miR-206-3p Inhibits 3T3-L1 Cell Adipogenesis via the c-Met/PI3K/Akt Pathway

Renqiao Tang, Feifei Ma, Wei Li, Shengrong Ouyang, Zhuo Liu, Jianxin Wu, Renqiao Tang, Feifei Ma, Wei Li, Shengrong Ouyang, Zhuo Liu, Jianxin Wu

Abstract

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.

Keywords: Akt signaling; adipogenesis; c-Met; miR-206-3p.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
miR-206-3p was downregulated during 3T3-L1 differentiation. (a) A mature miR-206-3p sequence is conserved among different species, and sequence alignment between miR-206 and c-Met mRNA 3′UTR of mouse and human is observed. The sequences in the red frame are miR-206 seed sequences of different species; (b) KEGG pathway analysis for predicted target genes of mmu-miR-206-3p. The pathways in the red frame are the top 3 most significant enrichment pathways, which include PI3K/Akt pathway; (c) miR-206 and c-Met expression levels were detected in microarray profile; and (d) verified by qPCR during 3T3-L1 differentiation. U6 snRNA was used as a reference gene. Data are shown as means ± SD, ** p < 0.01, *** p < 0.001.
Figure 2
Figure 2
Examination for the transfection efficiency and stability of miR-206-3p during 3T3-L1 differentiation. (a) Schematic diagram of the treatment schedule for cell induction and transfection during 3T3-L1 differentiation; (b) Expression of miR-206-3p was detected on D0, D2, D5, and D8 after MDI induction. U6 snRNA was used as a reference gene. Data are shown as means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
miR-206 inhibited the adipogenic differentiation of 3T3-L1 preadipocytes. (a, left) 3T3-L1 preadipocytes were transfected with miR-206 mimic or inhibitor, cells were fixed and stained with Oil Red O on D8 after MDI stimulation. Scale bar, 100 μm; (a, right) Lipid accumulation was quantified by extracting Oil Red O, and the absorbance was measured at 510 nm wavelength; (b) The mRNA expression of PPARγ, C/EBPα, C/EBPβ, FABP4, and c-Met on D8 after MDI stimulation were measured by RT-qPCR; (c) The protein levels of PPARγ, C/EBPα, C/EBPβ, FABP4, and c-Met on D8 after MDI stimulation were detected by Western blotting (left) and quantified by densitometry analysis (right). GAPDH protein levels served as a loading control. Data are shown as means ± SD, * p < 0.05, ** p <0.01, *** p < 0.001, n.s. stands for not significant.
Figure 4
Figure 4
c-Met overexpression can overcome the inhibition effect of miR-206 in 3T3-L1 cells. (a) The overexpression of c-Met was through the transfection of regular plasmid gene expression vector (pRP-Met) into 3T3-L1 preadipocytes; the expression of enhanced green fluorescent protein (EGFP) was monitored in the transduced cells byoptical microscopy. Scale bar, 100 μm; (b) c-Met expression was detected on the mRNA level (b, left) and protein level (b, right); (c) 3T3-L1 preadipocytes were co-transfected with miR-206 mimic and pRP-Met, cells were fixed and with Oil Red O stained lipid on D8 after MDI stimulation (up), and quantitative analysis with Oil Red O (bottom). Scale bar, 100 μm; (d, left) The protein levels of PPARγ, C/EBPα, C/EBPβ, FABP4, c-Met, Akt, and pAkt on D8 after MDI stimulation were detected by Western blotting and (right) quantified by densitometry analysis. GAPDH protein levels served as a loading control. Data are shown as means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. stands for not significant.
Figure 5
Figure 5
A schematic of the miR-206 regulatory mechanism during adipogenesis. The red frame is the results of our study and their position in the regulation of adipogenesis.

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