The Association Between The Methylation Level Of OPG/RANKL/RANK Gene And Alcohol-induced Osteonecrosis Of The Femoral Head

Purpose: Alcohol-induced osteonecrosis of the femoral head(Alcohol-induced ONFH)is a complex heterogeneous disease.The early stage is mostly hip pain,which can be relieved after rest.In the late stage,joint function is limited.In severe cases,it can lead to joint stiffness and inability to walk.Femoral head necrosis is caused by a variety of causes of poor blood flow to the femoral head,which causes further ischemia,necrosis,bone trabecular fracture,and collapse of the femoral head.The RANK/RANKL/OPG system is one of the most important regulators of bone resorption and remodeling,so it is an obvious candidate for studying epigenetic modifications related to bone pathology.RANK and its ligand RANKL located on the surface of osteoclasts and their precursors are necessary for the formation,differentiation,activity and survival of osteoclasts.RANKL is produced by osteoblasts and other types of cells in a soluble and membrane-bound form.On the contrary,OPG secreted by osteoblasts and some other types of cells acts as a decoy receptor by binding to RANKL,thereby preventing the activation of RANK.In clinical work,we found that only part of patients suffered femoral head necrosis when exposed to hormones and alcohol risk factors under the same conditions.It shows that hormone-induced and alcohol-induced osteonecrosis of the femoral head is a complex disease caused by the combined action of genetic and environmental factors,and has genetic susceptibility.Genetic factors and epigenetic modifications are one of the pathogenesis of the disease.However,the influence of epigenetic factors on the disease has not been systematically studied.The purpose of our research is to determine the methylation changes of ONFH induced by alcohol.Methods: In this experiment,we conducted a cross-sectional analysis of the blood of a Chinese male population(50 patients with alcoholic femoral head necrosis and 50 control groups).The Gold Mag-Mini Whole Blood Genomic DNA Purification Kit was used to extract DNA from the subject’s peripheral blood.Use the online tool epidesigner(http://www.epidesigner.com)to design PCR primers for OPG/RANKL/RANK amplicon sequences,and use Sequenom Mass ARRAY mass spectrometry methylation sequencing technology to detect 132 promoter regions of OPG/RANKL/RANK genes Cytosine-phosphate-guanine(CpG)DNA methylation status.Case-control research methods are used,and statistical methods such as chi-square test and linear regression analysis are used for data analysis.Results: Throughout the study,the chi-square test was used to analyze the methylation rate between the two groups,and it was found that 6 CpG sites were different,of which OPG1＿CpG＿2,OPG3＿CpG＿4,RANK1＿CpG＿6,RANK3＿CpG＿10,RANKL2＿CpG＿21,RANKL2＿CpG＿46 was higher in the group than in the control group,And OPG4＿CpG＿2 is lower than the control group.The results showed that in alcohol-induced ONFH patients,we detected the difference between the methylation level of 146 CpG sites and the healthy control group,of which the methylation level of 32 sites was not detected,and the remaining 114 sites There are 23 points of methylation levels that are different from alcohol-induced ONFH patients.We further used logistic regression to analyze the correlation between methylation status and alcohol-induced femoral head necrosis.Among them,OPG3＿CpG＿5(OR=0.36,95% CI: 0.14-0.91,p=0.031),RANK1＿CpG＿24.25.26.27(OR = 0.13,95% CI: 0.03-0.62,p = 0.011),alcoholic ONFH showed reduced risk,The methylation of the other 20 CpG sites increases the risk of alcoholic ONFH.The receiver operating characteristic(ROC)curve analysis shows that the methylation level of OPG/RANKL/RANK can effectively predict the presence of alcohol-induced ONFH.Conclusion: Our research on Chinese men suggests that several CpG sites of OPG/RANKL/RANK genes in peripheral blood cells of patients with alcohol-induced ONFH are in abnormal methylation status(high methylation tendency is more frequent).