CircECH1通过充当miR-365-5p海绵抑制猪前体脂肪细胞增殖

CircECH1 Inhibits Proliferation of Porcine Preadipocyte by Sponging MiR-365-5p

环状RNA(circRNA)是一种共价封闭RNA,在脂肪发育过程中具有重要作用。本研究旨在探讨猪环状RNA ECH1(circECH1)对前体脂肪细胞增殖的调控机制。本研究通过实时荧光定量PCR(qRT-PCR)分析、Sanger测序和RNase R酶消化法成功鉴定circECH1的稳定环状结构,其在马身猪各个组织中均有表达,并且其在脂肪组织中的表达量随日龄增加呈上升趋势。功能研究证明,干扰circECH1后,增殖相关基因PCNA、CDK1和MKi67极显著升高(P<0.01),增殖细胞数量极显著增加(P<0.01)。为进一步探究其分子机制,使用miRDB、miRWalk和RNAhybrid预测circECH1的下游靶基因。通过双荧光素酶报告基因分析和RNA结合蛋白免疫沉淀技术,验证circECH1能靶向结合miR-365-5p。在猪前体脂肪细胞中过表达miR-365-5p会增殖相关基因PCNA和CDK1的表达量极显著升高(P<0.01),增殖细胞数量极显著增加(P<0.01);干扰miR-365-5p后增殖相关基因PCNA、CDK1和MKi67的表达量极显著降低(P<0.01),增殖细胞数量极显著增加(P<0.01)。挽救实验结果显示,共转染si-circECH1和miR-365-5p inhibitor与单独转染miR-365-5p inhibitor相比,增殖相关基因PCNA、CDK1和MKi67的表达量显著升高(P<0.01),增殖细胞数量显著升高。本研究结果证明,circECH1存在于马身猪的脂肪组织中,并且通过海绵吸附miR-365-5p调控猪前体脂肪细胞增殖,为进一步了解调控猪前体脂肪细胞的分子机制提供理论依据。

Circular RNA(circRNA)is a covalently closed RNA and plays an important role in fat metabolism.This study was conducted to explore the regulation effect of circECH1(circular RNA enyl CoA hydrase 1,circECH1)on preadipocyte proliferation.In this study,with quantitative real-time PCR(qRT-PCR)analysis,Sanger sequencing,and RNase R digestion assay,circECH1 was proven to be capable of stable ring formation.It was expressed in all tissues of Mashen pig and its expression in adipose tissue increased with age.Functional studies showed that the expression of proliferation related genes(PCNA,CDK1 and MKi67)and the number of proliferating cells were significantly increased after circECH1 interference(P<0.01).In order to further explore the molecular mechanism,miRDB,miRWalk and RNAhybrid software were used to predict the downstream target miRNAs and genes of circECH1,and dual-luciferase reporter gene and RNA-binding protein immunoprecipitation assay revealed that circECH1 directly targeted miR-365-5p.Proliferation related genes(PCNA and CDK1)and the number of proliferating cells were significantly increased after overexpression of miR-365-5p in porcine preadipocyte(P<0.01).After transfected with miR-365-5p inhibitor,proliferation related genes(PCNA,CDK1and MKi67)and the number of proliferating cells were significantly decreased(P<0.01).In addition,after co-transfection with si-circECH1 and miR-365-5p inhibitor,compared with independent transfection with miR-365-5p inhibitor,both the expression of proliferation related genes(PCNA,CDK1 and MKi67)and the number of proliferative cells was significantly increased(P<0.01).The results of this study proved that circECH1 exists in porcine adipose tissue and regulates the proliferation of porcine preadipocyte by adsorption of miR-365-5p.The present study provides a theoretical basis for further understanding the molecular mechanism of porcine preadipocyte proliferation.