The Function Of MiR-638 In Lipid Metabolism In Vitro

BackgroundDisorders of lipid metabolism is a heavy burden on the Chinese population. A large number of epidemiological studies had shown that over the past decade, the way of life and diet changed in Chinese population, and led to increased total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), triglyceride (TG) levels, decreased high density lipoprotein cholesterol (HDL-C) levels through interacting with genetic factors.Cholesterol is an important component of the cell membrane, and plays a role in structural support and functional regulation in lipid bilayer, and is a precursor of steroid and bile acids. Most types of human cells lack the ability to perform catabolic effects of cholesterol.Thus, the homeostasis in intracellular cholesterol is maintained in many steps like cholesterol synthesis, esterification, uptake or efflux. MicroRNA (miRNA) is a highly conserved small noncoding single-stranded molecule, and is about 18-25 nucleotides in length. It plays an important role in regulation of genes, especially in post-transcriptional level. One miRNA usually regulates many target genes and participates in multiple steps in signal transduction pathway, making it a powerful biological regulator. Many studies had studied the role of miRNAs in the development and progression of cardiovascular disease through miRNA knockout mice models, and chemically synthesized mimics or inhibitors of miRNAs. Previous studies in our group had found that plasma miR-63 8 was associated with HDL levels. Compared with normal plasma HDL-C group, the expression of miR-638 in low HDL-C group was higher than that in normal group, and the difference was statistically significant. We also found that StarDIO is a target gene of miR-638 by using bioinformatics. These results provided a basis for further study on the effect of miR-638 on lipid metabolism-related gene expression.ObjectiveThis study was designed to further investigate the effect of miR-638 on lipid metabolism-related gene expression.MethodsIn order to further investigate the role of miR-638 in lipid metabolism, miR-638 mimics and inhibitors were transfected into HepG2 and macrophages. Appropriate concentrations and time points used to treat cells were determined through pre-experiment. Finally we found that the concentration of mimics/inhibitors at 20nM and lasted for 48 hours could effectively increase/decrease the expression level of intracellular miR-638. Then RT2 Profiler TM PCR Array chips contained 84 human lipoprotein signals and cholesterol metabolism related gene were used to investigate the effect of miR-638 on lipid metabolism. Real-time PCR and Western blot were also used to verify the results.ResultsTransfected miR-638 Mimcs in HepG2 cells and macrophages could significantly increase the expression of miR-638. Transfected miR-638 Inhibitors in HepG2 cells could significantly decreased the expression of miR-638. Results showed that transfected miR-638 Mimics in HepG2 cells and macrophages could reduce the RNA and protein expression levels of ATP-binding cassette A-1 (ABCA1), ATP-binding cassette G-1(ABCG1) and very low density lipoprotein receptor (VLDLR), and transfected miR-638 Inhibitors in HepG2 cells could increase the RNA and protein expression levels of ABCA1 and ABCG1.ConclusionsMiR-638 could influence the expression levels of genes related to lipid metabolism.BackgroundAtherosclerosis is the main basic pathological change in many cardiovascular and cerebrovascular diseases, including coronary heart disease and stroke, and it is seriously detrimental for people's health and even leads to death in China. Excessive cholesterol in plasma can accumulate under arterial intima and then form atherosclerotic plaque. Once plaque ruptures, formation of secondary thrombosis will block the arterial lumen and lead to myocardial ischemia or even necrosis. Cholesterol is one of the major components of the mammalian cell membrane and plays an important role in cell proliferation, bile acid and hormone biosynthesis. High density lipoprotein (HDL) can transport excess cholesterol from peripheral tissues and arterial walls to the liver, and promote its excretion through the bile pathway. This process, which is called cholesterol reverse transport (RCT), plays an important role in maintaining the homeostasis of cholesterol metabolism. Therefore, RCT is considered to be an important process for preventing atherosclerosis. Many studies had shown that microRNAs could participate in the process of regulating RCT. Previous studies in our group had found that plasma miR-638 was associated with HDL levels.Compared with normal plasma HDL-C group, the expression of miR-638 in low HDL-C group was higher than that in normal group, and the difference was statistically significant.These results provided the basis for further study of miR-638 in RCT.ObjectiveThis study aimed to investigate the effect of miR-638 on intracellular cholesterol efflux.MethodsHepG2 cells and macrophages were treated with miR-638 Mimics/Inhibitors and miR-638 Mimics/Inhibitors Negative Control, and NBD-cholesterol was incubated after loading cells with 100 ?g/mL of ox-LDL. ApoAI, HDL and standard serum were used to induce cholesterol efflux. Fluorescence intensity of both efflux fluid and cell lysate were measured by fluorescence microplate reader, and the NBD-cholesterol efflux rate was calculated according to the formula.ResultsIn HepG2 cells, miR-638 Mimics inhibited ABCA1-mediated cholesterol efflux to ApoAI and ABCG1-mediated cholesterol efflux to HDL, whereas miR-638 Inhibitors were able to promote ABCA1-mediated cholesterol efflux to ApoAI and ABCG1-mediated cholesterol efflux to HDL. In macrophages, miR-638 Mimics inhibited ABCA1-mediated cholesterol efflux to ApoAI.ConclusionsMiR-638 Mimics could inhibit intracellular cholesterol efflux, and miR-638 Inhibitors could promote intracellular cholesterol efflux.