among the three groups.Then,pairwise comparison was conducted among the three groups.It was found that the methylation level of MEG3 gene was only statistically different between the healthy control group and the HLA-B27 positive group.In addition,QRT-PCR results showed that the expression level of MEG3 gene in peripheral blood mononuclear cells of AS patients was significantly lower than that of healthy controls.Correlation analysis was conducted on the methylation level and m RNA expression level of MEG3gene and clinical characteristics of AS patients,and it was found that the methylation level of MEG3 promoter was positively correlated with BASDAI of AS patients,and significantly negatively correlated with the course of disease and C-reactive protein,but had no significant correlation with the course of disease,BASFI and ASDAS.There was no significant correlation between m RNA expression level and clinical features of AS patients.Conclusions:This study found that the methylation level of MEG3 gene promoter in AS patients was significantly higher than that in healthy controls,especially in HLA-B27 positive AS patients,and the expression level of MEG3 gene in peripheral blood mononuclear cells of AS patients was significantly lower than that in healthy controls,further verifying the results of DNA methylation.Hypermethylation of MEG3 gene promoter can participate in the occurrence and development of AS by reducing its expression level,and the methylation level and expression level of MEG3 gene are potential diagnostic markers of AS.