Study On The DNA Methylation In Spleen-stomach Damp Heat Syndrome And Liver Depression And Spleen Deficiency Syndrome In Chronic Hepatitis B

Chronic hepatitis B(CHB)is a major public health problem worldwide.CHB has shown its unique advantages in preventing and curing CHB with traditional Chinese medicine(TCM).To further improve the prevention and treatment capacity of TCM,in-depth elaboration of the biological basis of CHB syndrome seems to be the main link.The common TCM syndromes of CHB in Sichuan are spleen-stomach damp heat syndrome(SSDHS)and liver depression and spleen deficiency syndrome(LDSDS).The research group initially studied the biological basis of these two syndromes from mi RNA.Some literatures have shown that DNA methylation is involved in the occurrence and development of CHB,and DNA methylation seems to coincide with the formation of TCM syndromes,which may provide a new idea for the study of the biological basis of CHB SSDHS and LDSDS.Objective: This study based on the previous related mi RNA,multi-dimensional comprehensive observation of SSDHS and LDSDS the differences in the levels of DNA methylation,explore the syndrome is the biological basis of the above two kinds of CHB through DNA methylation and mi RNA regulation,affect epigenetic,thus regulate gene expression,form different syndrome,in order to preliminary confirmed whether DNA methylation differentially expressed for CHB syndrome difference is one of the major epigenetic mechanisms,for the above two kinds of CHB objectification and standardization of syndromes to provide some empirical basis.Methods:1.7 patients with CHB Spleen-stomach damp heat syndrome(SSDHS)(SSDHS group),7 patients with Liver depression and spleen deficiency syndrome(LDSDS)(LDSDS group)and healthy volunteer 6 cases(Health control(HC)).2.Collect all subjects' peripheral venous blood in the fasting and sterile environment in the morning,and use conventional detection methods to detect liver function-related indicators,two-half qualities and HBV-DNA content between groups.3.Use ELISA method to detect DNA methylation between groups expression levels of DNMT1,Me CP2 and HBV related indexes P53,E-cad,P16.4.The serum of peripheral venous blood was separated in time,DNA was extracted,and the DNA between groups was detected with Illumina Bead Array TM Infinium 850 K methylation chip differential expression of methylation,and GO enrichment and KEGG analysis of genes involved in differentially methylation sites.5.Use Target Scan7.2 and miRDB database to predict the target genes of the same mi RNAs in different syndromes,using Cytoscape3.6 construct a network map of DNA methylation genes and mi RNAs prediction target gene co-expression.Results:1.Compared with the HC group,the SSDHS and LDSDS groups showed no significant differences in the expression levels of DNMT1,p16,gender,age,disease course,biochemical indicators,two-half qualitative and HBV-DNA test results(P>0.05);E-cad expression levels were significantly different between the two groups(P<0.05),Me CP2 and P53 expression levels were not significantly different in the SSDHS group(P>0.05),there were significant differences in the LDSDS group(P <0.05).2.Illumina Bead Array TM Infinium 850 K methylation chip test results showed that there were 7868 significantly different methylation sites in CHB group and HC group,including 2310 high methylation sites and 5558 low methylation sites;After screening,the highest degree of high methylation difference was cg03278611 located in the gene NLGN4 Y,and the site with the lowest degree of low methylation difference was cg03670113 located in the gene TAZ;significantly different methylation sites related gene GO enrichment in protein deubiquitination,I-kappa B kinase/NF-kappa B signaling,fat particles,various enzymes,etc.;KEGG pathway mainly involved in steroid biosynthesis pathway,PPAR signaling pathway,etc.3.SSDHS group is characterized by the highest degree of hypermethylation difference is cg21898358 located in gene LILRA3,the highest degree of low methylation difference is cg09323788 located in gene CHTF18,differentially methylated sites related genes GO functions are mainly enriched in T cell co-stimulation,Rho protein signal transduction,Rho GTPases-related functions,etc.;KEGG pathway is mainly involved in ABC transporter,AMPK signaling pathway and tumor-related pathways;LDSDS group is characterized by the highest degree of hypermethylation difference is cg09857096 in gene BMPR1 B,and the highest degree of hypomethylation difference is cg09972436 in gene LCE3 C.The differentially methylated sites related genes GO functions are mainly enriched in dendritic spines,serine / threonine kinase,Rac GTPase,etc.KEGG pathway mainly involves metabolic pathways in phenylalanine metabolism,nitrogen metabolism,alanine,aspartic acid and glutamic acid metabolism;The most significant hypermethylation site between SSDHS group and LDSDS group is cg10765459 in gene COL6A2,the most significant hypomethylation site is cg07445365 in gene STK32 C.The differentially methylated sites related genes GO functions are mainly enriched in cells and tissues,KEGG pathway relates Erb B signaling pathway and other metabolic pathways,etc.4.The differentially methylated sites related genes in the SSDHS and LDSDS groups were compared with the predicted target genes of mi R-122-5p,mi R-1228-3p,and mi R-191-3p: the two groups of differentially methylated sites related genes and mi R-122-5p the target genes are predicted to have 6 genes that are coincident,of which the unique gene in the SSDHS group is RIMS1,and the unique gene in the LDSDS group is SLC41A1;the differential methylation sites related gene of the SSDHS group and the target gene of mi R-1228-3p are predicted to be 21 genes,the unique genes of this group are OTUB,AMPH,SEMA3 C,ZC3H12B,PCGF3,JAKMIP3.LDSDS group differentially methylated sites related genes and mi R-1228-3p predicted target genes have 17 genes coincide,the unique genes in this group are ARID1 B,NUDT7;SSDHS group differentially methylated sites related genes and mi R-191-3p predicted target genes have 2 genes are coincident,the unique gene in this group is MPP7.The differentially methylated sites related genes in the LDSDS group and mi R-191-3p predict that there are 3 genes that coincide with the target gene,the unique genes in this group are SPTB2 and CCNY.Conclusion:1.Me CP2 and P53 may participate in the formation of CHB LDSDS by affecting DNA methylation modification.E-cad may be one of the physical basis of CHB SSDHS and LDSDS.2.CHB has differential expression of DNA methylation,and its pathogenesis may be related to the methylation of NLGN4 Y,TAZ and other genes and protein deubiquitination,I-kappa B kinase/NF-kappa B signaling,steroid biosynthesis pathway,PPAR signaling pathway related.3.There are differential expressions of DNA methylation in the CHB SSDHS and LDSDS,and each has its own unique DNA methylation profile.LILRA3,CHTF18 and other methylation genes and T-cell co-stimulation-induced liver inflammation may be the molecular biological basis of the CHB SSDHS;the differential genes of methylated genes such as BMPR1 B and LCE3 C and dendritic spines changes and abnormal substance metabolism may be the molecular biological basis of CHB LDSDS.Abnormal substance metabolism may be related to liver gas deficiency.It suggests that DNA methylation may be one of the epigenetic mechanisms for the difference between CHB SSDHS and LDSDS.4.The occurrence of some identical mi RNAs in different syndromes may be related to the differences in methylation levels of different target genes,and the specific regulatory mechanism still needs to be further confirmed.