MicroRNA-125a-5p Mediates 3T3-L1 Preadipocyte Proliferation and Differentiation

Abstract

Excessive accumulation of adipose tissue is a main cause of obesity or overweight, which is significantly involved in increasing the risk of diseases. Recently, numerous studies have proved that microRNAs (miRNAs) play important roles in adipogenesis by negatively regulating gene expression at posttranscriptional levels. In this study, we showed that miR-125a-5p was expressed at lower levels in the adipose tissues of high-fat diet (HFD)-fed mice than the normal chow (NCW)-fed mice. MiR-125a-5p expression were strongly up-regulated by nearly five-fold, when 3T3-L1 preadipocyte were induced and differentiated into mature adipocytes. Functional analysis indicated that overexpression of miR-125a-5p promoted 3T3-L1 preadipocyte proliferation and inhibited its differentiation. By contrast, inhibition of miR-125a-5p repressed 3T3-L1 preadipocyte proliferation and accelerated its differentiation. Furthermore, a dual-luciferase reporter assay demonstrated that signal transducer and activator of transcription 3 (STAT3) is a direct target gene of miR-125a-5p during 3T3-L1 preadipocyte differentiation. Further analysis confirmed that the process of miR-125a-5p inhibiting 3T3-L1 preadipocyte differentiation might be associated with the regulation of fatty acid metabolism related genes. Taken together, our results indicated that miR-125a-5p might promote 3T3-L1 preadipocyte proliferation, whereas inhibiting 3T3-L1 preadipocyte differentiation by negatively regulating STAT3.

Keywords: 3T3-L1; STAT3; miR-125a-5p; obesity; preadipocyte.

Figure 1

miR-125a-5p is associated with adipogenesis. (A) Mature sequence of miR-125a-5p was conserved among species including mouse (Mmu), human (Hsa), Rat (Rno), swine (Ssc); (B) the expression of miRNA-125a-5p using Illumina Genome Analyzer II and adipocyte volume in different adipose tissues of swine, including retroperitoneal adipose (RAD), greater omentum (GOM) and mesenteric adipose (MAD); (C) the expression of miR-125a-5p in different tissues of mice. Additionally, after kunming mice were fed high-fat diet (HFD) or normal chow (NCW) diet, (D) body weight, (E,F) serum levels of TG and TC and the mRNA levels of (G) AP2 and (H) miR-125a-5p were measured; (I) The expression levels of miR-125a-5p during 3T3-L1 preadipocyte differentiation. Results are presented as means ± SEM. n = 3. * p < 0.05; ** p < 0.01.

Figure 2

miR-125a-5p promotes 3T3-L1 preadipocyte proliferation. After 3T3-L1 preadipocyte were transfected, respectively, with miR-125a-5p mimics, inhibitors or negative control (NC), (A) the expression levels of miR-125a-5p were measured by performing qRT-PCR; (B) cells proliferation were evaluated by CCK8 (C,D) EdU analysis, respectively. Moreover; (E) the mRNA levels of CDK2, CDK4, CDK6 and p21 were measured by qRT-PCR. Scale bar, 10 μm. Results are presented as means ± SEM. n = 3. * p < 0.05; ** p < 0.01.

Figure 3

miR-125a-5p inhibits 3T3-L1 preadipocyte differentiation. After differentiation, cells were transfected with miR-125a-5p mimics, inhibitors or NC for eight days. (A) The expression levels of miR-125a-5p were measured by qRT-PCR; (B) Oil Red O staining of terminally differentiated adipocytes; (C) the content of triglycerides in terminally differentiated adipocytes; (D) the expression of adiponectin was evaluated by performing immunofluorescence analysis; (E) the mRNA levels of C/EBPα, PPARγ and FABP4 were measured by qRT-PCR; (F) sequence alignment of miR-125a-5p with 3′ UTR of Mmu, Hsa, Rno and Ssc STAT3 mRNA. Binding site and seed region of miR-125a-5p are indicated in red. Additionally, the recombinant double fluorescent reporter plasmid contains either a wild-type (WT) 3′ UTR of STAT3 or mutant (WT) 3′ UTR of STAT3, respectively; (G) The expression levels of STAT3 in adipose tissues from HFD- or NCW-fed mice; (H) The expression levels of STAT3 in cells transfected with miR-125a-5p mimics, inhibitors or negative control (NC); (I) Luciferase assays revealed the repressive effect of miR-125a-5p on the activity of STAT3; (J) The expression levels of genes related to fatty acid oxidation and fatty acid synthesis in differentiated cells transfected with miR-125a-5p mimics, inhibitors, NC. Scale bar, 10 μm. Results are presented as means ± SEM. n = 3. * p < 0.05; ** p < 0.01.