miR-206-3p Inhibits 3T3-L1 Cell Adipogenesis via the c-Met/PI3K/Akt Pathway
Feifei Ma，Zhuo Liu
MicroRNAs (miRNAs) are important post-transcriptional regulators during adipocyte adipogenesis. MiR-206-3p, a tissue-specific miRNA, is absent in white adipocytes. In this study, we examined the roles of mmu-miR-206-3p in the adipogenic differentiation of 3T3-L1 preadipocytes. The miR-206-3p expression has shown an apparent decreasing trend after induction, and sustained low expression throughout the differentiation of 3T3-L1 cells. miR-206-3p blocked the adipogenic differentiation of 3T3-L1 cells by attenuating c-Met expression; the inhibition effect of miR-206 to the adipogenic differentiation can be counteracted by restoring c-Met expression. In addition, miR-206-3p decreased the phosphorylation of Akt, which is the downstream effector of c-Met in the PI3K/Akt signaling pathway. These data indicate that miR-206-3p inhibits adipocyte adipogenesis through silencing c-Met and subsequently inactivating the PI3K/Akt signaling pathway.A mature miR-139-5p sequence is highly conserved in many mammals including mice, humans, and chickens. The alignment of mouse and human c-met 3′UTRs with the miR-206 “seed” region revealed a high degree of evolutionary conservation (Figure 1a). Pathway analysis for the predicted target genes of mmu-miR-206-3p revealed that the main functions of mmu-miR-206-3p were related to cancer and the PI3K/Akt pathway (Figure 1b). Time course analysis uncovered that miR-206 showed a decreased trend of expression in microarray assays during 3T3-L1 cells adipogenic differentiation (Figure 1c, up). 3T3-L1 cells were collected at different time points (i.e., 0, 2, 4, 6, and 8 days) after 1-methyl-3-isobutylxanthine, dexamethasone, and insulin (MDI) stimulation to confirm the changes of miR-206 during adipogenic differentiation. Stem-loop quantitative polymerase chain reaction (qPCR) results show that miR-206 gradually decreased after MDI stimulation, and were maintained at low level throughout adipogenesis (Figure 1d, up). Meanwhile, c-Met, the target gene of miR-206, has shown sustained increasing trends both in microarray profile (Figure 1c, bottom) and qPCR verification (Figure 1d, bottom). These results suggest that miR-206 is likely associated with adipogenesis.In the present work, a time course analysis was performed based on the transcription profile of a microarray data series (GSE20696) and miRNA expression profile (GSE20698) using the Short Time-Series Expression Miner software. GSE20696 and GSE20698 are expression profiles of 3T3-L1 adipogenesis, first published by Mikkelsen et al.  from Broad Institute, MA, USA. miRNA targets were predicted by miRecords (available online: http://c1.accurascience.com/miRecords/) . The functional annotation of relevant gene clusters was performed using the DAVID online tool (available online: http://david.abcc.ncifcrf.gov/) .This work was supported by the Beijing Natural Science Foundation (Z140001) and by the National Basic Research Program of China (2013CB530605) and by the National Science Foundation of China (81400365). This work was also supported, in part, by the CAMS Central Public Welfare Scientific Research Institute Basal Research Expenses (2016ZX310182-3) and CAMS Initiative for Innovative Medicine (CAMS-I2M).