The Molecular Mechanism Of The Effection Of Microrna-101 On Regulating Cell Cholesterol Efflux Under Non-Inflammatory And Inflammatory Conditions

Aims:To investigated the role of microRNA-101 (miR-101) in regulating cholesterol efflux under non-inflammatory and inflammatory conditions in human THP-1-derived macrophages (THP-1) and HepG2 hepatoblastoma cells(HepG2) in order to provide experimental basis of the functions of miR-101 in the development of atherosclerosisMaterials and Methods:Cell models were built with over-expression of miR-101 or Anti-miR-101 by infection with lentiviral vectors. A luciferase reporter assay was used to examine miR-101 binding to the 3’untranslated region (3’UTR) of ATP binding cassette transporter Al(ABCAl). The groups included wild-type (WT)+Con-miR group, the site-directed mutations (SDM)+miR-101 group and WT+miR-101 group. THP-1 and HepG2 were divided into groups including control group, miR-101 group and Anti-miR-101 group under non-inflammatory condition and control group, miR-101 group, Anti-miR-101 group, IL-6 group(0.2%BSA plus 20ng/ml IL-6), TNF-α group(0.2%BSA plus 25ng/ml TNF-α), IL-6+miR-101/ anti-miR-101 group and TNF-a+anti-miR-101 group under inflammatory condition. Every groups were in the presence or absence of low density lipoprotein (LDL) conditions(0.2%BSA plus 2μg/ml LDL). Cells were loaded with BODIPY-cholesterol and stained with oil red O to assess cholesterol efflux, cholesterol contents were assessed by quantitative intracellular cholesterol assay(enzymic method). miR-101 or ABCAl protein were examined using quantitative real-time polymerase chain reaction or Western Blotting.Results:The 3’UTR activity of ABCA1 in WT+miR-101 group was significantly lower than that in WT+Con-miR group and SDM+miR-101 group at site 2(P<0.05). In the absence or presence of LDL, the expression of ABCAl protein and apoA-I mediated cholesterol efflux were significantly decreased in THP-1 and HepG2 with over-expression of miR-101 under non-inflammatory condition (P<0.05). In the absence or presence of LDL, the expression of ABCA1 protein and apoA-I mediated cholesterol efflux were significantly increased in THP-1 and HepG2 with over-expression of anti-miR-101 under non-inflammatory condition (P<0.05). Over-expression of miR-101 promoted while anti-miR-101 reduced intra-cellular cholesterol accumulation in THP-1 and HepG2 (P<0.05). Under inflammatory condition, compared with those of control group, the expression of miR-101 of HepG2 in IL-6 group and TNF-a group was significantly increased, and that of miR-101 of THP-1 in IL-6 group was also significantly increased (P<0.05). Compared with IL-6 group and TNF-a group, the expression of ABCA1 protein of THP-1 and HepG2 in IL-6+anti-miR-101 group and TNF-a+anti-miR-101 group significantly increased (P<0.05).Conclusions:miR-101 can inhibit ABCA1 expression and cholesterol efflux by binding at the 3’UTR activity of ABCA1. Inflammation can increase expression of miR-101 and enhance effection of miR-101 on increasing intracellular cholesterol content. Down-regulating of miR-101 can decrease effection of inflammation on suppressing ABCA1 expression. miR-101 may play an important role in the development of atherosclerosis.