miR-17-92 cluster accelerates adipocyte differentiation by negatively regulating tumor-suppressor Rb2/p130

Abstract

Adipogenesis involves cell proliferation and differentiation, both of which have been shown to be regulated by micro (mi)RNA. During mouse preadipocyte 3T3L1 cell differentiation, we found that miR-17-92, a miRNA cluster that promotes cell proliferation in various cancers, was significantly up-regulated at the clonal expansion stage of adipocyte differentiation. Stable transfection of 3T3L1 cells with miR-17-92 resulted in accelerated differentiation and increased triglyceride accumulation after hormonal stimulation. By using a luciferase reporter assay, we demonstrated that miR-17-92 directly targeted the 3 UTR region of Rb2/p130, accounting for subsequently reduced Rb2/p130 mRNA and protein quantities at the stage of clonal expansion. siRNA-mediated knock-down of Rb2/p130 at the same stage of clonal expansion recapitulated the phenotype of overexpression of miR-17-92 in the stably transfected 3T3L1 cells. These data indicate that miR-17-92 promotes adipocyte differentiation by targeting and negatively regulating Rb2/p130. G iven the global obesity epidemic, deciphering the mechanisms underlying adipocyte differentiation in mammals poses an important challenge. The mouse 3T3L1 preadipocyte cell line has been an ideal in vitro system for unraveling the molecular events of adipocyte differentiation (1, 2). The entire process of 3T3L1 preadipocyte cell differentiation involves several cellular stages. First, proliferating preadipocytes become growth-arrested by contact inhibition, which can be reversed by hormonal induction and reentry into the cell cycle. After several rounds of clonal expansion, the cells again become quiescent with the initiation of coordinate transcriptional activation of adipocyte genes such as C/Ebp␣, Ap2, Lpl, Srebp, and Ppar␥. Expression of these genes leads to terminal adipocyte differentiation (3).Among the above cellular programs, clonal expansion is one of the key events taking place in early adipogenesis (4, 5). Blocking cell cycle reentry with a DNA synthesis inhibitor prevents adipocyte differentiation, suggesting that an active clonal expansion is required for the differentiation process (4, 6). Clonal expansion is critically regulated by retinoblastoma family (pRB) genes through interactions with the E2F transcription factors. A significant role of two members of the pRB family p130 and p107 in early adipocyte differentiation is suggested by the dramatic change in their expression within the first 24 h (i.e., clonal expansion) of hormonal induction. In growth-arrested preadipocytes, there are significant levels of p130 with little or no detectable p107. However, this trend is completely reversed at 24 h, with a significant decrease in p130 accompanied by a marked increase in p107. This dramatic regulation has been designated as the p130:p107 switch (6). The functional significance of the p130:p107 switch has now been demonstrated by disrupting the p130 expression pattern during the mitotic clonal expansion phase, resulting in complete inhibition of adipocyte differentiation (7).In...

Down-regulation of Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) Expression Is Necessary for Adipocyte Differentiation

“…We show that it causes a modest caspase activation in 3T3-L1 cells that does not result in a long term cell mortality as confirmed by the absence of effect on postclonal expansion, a phenomenon reported to be necessary for the differentiation of 3T3-L1 cells (33). It is known that during adipocyte differentiation, the early expression of C/EBP␤ induces C/EBP␣ and PPAR␥ (34), which are responsible for the coordinated regulation of expression of several genes leading to terminal differentiation.…”

Macro-management of microRNAs in cell cycle progression of tumor cells and its implications in anti-cancer therapy

“…As a potential mechanism of cancer cell cycle regulation by miR-17-92, it was reported that c-mycinduced miR-17 and miR-20a cooperate with the RB/E2F signaling pathway to control the accurate timing of the G 1 /S transition [18] . Cloonan et al reported that miR-17-5p regulates more than 20 G 1 /S transition-related genes [19] , and RBL2 was identified as a functional downstream gene of miR-17-5p [20,21] . It should be noted that the targets of miR-17-92 depended on the cellular context, which results in diverse functions of the miR-17-92 cluster in different cells.…”

“…It has been reported that the miR-17-92 cluster is frequently amplified [14,15] , and let-7 is frequently down-regulated [9,104] in various cancers. miR-17-92 exerts oncogenic functions through positively regulating cell cycle-related proteins, including p21, p63, p57, and RBL2 [18][19][20][21] , whereas let-7 exerts its tumor suppressive function by down-regulating RAS, cyclins and CDK4 [8][9][10][11] . miR-21 is activated by RAS signaling and suppressed by several negative regulators of RAS signaling and the PI3K/AKT pathway, which in turn potentiates RAS activation and PI3K/AKT activities [72] .…”

Expression of microRNA-26b, an obesity-related microRNA, is regulated by free fatty acids, glucose, dexamethasone and growth hormone in human adipocytes

“…Emerging evidence has suggested that a number of miRNAs are important in the regulation of adipocyte differentiation, including the miR-143 (9) and miR-1792 clusters (10). miR-26b is an intronic miRNA encoded in the carboxy-terminal domain, RNA polymerase II, polypeptide A, small phosphatase 1 (CTDSP1) gene that is important in a variety of different diseases, including lung carcinoma, breast cancer and cardiac hypertrophy (11)(12)(13).…”