| ObjectiveThe study aimed to analyze the clinical characteristic of the hyperlipidemia individuals and identify the differently expressed genes(DEGs)involved in hyperlipidemia by comparing the gene expression profiles between hyperlipidemia and the control with peripheral blood mononuclear cell(PBMC),and also explore the cellular biological function and signaling pathways involved in the formation of hyperlipidemia;in addition,the study also tried to analyze the clinical characteristic of the hyperlipidemia individuals with different kind of TCM constitutions and compare the miRNA expression profiles with blood serum to identify the differently expressed miRNAs(DEMs)involved in hyperlipidemia with different TCM constitutions,as well as explore signaling pathways involved in the formation of TCM constitution and atherosclerotic diseases.MethodsPart 1:A cross-sectional study design and random sampling method was used in the study.According to the diagnostic,inclusion and exclusion criteria,20 individuals with hyperlipidemia and 19 controls without hyperlipidemia were included in the study.General clinical datas and PBMC were collected from individuals with hyperlipidemia(n=20)or non-hyperlipidemia control(n=19).Clinic datas was analysed by SPSS.Qualitative data was analysed using Fisher exact method as the total sample was less than 40,presented as N(%);measurement data was analysed using Student's t-test,presented as(meanąSD).The difference is significant when P<0.05.For microarray datas,after extrat the total RNA from PBMC,gene expression profiling was determined using the Agilent SurePrint G3 Human Gene Expression v3 Microarray.After further screening by PCA(principal component analysis),the microarray datas from 19 hyperlipidemia and 8 non-hyperlipidemia were finally included for DEGs analysis after cluster analysis,correlation analysis and principal component analysis(PCA).Based on fold change?2 or<0.5 and P-value<0.05,DEGs were identified.The analysis of GO and KEGG pathway were carried out using DAVID 6.8.Gene-gene interactive network graph was made in Cytoscape(Version 3.7.2),based on String datebase.Some DEGs were verified using qRT-PCR(Quantitative Real-time-PCR).Part 2:A cross-sectional study design and stratified random sampling method was used in the study.According to the diagnostic,inclusion and exclusion criteria,347 individuals with hyperlipidemia were screening,including 234 hyperlipidemia individuals with balanced constitution(HBC),53 strong constitution(HSC)and 60 asthenic constitution(HAC).Using Simple random sampling,hyperlipidemia individuals with HBC or HAC or HSC(n=10 each)were included in the study.General clinical data and blood serum was collected from hyperlipidemia individuals with HBC or HAC or HSC(n=10 each).Clinical data were analyzed by SPSS.Qualitative data,such as gender,were analysed using Fisher exact method as the total sample was less than 40,presented as N(%);measurement data,such as age,were analysed using one-way anova,presented as(meanąSD).Bonferroni method was used for pairwise comparison.The difference is significant when P<0.05.After extrat the total RNA from blood serum,mi RNA expression profile was determined using the miScript miRNA PCR Array.Based on further screening by PCA(principal component analysis),the microarray datas from HBC(N=9),HAC(N=8)and HSC(N=7)were finally included for DEMs analysis.Based on fold change?2 or?0.5 and P-value<0.05,DEMs were identified.The target genes were predicted using QIAGEN 3.5 and analysis of KEGG pathway was carried out using DAVID 6.8.Venny analysis was also performed using VENNY.MiRNA-pathway interactive network graph was made in Cytoscape(Version 3.7.2),based on KEGG datebase.ResultsResult 1:There were no significant difference in gender,smoking history,drinking histories and TCM constitution classification between hyperlipidemia individuals and the controls(P>0.05).Comparing to the controls,there was an older age,higher BMI,higher level of high-sensitivity C-reactive protein,and blood glucose in hyperlipidermia individuals(P<0.05).Analysis of gene expression by microarrays showed that there were significant differences in gene expression profile,and 4795 genes,including 2101 upregulated and 2694 downregulated,were differentially expressed in hyperlipidemia compared with controls.Enrichment of functions and signaling pathways of the target genes revealed that there were 3242 GO functions and 18 KEGG signaling pathways involved in hyperlipidemia,most of which involved in infection immunity and inflammation,including chemokine receptor activity,G-protein coupled chemoattractant receptor activity,specific granule lumen,chemokine-mediated signaling pathway,neutrophil mediated immunity,herpes simplex virus 1 infection,cytokine-cytokine receptor interaction and so on.Difference in the expression of some genes(ATP1A1?DFNB31?FAM53B?HBEGF?MAPK14?PIK3CB?ABCA1),were confirmed by RT-PCR.These DEGs mainly involved in the biological process of response to mechanical stimulus,positive regulation of Wnt signaling pathway,cyclic purine nucleotide metabolic process and epithelial cell migration,which may be involved in regulating the formation of hyperlipidemia.Result 2:There were significant differences in history of diabetes and hyperuricemia,high level of uric acid and FT3,white blood cell count,red blood cell count and hemoglobin among the three groups(P<0.05).After pairwise comparison with Bonferroni method,we found that compared to HBC and HAC,there was a high proportion of diabetes and hyperuricemia in HSC(P<0.05);compared to HSC and HBC,there was a lower level of FT3 in HAC(P<0.05);compared to HBC,there was a lower level of white blood cell count and red blood cell count in HAC(P<0.05);compared to HSC,there was a lower level of hemoglobin in HAC(P<0.05);compared to HAC,there was a higle level of uric in HSC(P<0.05).We also found that 25 down-regulated DEMs were screened between HSC and HBC,and 8 DEMs,including 7 up-regulated and 1 down-regulated DEMs,between HAC and HBC,and 6 down-regulated DEMs between HSC and HAC.Among them,only the hsa-miR-338-3p was identified as the common DEM among three groups.Compared to the HBC,hsa-miR-338-3p was down-regulated in HSC but up-regulated in HAC.ROC(receiver operating characteristic curve)revealed that hsa-miR-338-3p could be used as seral biomarkers to identify TCM constitutions(AUC were 0.908,0.99,0.607 in HSC,HAC,HBC,respectively,P<0.05).In addition,total 198 target genes that were r by the hsa-miR-338-3p were significantly enriched in 11 KEGG pathways,most of which,including Ras signaling pathway,insulin secretion,glycerophospholipid metabolism,involved in inflammation process and glucose and lipid metabolism,which could be associated with the development of atherosclerosis and cardiovascnlar and cerebrovascular diseases in hyperlipidemia.In addition,we also found that hsa-miR-145-5p?hsa-miR-183-3p?hsa-miR-210-3p were the unique DEMs in HAC.Compared to HBC and HSC,respectively,hsa-miR-145-5p?hsa-miR-183-3p?hsa-miR-210-3p were significant up-regulated in HAC,which could be used as the seral biomarkers to identify HAC.In addition,ROC revealed that hsa-miR-145-5p(AUC=0.813,P=0.014),hsa-miR-183-3p(AUC=0.969,P<0.001)and hsa-miR-210-3p(AUC=0.984,P<0.001)had a high accurate in identification of HAC.Conclusion1.Our results showed that an older age,higher BMI,higher level of high-sensitivity C-reactive protein and blood glucose probably were the risk factors of hyperliperdemia;and there was a significant difference in gene expression profiles in hyperlipidemia compared with those of matched healthy controls;2.Genes,ATP1A1?DFNB31?FAM53B?HBEGF?MAPK14?PIK3CB?ABCA1,and its biological process may be involved in the formation of hyperlipidemia;3.hsa-miR-338-3p probably was a biomarker for identification of TCM constitution in hyperlipidemia,and its target genes involving in inflammation process and glucose and lipid metabolism may be associated with the development of atherosclerosis and cardiovascnlar and cerebrovascular diseases in hyperlipidemia;4.hsa-miR-145-5p?hsa-miR-183-3p?hsa-miR-210-3p could be used as the seral biomarkers to identify HAC;5.Strong constitution may be an early warning factor of atherosclerotic disease in people with hyperlipidemia since there were several risk factors of atherosclerotic disease,including diabetes,hyperuricemia and down-regulated hsa-miR-338-3p in HSC. |
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