We sought to identify and characterize microRNA (miRNAs) that posttranscriptionally regulate the expression of scavenger receptor class B type I (SR-BI) and SR-BI-linked selective high-density lipoprotein (HDL) cholesteryl ester (CE) transport and steroidogenesis. Four miRNAs (miRNA-125a, miRNA-125b, miRNA-145, and miRNA-455) with a potential to regulate SR-BI were identified in silico and validated by quantitative real-time PCR (qRT-PCR), Western blot analysis, and SR-BI 3= untranslated region (UTR) reporter assays. In vitro treatment of primary rat granulosa cells and MLTC-1 cells with cyclic AMP (cAMP) or in vivo treatment of rat adrenals with adrenocorticotropic hormone (ACTH) decreased the expression of miRNA-125a, miRNA125b, and miRNA-455 and reciprocally increased SR-BI expression. Using luciferase constructs containing the 3= untranslated region of SR-BI combined with miRNA overexpression and mutagenesis, we have provided evidence that steroidogenic SR-BI is a direct target of miRNA-125a and miRNA-455. Moreover, the transfection of Leydig tumor cells with precursor miRNA 125a (pre-miRNA-125a) or pre-miRNA-455 resulted in the suppression of SR-BI at both the transcript and protein levels and reduced selective HDL CE uptake and HDL-stimulated progesterone production. Transfection of liver Hepa 1-6 cells with pre-miRNA125a significantly reduced SR-BI expression and its selective transport function. In contrast, overexpression of miRNA-145 did not affect SR-BI expression or selective HDL CE uptake mediated by SR-BI in steroidogenic cell lines. These data suggest that a trophic hormone and cAMP inversely regulate the expression of SR-BI and miRNA-125a and miRNA-455 in steroidogenic tissues/cells and that both miRNA-125a and miRNA-455, by targeting steroidogenic SR-BI, negatively regulate selective HDL CE uptake and HDL CE-supported steroid hormone production.
Circulating lipoproteins, particularly high-density lipoprotein (HDL), deliver cholesteryl esters (CEs) to cells via the "selective" CE pathway, a process in which the HDL core CE is taken into cells without parallel uptake and degradation of the HDL particle itself (5, 53, 55). The HDL CE selective pathway plays a major role in plasma cholesterol metabolism by delivering HDL CE to the liver in the final steps of reverse cholesterol transport for its excretion in bile (67) or for bile acid synthesis (52). Selective uptake of HDL CE also occurs prominently in steroidogenic cells of the adrenal gland and ovary and under certain physiological conditions in testicular Leydig cells, where it provides cholesterol for steroid biosynthesis and for the accumulation of cytoplasmic CE storage droplets (5,32,55,(60)(61)(62)74).Scavenger receptor class B type I is a physiologically relevant HDL receptor (1, 2, 65) which binds HDL particles and mediates selective uptake of HDL CE in vitro (1,3,19,30,62,74) and in vivo (36,39,66,76). Scavenger receptor class B type I (SR-BI) also facilitates the bidirectional flux of free cholesterol (FC) (35) and phospholipids between...