MicroRNA-148a Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting PTEN.

Xiayang Jin,Na Ke,Jiyuan Shen,Mengli Zhao,Xinmiao Wu,Yuzhu Luo,Lirong Qiao,Liyan Hu,Yize Song,Jiqing Wang,Yujie Lu,Zhiyun Hao

doi:10.3390/ani11030820

Abstract

Simple SummaryThe miR-148a has been shown to play an important role in preadipocyte differentiation. Herein, we explored the role of miR-148a in ovine adipocyte development, using Oil Red O staining, CCK-8, EdU, flow cytometry and RT-qPCR. The results showed that miR-148a suppressed proliferation and facilitated the differentiation of preadipocytes. The dual fluorescent reporter vector experiments showed that miR-148a directly targeted PTEN. Meanwhile, we demonstrated that PTEN significantly inhibited the differentiation of preadipocytes. In conclusion, our research provides new insights that miR-148a inhibits ovine preadipocyte proliferation and accelerates differentiation by the negative regulation of PTEN.MicroRNAs (miRNAs) have been found to be involved in lipid deposition and metabolism. However, there have been no reports on the roles of miR-148a in the proliferation and adipogenesis of preadipocytes in sheep. In this study, the expression of miR-148a was profiled in the eight tissues of Tibetan ewes and differentiated preadipocytes, and the role of miR-148a in differentiation and proliferation of ovine preadipocytes was investigated using Oil Red O staining, CCK-8, EdU staining, cell cycle detection, and RT-qPCR. The effect of PTEN on the differentiation of ovine preadipocytes was also investigated. The miR-148a was widely expressed in the eight tissues investigated and had significantly increased expression in liver, spleen and subcutaneous adipose tissues, and the heart. The expression of miR-148a continued to increase with the differentiation of ovine preadipocytes. The over-expression of miR-148a significantly promoted differentiation but inhibited the proliferation of ovine preadipocytes. The inhibition of miR-148a had the opposite effect on the differentiation and proliferation of ovine preadipocytes with over-expressed miR-148a. The results from the dual luciferase reporter assays showed that miR-148a mimic significantly decreased the luciferase activity of PTEN-3′UTR dual luciferase reporter vector, suggesting that PTEN is a target gene of miR-148a. In over-expressed-PTEN preadipocytes, the number of lipid droplets remarkably decreased, and the expression levels of adipogenesis marker genes PPARγ, FASN, FATP4, GLUT4, C/EBPβ and LPL were also significantly down-regulated. These results suggest that miR-148a accelerated the adipogenic differentiation of ovine preadipocytes by inhibiting PTEN expression, and also inhibited the proliferation of ovine preadipocytes.

Highlights

Adipose tissue plays important roles in providing energy, maintaining body temperature, and protecting internal organs in animal bodies [1]
The Expression Profile of miR-148a. It was deduced from the reverse transcription quantitative PCR (RT-qPCR) analysis in the eight different tissues of Tibetan ewes that miR-148a was robustly expressed in liver, spleen, and subcutaneous a5doipf 1o7se tissues and the heart, while it had a weak expression in kidney, lung, longissimus dorsi muscle and testis (Figure 1A)
The over-expression of miR-148a promoted the accumulation of lipid droplets and increased the expression levels of aP2, PPARγ, fatty acid synthase (FASN), GLUT4 and lipoprotein lipase (LPL). aP2 is mainly expressed in adipose tissue and mature adipocytes and regulates fatty acid uptake and intracellular transport in adipocytes [29]

Summary

Introduction

Adipose tissue plays important roles in providing energy, maintaining body temperature, and protecting internal organs in animal bodies [1]. It has been reported that moderate fat deposition improves the tenderness and flavor of mutton in sheep [3]; an in-depth understanding of the molecular mechanisms that regulate fat tissue development provides the opportunity to improve meat quality in sheep. The development of adipose tissue has been found to be closely associated with the proliferation and differentiation of preadipocytes [4]. It is well-known that the proliferation and differentiation of preadipocytes are regulated by functional genes and non-coding RNAs [5,6]. It has been found that miRNAs are involved in the proliferation and adipogenesis of preadipocytes. The function of other miRNAs has been elucidated in adipogenesis, including miR-204-5p [12], miR-145 [13], miR20a-5p [14], miR-106a [15], and miR-127 [16]

Methods

Results

Conclusion