MiR-433-3p靶向调节绵羊BCKDHB表达

doi:10.3864/j.issn.0578-1752.2017.22.015

摘要/Abstract

摘要： 【目的】通过理论预测与试验验证，旨在揭示miR-433-3p对BCKDHB的调节机制。【方法】利用TargetScan、miRanda和DIANA-microT 3个在线软件，以BCKDHB 的mRNA和蛋白的相对表达量，发现过表达组BCKDHB mRNA3′-UTR mRNA的表达量的序列预测与BCKDHB有靶标关系的相关miRNAs。为了验证理论上的预测结果，用设计好的BCKDHB 3′-UTR的特异性引物进行PCR扩增，得到目的片段并进行割胶回收和纯化，并将Pmir-GLO与目的片段同时使用Xho I>和Xba I两个限制性内切酶进行双酶切，再用T4连接酶连接双酶切之后的目的片段和Pmir-GLO，成功构建BCKDHB 3′-UTR的双荧光素酶报告载体。从公司购买miR-433-3p的过表达载体mimics和阴性对照载体NC，设置miR-433-3p过表达、阴性对照、空白对照3个组，分别将2组载体和双荧光素酶报告载体利用lipofectamine^TM3000转染试剂共转染至miR-433-3p过表达、阴性对照组的HEK-293T细胞中，空白对照组中的HEK-293T细胞正常培养，之后分别检测3组细胞中的荧光活性，得到萤火虫荧光素酶活性和海肾荧光素酶活性，以海肾荧光素酶活性为内参计算萤火虫荧光素酶的相对活性。便于了解miR-433-3p和BCKDHB在绵羊前体脂肪细胞中的调控机制，对采取的绵羊尾部前体脂肪细胞进行离体培养。用过表达miR-433-3p的方法探索miR-433-3p在绵羊前体脂肪细胞中对BCKDHB的调控，提取过表达miR-433-3p前后细胞的总RNA和总蛋白，利用RT-qPCR检测过表达miR-433-3p前后的miR-433-3p和BCKDHB、以及利用Western blotting技术检测BCKDHB在过表达前后的蛋白水平。为了解绵羊前体脂肪细胞分化过程中BCKDHB和miR-433-3p表达量的变化，用RT-qPCR检测前体脂肪细胞分化过程中BCKDHB和miR-433-3p的时序表达。为增加结果的可信度，还对分化过程中不同时段的细胞进行了照片采集和油红O染色。【结果】miR-433-3p在BCKDHB 3′-UTR的第8—28个碱基处存在理论上的结合位点。通过比较过表达组，阴性对照组，对照组的相对荧光活性发现过表达miR-433-3p后，BCKDHB重组双荧光载体的相对荧光活性降低（P<0.01），说明miR-433-3p可以与BCKDHB 3′-UTR特异性结合，验证了预测结果的准确性。在绵羊前体脂肪细胞中过表达miR-433-3p后，通过比较过表达组和阴性对照组BCKDHB和蛋白的相对表达量低于阴性对照组（P<0.05），说明miR-433-3p在绵羊前体脂肪细胞中对BCKDHB有负调控作用。在诱导绵羊前体脂肪细胞分化为成熟脂肪细胞的过程中，从采集到的图片和油红O染色的结果发现此过程中脂滴聚积得越来越多，油红O染色验证了脂滴的聚集。另外，在分化过程中检测到miR-433-3p和BCKDHB mRNA的表达量呈现负相关关系。【结论】这些结果充分说明miR-433-3p通过与BCKDHB 3′-UTR的结合负调节该基因及其编码蛋白的表达，为进一步研究BCKDHB调节绵羊脂肪代谢的分子机理提供了科学依据。

关键词: miR-433-3p, BCKDHB, 绵羊, 前体脂肪细胞, 细胞分化

Abstract: 【Objective】 This study aims to reveal the target relationship between miR-433-3p and BCKDHB by theoretical prediction and experimental verification approaches. 【Method】The binding site between miR-433-3p and BCKDHB was predicted by three bioinformatics softwares including TargetScan、miRanda and DIANA-microT based on the sequence of BCKDHB. In order to get the target fragment the primersof BCKDHB 3′-UTR were designed for PCR amplification. Double digestion with Xho I and Xba I was performed with vector Pmir-GLO and target fragments, which were then ligated with T4 enzyme to obtain the recombinant dual-luciferase expression plasmid. Two vectors of mimics and NC were commercially available. One experimental (overexpressing miR-433-3p) and two control groups (negative and blank) were set. By using Lipofectamine^TM3000 reagent the dual-luciferase vector was co-transfected with the commercially obtained vectors into HEK-293T cells of overexpressing-miR-433-3p and negative groups, respectively. And the cells of blank group were cultured normally. Subsequently, their relative luciferase activities were detected, with renilla luciferase activity as the control. Ovine preadipocytes were cultured in vitro to study the interacting relationship of miR-433-3p and BCKDHB in the differentiated preadipocytes. MiR-433-3p overexpression was adopted to study its regulating function to BCKDHB by detecting RNA quantity of miR-433-3p and BCKDHB as well as protein quantity of BCKDHB before and after miR-433-3p overexpression in ovine preadipocyte. RT-qPCR was carried out to detect the temporal expression dynamics of BCKDHB and miR-433-3p in the differentiation process of ovine preadipocytes . In order to increase the reliability of the results, we also carried out photo collection and oil red O staining at different stages during the differentiation of ovine preadipocytes.【Result】 The results showed that miR-433-3p had a theoretical binding site at the 8~28 bases of BCKDHB 3′-UTR. We found miR-433-3p overexpression significantly (P<0.01) decreased luciferase activity of the recombinant double fluorescent plasmid by comparing the relative fluorescence activity of the overexpression, the negative and the blank groups, indicating that miR-433-3p could bind to the 3′-UTR, which verified the correctness of our prediction. Overexpressed miR-433-3p inhibited the expressions of BCKDHB at both mRNA (P<0.01) and protein (P<0.05) levels in ovine preadipocyte. The expression of miR-433-3p correlated negatively with that of BCKDHB in the process of ovine preadipocyte differentiation. Cellular photos and the oil red O analysis showed lipid droplets accumulated in the differentiation of ovine preadipocytes.【Conclusion】 These results indicated that miR-433-3p negatively regulated the expression of BCKDHB and its coding protein by binding to the 3′-UTR, which provided a scientific basis for further research on the molecular mechanism of BCKDHB in regulating fat metabolism in sheep.

Key words: miR-433-3p, BCKDHB, ovine, preadipocytes, cell differentiation

闫晓茹，师涛，潘洋洋，景炅婕，程俐芬，曹宁贤，乔利英，刘文忠. MiR-433-3p靶向调节绵羊BCKDHB表达[J]. 中国农业科学, 2017, 50(22): 4389-4397.

YAN XiaoRu, SHI Tao, PAN YangYang, JING JiongJie, CHENG LiFen, CAO NingXian, QIAO LiYing, LIU WenZhong. Regulation of the BCKDHB Gene Expression by miR-433-3p in Ovine Preadipocytes[J]. Scientia Agricultura Sinica, 2017, 50(22): 4389-4397.

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