Those pre-miRNAs are exported to the cytoplasm and cleaved by Dicer to produce miRNA/miRNA* duplexes. miRNAs associate with Argonaute (Ago) selleck catalog proteins to form the core effector complex known as RISC (for RNA-induced silencing complex). This complex is capable of recognizing mRNAs and inhibiting protein translation. In 2006, Schmitter and colleagues analyzed HEK293 cell lines depleted of Dicer or individual Ago proteins (20). Their results indicated that Ago2 was the most important Ago protein acting in the miRNA pathway in HEK293 cells and that knocking down Ago2 had a similar effect to that of Dicer. More recently, Su et al. demonstrated that mammalian Agos all contribute to miRNA silencing, and individual Agos have overlapping functions in this process (22).
In addition to the role of Ago2 in the action of miRNAs, Diederichs and colleagues have shown that Ago2 also plays an important role in the processing of the mature form of several miRNAs (3). A number of miRNAs have been reported to be expressed and regulated during adipocyte differentiation. Esau et al. (5) were the very first ones to show that miRNA-143 regulated adipocyte differentiation. More recently, Xie et al. (26) showed that miRNA-103 and miRNA-143 are induced during adipogenesis and accelerate fat cell development. Oskowitz and colleagues analyzed miRNA expression during human multipotent stromal cells differentiation (18). They identified 19 miRNAs that were upregulated during osteogenesis and 20 additional miRNAs induced during adipogenesis.
Overexpression of miRNA-27 inhibits adipocyte formation by blocking expression of peroxisome proliferator-activated receptor (PPAR)�� and C/EBP��, without affecting myogenic differentiation (16). miRNA let-7 (24) also plays a role in adipogenesis. Expression of several others have been described as expressed and regulated in the adipogenic differentiation of adipose-derived stem cells (25) or in human omental and subcutaneous adipose tissue (12). MATERIALS AND METHODS Cell culture. Maintenance and adipogenesis of 3T3-L1 preadipocytes were as described previously using methylisobutylxanthine, dexamethasone, and insulin (MDI) (7). For adipogenesis, cells that had been confluent for 2 days (day 0) were treated with 10% fetal calf serum (FCS), 1 ��M dexamethasone, 0.5 mM methylisobutylxanthine, 1 ��g/ml insulin, and 5 ��M troglitazone.
On day 2, cells were fed with 1 ��g/ml insulin Cilengitide in 10% FCS media, and on day 4 and every 2 days thereafter, cells were refed with 10% FCS. Lipid accumulation in adipocytes was visualized by staining with Oil Red-O (4). Marrow-derived ST2 cells were incubated at 37��C and 5% CO2 in ��-MEM supplemented with 10% FCS (Atlanta Biologicals, Lawrenceville, GA). Plasmids and transfections. A genomic fragment of 2.