Study On DNA Methylation, MiRNA And IncRNA Expression Profile Of Phlegm - Dampness

Experiment 1. Study on phlegm dampness constitution DNA methylation expression profileObjective:1) Detect the DNA methylation level of PBMC from balanced constitution and phlegm dampness constitution subjects, and construct DNA methylation expressionprofile test on the genome-wide level.2) Screen the specific DNA methylation loci of phlegm dampness constitution, explore the DNA methylation mechanism in the formation of phlegm dampness constitution and provide potential biomarkers for microscopic identification of phlegm dampnessconstitution.Method:1) Collect peripheral blood samples of 8 phlegm dampness disease-free volunteers and 4 balanced constitution healthy volunteers, extract DNA from PBMCs to conduct microarray detect with Illumina Human Methylation 450K BeadChip.2)Through the analysis of expression data to screen differentially expressed DNA methylation loci.3)Conduct function enrichment analysis of the genes annotated by the specific DNA methylation loci.Results:1) There’s no significant difference in the basis condition of gender, age, BMI, etc. between the phlegm dampness constitution and balanced constitution groups.2)A number of 288 specific DNA methylation loci were screened between the two different TCM constitution groups, includting 187 hypermethylation loci and 101 hypomethylation loci. These 288 loci can be annotated to 256 genes, with 169 hypermethylation genes and 87 hypomethylation genes.3) According to Δβ the greatest difference locus is cg21498547 （Δβ=0.495）, and it can be annotated to gene DLGAP2 which is associated to non-insulin-dependent diabetes indicated by Ingenuity Pathway Analysis （IPA）. ThroughManhattan plot we screened out differentially expressed loci （p<le-04）, according to Δβ sort the most obvious difference is CpG sites cg17796323 annotated to gene N4BP3. Amoung the total 288 different loci, there are two loci cg22151644 and cg 18473521 are annotated to gene HOXC4, published literatures indicate that HOXC4 is related to obesity and fat accumulation.4) IPA revealeshypermethylated genes such as B4GALNT1, ST6GALNAC5, DHCR24, MVK, STARD3, PRKCZ are closely related to lipid metabolism, additionally IPA indicates the molecular and cellular functionsof hypermethylated genes are mainly related to immune and inflammatory responses, which is consistent with previous studies.Conclusion:1) Phlegm dampness constitution and balanced constitution exhibit systematic differences in the DNA methy lation profilewith the genome-wide level.2) The two groups show no significant difference in gender, age, BMI, etc. and all the subjects are disease-free, in such situation phlegm dampness exhibit signifit expression difference in DNA methylation, which indicates phlegm dampness is an independent risk factor dirrerent from obesity.3) The phlegm dampness constitution subjects showsignificant DNA methylation expression difference even in the disease-free condition, and the genes annotated by the specific loci can be enriched to metabolic related pathways, indicates that phlegm dampness constitution has molecular mechanism basis for the predisposion of metabolic disorders.4) This study has constructed DNA methylaiton expression profile of phlegm dampness constitution, and screened differentially expressed DNA methylation loci, it laid the foundation for the further research of microscopic identification and molecular mechanisms of phlegm dampness constitution.Experiment 2. Study on phlegm dampness constitution miRNA expression profileObjective:1)Detect the miRNA expression level of PBMC from balanced constitution and phlegm dampness constitution subjects, and construct miRNA expressionprofiletest onthe genome-wide level.2) Screen the specific miRNA of phlegm dampness constitution, explore the miRNA regulation mechanismin the formation of phlegm dampness constitution and provide potential biomarkers for microscopic identification of phlegm dampness constitution.Method:1) Collect peripheral blood samples of3 balanced constitution healthy volunteers （group P）, 3 phlegm dampness disease-free volunteers （group T） and 3 phlegm dampness constitution metabolic syndromepatients （group M）.Extract total RNA from PBMCs to conduct microarray detect with Human miRNA OneArray（?）.2) Through the analysis of miRNA expression data to screen differentially expressed miRNAs.3)Conduct function enrichment analysis of the miRNA target genes to predict the biofunctions of different miRNAs.Results:1) A number of 73 specific miRNAs were screened between group T and group P, includting 32 upregulated different miRNAs and 41 downregulated different miRNAs. Group M and group T show 14 specific mirNAs includting 8upregulated different miRNAs and 6 downregulated different miRNAs.2) The most upregulated different miRNA is hsa-miR-1228-5p, and the most downregulated different miRNA is hsa-miR-1228-5p, they could work as the potential biomarkers to identify phlegm dampness constitution.3) Through biofunction analysis of the different miRNA target genes, multiple tumour related GO biological process and KEGG pathway terms are enriched.Conclusion:1) Phlegm dampness constitution and balanced constitution exhibit systematic differences in the miRNA profile with the genome-wide level.2) The phlegm dampness constitution subjects showsignificant miRNA expression difference even in the disease-free condition, and the genes annotated by the specific loci can be enriched to metabolic related pathways, indicates that phlegm dampness constitution has molecular mechanism basis for the predisposion of metabolic disorders.3) This study has constructed miRNA expression profile of phlegm dampness constitution, and screened differentially expressed miRNA, it laid the foundation for the further research of microscopic identification and molecular mechanisms of phlegm dampness constitution.Experiment3. Study on phlegm dampness constitution incRNA expression profileObjective:1) Detect the IncRNA expression level of PBMC from balanced constitution and phlegm dampness constitution subjects, and construct Inc RNA expressionprofile test onthe genome-wide level.2) Screen the specific IncRNA of phlegm dampness constitution, explore the IncRNA regulation mechanism in the formation of phlegm dampness constitution and provide potential biomarkers for microscopic identification of phlegm dampness constitution.3) Through comparing the different expression of lncRNAs and mRNA biofunction enrichment between group M and group T, to explore variance between phlegm dampness constitution and metabolic disease of molecular mechanism.Method:1) Collect peripheral blood samples of 8 balanced constitution healthy volunteers （group P）,10 phlegm dampness disease-free volunteers （group T） and 10 phlegm dampness constitution metabolic syndromepatients （group M）. Extract total RNA from PBMCs to conduct microarray detect with Agilent Human LncRNA, which could test lncRNA as well as mRNA simultaneously.2) Through pairwise comparisons among the three groupsanalysis of lncRNA expression data to screen differentially expressed lncRNAs between each two groups.3) Conduct function enrichment analysis of the lncRNA target genes of each two groups to predict the biofunctibns of different IncRNAs.Results:1) A number of 597 specific IncRNAs and 311 mRNAs were screened.between group T and group P; Group M and group T show 705 specific mRNAs and 2045 IncRNAs; A number of 854 specific IncRNAs and 275 mRNAs were screened between group M and group T.2) The most upregulated different lncRNA between group T and group P is TCONS₀0015846, and the most downregulated different lncRNA is NONHSAT009226, they could work as the potential biomarkers to identify phlegm dampness constitution.3) Through biofunction analysis of the phlegm dampness constitution different lncRNA target genes, multiple glucose and lipid metabolism, inflammation and immune responses related GO biological process and KEGG pathway terms are enriched.Conclusion:1) Phlegm dampness constitution and balanced constitution exhibit systematic differences in the lncRNA profile with the genome-wide level.2) The phlegm dampness constitution subjects showsignificant lncRNA expression difference even in the disease-free condition, and the genes annotated by the specific loci can be enriched to metabolic related pathways, indicates that phlegm dampness constitution has molecular mechanism basis for the predisposion of metabolic disorders.3) Group P compared with group M show even more significant difference than group T, and the target genes of group M and group T different IncRNAs are enriched to not only the common GO and KEGG terms such as inflammation and immune response, glocuse and lipid metabolism, and the group M also showes a higher risk of developing cardiovascular disease,indicates that phlegm dampness constitution is an intermediate state between health and metabolic diseases.4) This study has constructed lncRNA expression profile of phlegm dampness constitution, and screened differentially expressed lncRNA, it laid the foundation for the for ther research of microscopic identification and molecular mechanisms of phlegm dampness constitution.