miR-135a-5p inhibits 3T3-L1 adipogenesis through activation of canonical Wnt/β-catenin signaling

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   Figure 1

   The relative expression level of miR-135a-5p during 3T3-L1 preadipocyte differentiation. Adipogenic differentiation was induced by adipogenic medium containing INS, DEX, and IBMX (see Materials and methods). Total cellular RNA was harvested at the designated time points, and the relative expression of miR-135a-5p was detected by the stem-loop QPCR method using U6 snRNA as the control gene (n=3; *P<0.05).
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   Figure 2

   Inhibition of 3T3-L1 preadipocyte differentiation and adipogenesis by miR-135a-5p. 3T3-L1 preadipocytes were transfected with equal amounts of miR-135a-5p mimics or NC, and then subjected to adipogenic differentiation 24 h after transfection (designated as day 0). The positive group consists of untransfected normal differentiated 3T3-L1 cells, and the negative group consists of undifferentiated 3T3-L1 preadipocytes. (A) Oil Red O staining showed the lipid droplets accumulation on day 10 of 3T3-L1 preadipocyte differentiation. (B) Quantification of Oil Red O (left) and measurement of triglyceride content of cells (right) on day 10 of differentiation. (C) The relative mRNA expression levels of Pparg and Cebpa among treated cells were analyzed at the given time points and other adipogenic marker genes Ap2 and Fas (D) were also detected on day 9 of differentiation by SYBR QPCR (n=3; *P<0.05, **P<0.01).
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   Figure 3

   Apc is the real target of miR-135a-5p. (A) Two potential binding sites for miR-135a-5p in mouse Apc 3′-UTR were predicted by TargetScan and RNAhybrid software, and the first binding site is conserved among mammals (left). Four nucleotides in the seed region, underlined were mutated to abolish the interaction between miR-135a-5p and Apc 3′-UTR (right). (B) Apc-w, Apc-m1, Apc-m2, or Apc-m3, four psi-CHECK2 vectors, were used in co-transfection of BHK cells with miR-135a-5p mimics or NC respectively. Whole cellular lysates were obtained 24 h after transfection and the relative luciferase activity was then measured. (C) Endogenous Apc mRNA level in 3T3-L1 preadipocytes was detected 24 h after transfection with miR-135a-5p mimics or NC, and (D) APC protein level was also monitored by western blot analysis 48 h after transfection with miR-135a-5p mimics, NC, miR-135a-5p inhibitor, or NC inhibitor. β-actin was used as a loading control. The positive group consisted of untransfected, 3T3-L1 preadipocytes (n=3; *P<0.05, **P<0.01).
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   Figure 4

   Activation of the canonical Wnt/β-catenin signaling pathway by miR-135a-5p. (A) The mRNA expression levels of Ccnd1 and Cmyc in 3T3-L1 preadipocytes were evaluated 24 h after transfection with miR-135a-5p mimics or NC. (B) Nuclear protein was collected 48 h after transfection, the β-catenin protein level in 3T3-L1 preadipocytes was assessed by western blot assay and histone H3 was used as an internal control. The positive group consists of untransfected 3T3-L1 preadipocytes (n=3; **P<0.01).
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   Figure 5

   Inhibition of APC with siRNA reduced 3T3-L1 adipogenic differentiation and adipogenesis. (A) The protein level of APC was monitored in 3T3-L1 preadipocytes 48 h after transfection with siApc-1, siApc-2, or Apc NCi oligonucleotides. (B) 3T3-L1 preadipocytes, transfected with equal amounts of siApc-1 or Apc NCi, were incubated with adipogenic medium for 4 days and then stained with Oil Red O. (C) Quantification of Oil Red O (left) and measurement of triglyceride content of cells (right) on day 4 of 3T3-L1 preadipocyte differentiation. (D) 3T3-L1 preadipocytes were treated as described in (B). And the mRNA expression levels of adipogenic marker genes (Pparg, Cebpa, and Fas) and (E) the targets (Ccnd1 and Cmyc) of the canonical Wnt/β-catenin signaling pathway were measured by QPCR analysis on day 4 of differentiation. The positive group consists of untransfected normal differentiated 3T3-L1 cells (n=3; *P<0.05, **P<0.01).
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   Figure 6

   Model of how miR-135a-5p impairs 3T3-L1 preadipocyte differentiation and adipogenesis by activating the canonical Wnt/β-catenin signaling pathway. miR-135a-5p inhibited Apc, activated the canonical Wnt/β-catenin signaling pathway, and blocked the expression of adipogenic marker genes such as Pparg and Cebpa, leading to impaired adipogenesis.

• © 2014 Society for Endocrinology