The Function Of MicroRNA-33in HDL Abnormalities Associated With Metabolic Syndrome

BackgroundMetabolic syndrome (MS) in humans is always associated with the reduction of high-density lipoprotein cholesterol (HDL-C) and the elevation of triglyceride (TG). However, the reduction of HDL-C would not happen in the mouse model of MS. Until now, there is no clear explanation for these. In our previous experiments, we observed that the gene expression of sterol-regulatory element binding protein-lc (SREBP-1c) and ATP-binding cassette transporter A1(ABCA1) in hepatocytes were changed when we co-cultured the hepatocytes with adipocytes. Combined with the fact that in the intron of SREBP-lc, a microRNA called miR-33could regulate the expression of ABCA1gene, we speculate that the relevant factors of MS might affect SREBP1c-miR33b, resulting in HDL-C decrease, accompanied by TG increase. In addition, there is a lack of miR-33b in the sequence of mouse SREBP1gene, and this may be a reason for differences of dyslipidemia between mice and men.ObjectiveExplore the effects of MS relevant factors on SREBP1/miR-33b in hepatocytes, and its role in regulating hepatocyte HDL metabolism.Methods Primary human hepatocytes were treated with100nM insulin and200ng/ml TNF-alpha, respectively. The untreated cells were used as control group. After24hours, the total RNA and protein were extracted. The relative gene expression of miR-33b, SREBP-1c, SREBP-2, ABCAl, ABCG1and APOA1were measured by real-time polymerase chain reaction (real-time PCR). The relative protein expression of ABCA1and ABCG1were measured by Western Blot. The efflux rates of3H-cholesterol in hepatocytes were detected by means of liquid scintillation counter. Primary human hepatocytes were transfected with50nM miR-33b mimic or100nM miR-33b inhibitor by liposome LipofectamineTM2000. After6hours, those cells were treated with100nM insulin and after24hours, the gene expression of miR-33b, SREBP-lc and ABCAl were detected by real-time PCR.Results1. Compared with the control group, the gene expressions of miR-33b and SREBP-1c in primary human hepatocytes of insulin intervention group were significantly up-regulated, while ABCA1, ABCG1and APOA1genes were down-regulated. In consistent with that, the expressions of ABCA1and ABCG1protein levels were also decline, while the cholesterol efflux rates form hepatocytes were decreased. All the changes were statistically significant (P<0.05). However, there is no significant change in SREBP-2gene expression (P>0.05). 2. Compared with the control group, the gene expressions of miR-33b and SREBP-1c in primary human hepatocytes of TNF-alpha group were significantly down-regulated; meanwhile, the gene expressions of ABCA1and APOA1were also down-regulated, while the expression of ABCG1gene was increased. All the changes were statistically significant (P<0.05). However, there is no significant change in SREBP-2gene expression (P>0.05).3. Overexpression of miR-33b could down-regulate ABCA1in primary human hepatocytes and inhibition of miR-33b could up-regulate ABCA1; The stimulation of insulin would further repress ABCA1when we overexpressed miR-33b, however, the depressant effect of insulin on ABCA1disappeared when we slience miR-33b (all P<0.05).4. Neither overexpression nor inhibition of miR-33b could affect the gene expression of SREBP-1c in primary human hepatocytes,P>0.05.ConclusionsHigh level of Insulin can increase the gene expressions of SREBP-1c and miR-33b in primary human hepatocytes simultaneously, thus inhibiting ABCA1and ABCG1mRNA and protein expressions. As a result, the cholesterol efflux mediated by apoA-â…  is repressed.