| Background and Objectives: Coronary heart disease（CHD）is a serious health problem all over the world,and its morbidity and mortality are increasing year by year.The occurrence and development of CHD is extremely complex,and its underlying molecular mechanism remains unclear,among which abnormal lipid metabolism is the main risk factor for CHD.Therefore,it is of great significance to find the key molecules regulating lipid metabolism for the prevention and treatment of CHD.Studies have shown that serum/glucocorticoid regulated kinase 3（SGK3）is highly expressed in endothelial cells stimulated by oxidized low density lipoprotein（ox-LDL）.However,the expression of SGK3 in peripheral blood and its role in lipid metabolism remain unclear.In this study,we analyzed the expression of CHD common differentially expressed genes by chip screening combined with peripheral blood leukocyte detection.The role of SGK3 in lipid metabolism was investigated in vitro experiments.Methods:（1）Differential gene screening:In June 2018,peripheral blood of 5 patients with ST-segment elevation myocardial infarction（STEMI）indicated by coronary angiography and 3 controls with negative coronary angiography were collected in Taihe hospital,Shiyan city,Hubei province.Plasma exosomes were extracted and differentially expressed mRNA was screened by plasma exosome transcriptome sequencing.（2）Expression detection of exosome differentially expressed genes in peripheral blood leukocytes: Peripheral blood samples from 293 CHD patients and 272 controls who were treated at Taihe hospital,Shiyan city,Hubei province from June 2019 to September 2020 were collected.Trizol method was used to extract total RNA from residual blood leukocytes after routine detection,and real-time fluorescence quantitative PCR（qPCR）was used to detect the expression of SGK3,RPL34,RPS12,MAP6D1 and PPP2 CB mRNA in peripheral blood leukocytes with significantly different expression in exosomes.Five differentially expressed genes were correlated with clinical parameters by Pearson correlation analysis and Logistic regression analysis.（3）In vitro mechanism exploration: 10 samples of hyperlipidemia from CHD patients were collected and mixed hyperlipidemia was prepared.THP-1 macrophages and Hep G2,Huh7 and LO2 hepatocytes were treated with 5% and 10% mixed hyperlipidemia,the expression levels of SGK3 mRNA and protein in THP-1 macrophages and Hep G2,Huh7 and LO2 hepatocytes were detected by qPCR and Western blot 24 h later;The expression of SGK3 in cells was detected by immunofluorescence assay;Hep G2 cells were infected with CRISP/Cas9 lentivirus to knock out SGK3,and the expression levels of SGK3 mRNA and protein in Hep G2 cells were detected after 10% mixed hyperlipidemia stimulation for 48 h;Lipid deposition was detected by oil red O staining;The LDL-uptake method was used to detect the endocytosis of LDL and the expression of cellular LDL receptors.Results:（1）According to the condition of |log FC（fold change）|>0.5,P<0.05,a total of 194 differentially expressed genes were screened in plasma exosomes in STEMI patients and control group,of which 173 genes were up-regulated and 21 genes were down-regulated.（2）The screening results of exosome microarray showed that the mRNA expression levels of SGK3,MAP6D1 and PPP2 CB in plasma exosomes of STEMI patients were significantly up-regulated,and the mRNA expressions of RPL34 and RPS12 were significantly down-regulated（all P<0.001）.Compared with the controls,the quantitative detection results of peripheral blood leukocytes showed that SGK3,RPL34,MAP6D1,and PPP2 CB mRNA were significantly down-regulated（P<0.001）,and the expression of RPS12 mRNA was significantly up-regulated（P<0.001）.The variation trend of differential gene expression in exosomes was not completely consistent with that in leukocytes.Correlation analysis showed that SGK3 mRNA expression was positively correlated with TC,HDL-C and LDL-C（R=0.157,P=0.001;R= 0.135,P=0.007;R=0.141,P= 0.005）.The expression levels of RPL34,MAP6D1 and PPP2 CB were significantly positively correlated with the content of TC（R=0.138,P=0.003;R= 0.131,P= 0.005;R= 0.077,P = 0.004）;the expression level of RPS12 was significantly positively correlated with TG（R=0.106,P=0.023）.After adjusting for risk factors such as gender,age,hyperlipidemia,diabetes and hypertension,Logistic regression analysis showed that: low expression of SGK3,RPL34,MAP6D1 and PPP2 CB were independent risk factors for CHD（OR=0.668,95%CI=0.483~0.923,P=0.014;OR=0.631,95%CI=0.531~0.75,P<0.001;OR=0.733,95%CI=0.62~0.867,P<0.001;OR=0.731,95%CI=0.614~0.87,P<0.001）;the high expression of RPS12 was an independent risk factor for CHD（OR=1.214,95% CI=1.073~1.373,P=0.002）.（3）In vitro mechanism studies showed that: Compared with the control group,the expression levels of SGK3 mRNA and protein in THP-1 macrophages and Hep G2,Huh7,LO2 hepatocytes were significantly upregulated in 10% mixed hyperlipidemia（all P<0.05）;Immunofluorescence results showed that SGK3 was widely expressed in the studied cells;The expression of SGK3 in the nucleus and cytoplasm of THP-1 and Hep G2 cells was increased after 10% mixed high-fat stimulation for 24 hours;after knockout of SGK3,the mRNA and protein levels of SGK3 were significantly decreased（P<0.01）;Compared with the control group,10% hyperlipidemide induced lipid deposition in Hep G2 cells and increased uptake of LDL.When SGK3 was knocked out,intracellular lipid deposition was significantly reduced,and LDL uptake and LDL receptor expression were significantly reduced.Conclusion: The variation trend of gene expression in plasma exosomes of patients with cardiovascular disease was not completely consistent with that in peripheral blood leukocytes.SGK3 mRNA expression was significantly up-regulated in plasma exosomes and down-regulated in peripheral blood leukocytes of cardiovascular patients.The expression level of SGK3 was positively correlated with the contents of TC,HDL-C and LDL-C.In vitro studies have shown that SGK3 may increase LDL uptake and cholesterol accumulation after being stimulated by hyperlipidemide.SGK3 may be a key gene for lipid metabolism regulation.This finding provides a new idea for CHD gene diagnosis and therapeutic target research. |
PDF Full Text Request