miR-196a-5p对3T3-L1前脂肪细胞增殖和分化的影响效应

The Effect of miR-196a-5p on Proliferation and Differentiation of 3T3-L1 Preadipocyte

目的:探究miR-196a-5p对小鼠前体脂肪细胞增殖、分化的影响及其潜在的分子机制。方法:(1)构建小鼠肥胖模型,RT-PCR检测脂肪组织中miR-196a-5p表达量;(2)鸡尾酒法诱导3T3-L1前脂肪细胞分化,RT-PCR检测分化过程中miR-196a-5p的表达变化;(3)合成miR-196a-5p mimics和inhibitors转染3T3-L1细胞,以CCK8、EdU试剂盒检测miR-196a-5p对3T3-L1前脂肪细胞增殖的影响作用;(4)运用油红O染色、甘油三酯测定评估miR-196a-5p对3T3-L1细胞分化的影响;(5) RT-PCR检测miR-196a-5p对前脂肪细胞增殖、分化相关基因的影响;(6)结合前人文献,运用生物信息软件、萤光素酶报告系统对miR-196a-5p调控脂肪细胞分化的靶基因进行筛选和验证。结果:(1) miR-196a-5p在肥胖小鼠脂肪组织中高表达,在3T3-L1前脂肪细胞分化过程中先升高后下降;(2)与阴性对照组相比,mimics转染抑制了3T3-L1细胞增殖,inhibitors转染促进了3T3-L1细胞增殖;(3)与阴性对照组相比,mimics组积累了大量油红着色的脂滴,甘油三酯含量增多,而inhibitors组的脂滴少而小,甘油三酯含量相对降低;(4)与阴性对照组相比,mimics转染抑制了增殖标志基因Cyclin D1、Cyclin E、CDK2和CDK4表达,促进了分化标志基因PPARγ、C/EBPα、LPL、aP2等的表达,inhibitors转染则表现出与mimics转染相反的作用;(5) miR-196a-5p可显著抑制野生型MAP4K3和MAPK1 3’UTR萤光素酶活性,而突变绑定位点可废除该抑制效应。结论:miR-196a-5p不仅可抑制3T3-L1前脂肪细胞增殖,还可促进其诱导分化、沉积脂滴;miR-196a-5p可能通过靶向调节MAP4K3和MAPK1来介导3T3-L1前脂肪细胞分化。

Objective: To investigate the effect of miR-196a-5p on proliferation and differentiation of mouse adipocyte,and explore its potential molecular mechanisms. Methods:(1) Utilizing RT-PCR,miR-196a-5p expression levels in adipose tissues from obese or normal mice were measured;(2) The miR-196a-5p expression level during preadipocyte differentiation were measured by RT-PCR method,after 3T3-L1 cells were induced to differentiate by cocktail method;(3)After miR-196a-5p mimics or inhibitors were transfected into 3 T3-L1 cells,CCK8 and EdU detection were performed to evaluate the effect of miR-196 a-5 p on its proliferation;(4) Measuring the effect of miR-196a-5 p on 3T3-L1 cells differentiation by Oil red O staining and triglyceride assay;(5) Detecting the effect of miR-196 a-5 p on the expression levels of 3T3-L1 cells proliferation and differentiation related genes;(6) Based on previous reports,using bioinformatics and luciferase reporter assays to identify targets that miR-196a-5p regulates preadipocyte differentiation. Result:(1)miR-196a-5p not only were highly expressed in adipose tissues of obese mice,but also were expressed dynamically during 3T3-L1 cells differentiation;(2)When compared with negative control,mimics transfection inhibited 3T3-L1 cells proliferation,inhibitors transfection promoted its proliferation;(3)When compared with negative control,mimics or inhibitors transfection increased or decreased lipid accumulation and triglyceride content,respectively;(4)When compared with negative control,mimics transfection repressed proliferation related markers( Cyclin D1,Cyclin E,CDK2 and CDK4) and promoted differentiation related markers( PPARγ,C/EBPα,LPL and aP2),however,inhibitors transfection had an opposite effect than that of mimics transfection;(5) The miR-196 a-5 p mimics significantly suppressed a luciferase reporter gene whose expression was regulated by the mouse MAP4 K3 and MAPK1 mRNA 3’UTR,whereas mutation of the miR-196a-5p binding site in murine MAP4K3 and MAPK1 3’ UTR completely abolished this response. Conclusions: miR-196 a-5 p might inhibit 3T3-L1 preadipocyte proliferation, and enhance its differentiation. The regulation of preadipocyte differentiation may be mediated by targeting MAP4K3 and MAPK1.