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Currently, while numerous studies have correlated mediator complex (MED) alterations with several diseases, fewer studies have investigated MED's role in coronary heart disease (CHD) initiation and progression. A number of studies have indicated that several subunits like MED1, MED13, and MED15 exert important roles in glucose and lipid metabolism. Our previous gene chip study in the patients with CHD suggested that mediator complex subunit 6 (MED6) gene had low expression in the peripheral blood cells. We wonder whether MED6 is also involved in lipid metabolism and therefore promote the initiation and progression of CHD. The aim of this study is to examine the function of MED6 in lipid metabolism in rat myocardial cells (H9C2).
RNA interference (RNAi) vector targeting MED6 and green fluorescent protein(GFP) empty vector were transfected into H9C2 to specifically silence the expression of MED6, GFP empty vector was used as the negative control. Transfection efficiency of siRNA was tested by an inverted fluorescence microscope after transfected of 24h. The total RNA and protein were extracted from H9C2 at 48h post-transfection. Real-time Quantitative PCR and Western Blot were then performed to detect the expression of MED6 gene on the transcription and the translation levels after siRNA transfection. Culture medium was collected before transfection, 12h, 24h, 36h and 48h after transfection. The level of low density lipoprotein (LDL) in the supernatant of H9C2 cells were measured by ELISA.
The result of green fluorescence showed that the vectors were transfected into H9C2 cells successfully, and had no difference in transfection efficiency. The results of Real-time Quantitative PCR using 2-ΔΔCt method showed the relative expression of RNAi vector plasmids compared with the GFP control cells was 0.36±0.04. Western Blot showed the relative MED6 protein expression of RNAi vector plasmids compared with the GFP control cells was 0.51±0.03. ELISA showed that there were no significant difference between the two cell populations in LDL content before transfection (respectively 43.06±1.37nmol/L, 42.01±4.09nmol/L). But the LDL content in control cells remained relatively constant over time (respectively 46.67±2.66nmol/L, 45.25±3.33nmol/L, 40.96±3.81nmol/L, 42.67±2.28nmol/L), whereas the content in siRNA transfected cells increased over time (respectively 48.54±2.26nmol/L, 50.83±3.63nmol/L, 53.12±2.16nmol/L, 62.68±7.85nmol/L), which suggested that MED6 gene is involved in lipid metabolism, and accompanying the mRNA and protein levels of MED6 decreased with the LDL content increased after siRNA transfection.
Silencing the expression of MED6 gene increased LDL production in rat myocardial cells. According to this finding, MED6 could be a new target to regulate lipid biosynthesis in the related pathologies. As the disorder of lipid metabolism is an independent risk factor for CHD, the expression of MED6 may takes effect on the progression of CHD in part.