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1、MiR-433-3p 靶向调节绵羊 BCKDHB 表达 闫晓茹 师涛 潘洋洋 景炅婕 程俐芬 曹宁贤 乔利英 刘文忠 山西农业大学动物科技学院 山西省畜禽繁育工作站 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 Target Scan、miRanda 和 DIANA-micro T 3 个在线软件, 以 BCKDHB 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 Pm
2、ir-GLO 与目的片段同时使用 Xho 和 Xba 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lipofectamineTM3000 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
3、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 m RNA 的表达量、以及利用 Western blotting 技术检测
4、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 BCKDHB3-UTR 的第 828 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 3-UTR 重组双荧光载体的相对荧光活性降低 (P0.01) , 说明 miR-43
5、3-3p 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 的 m RNA 和蛋白的相对表达量, 发现过表达组 BCKDHB m RNA 和蛋白的相对表达量低于阴性对照组 (P0.05) , 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
6、BCKDHB m RNA 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; 作者简介：闫晓茹;E-mail:。作者简介：刘文忠;Tel:0354-6288337;E-mail:收稿日期：2017-06-20基金：国家自然科学基金 (31372292) Regulation of the BCKDHB Gene Expression by miR-
7、433-3p in Ovine PreadipocytesYAN XiaoRu SHI Tao PAN YangYang JING JiongJie CHENG LiFen CAO NingXian QIAO LiYing LIU WenZhong College of Animal Science and Veterinary Medicine, Shanxi Agricultural University; Division of Animal and Poultry Breeding, Department of Agriculture of Shanxi Province; Abstr
8、act： 【Objective】This study aims to reveal the target relationship between miR-433-3 p and BCKDHB by theoretical prediction and experimental verification approaches. 【Method】The binding site between miR-433-3 p and BCKDHB was predicted by three bioinformatics softwares including Target Scan、miRanda a
9、nd DIANA-micro T based on the sequence of BCKDHB. In order to get the target fragment the primers of BCKDHB 3-UTR were designed for PCR amplification. Double digestion with Xho and Xba was performed with vector Pmir-GLO and target fragments, which were then ligated with T4 enzyme to obtain the recom
10、binant dual-luciferase expression plasmid. Two vectors of mimics and NC were commercially available. One experimental (overexpressing miR-433-3 p) and two control groups (negative and blank) were set. By using LipofectamineTM3000 reagent the dual-luciferase vector was co-transfected with the commerc
11、ially obtained vectors into HEK-293 T cells of overexpressing-miR-433-3 p 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 wer
12、e cultured in vitro to study the interacting relationship of miR-433-3 p and BCKDHB in the differentiated preadipocytes. Mi R-433-3 p overexpression was adopted to study its regulating function to BCKDHB by detecting RNA quantity of miR-433-3 p and BCKDHB as well as protein quantity of BCKDHB before
13、 and after miR-433-3 p overexpression in ovine preadipocyte. RT-qPCR was carried out to detect the temporal expression dynamics of BCKDHB and miR-433-3 p in the differentiation process of ovine preadipocytes. In order to increase the reliability of the results, we also carried out photo collection a
14、nd oil red O staining at different stages during the differentiation of ovine preadipocytes.【Result】The results showed that miR-433-3 p had a theoretical binding site at the 828 bases of BCKDHB 3-UTR. We found miR-433-3 p overexpression significantly (P0.01) decreased luciferase activity of the reco
15、mbinant double fluorescent plasmid by comparing the relative fluorescence activity of the overexpression, the negative and the blank groups, indicating that miR-433-3 p could bind to the 3-UTR, which verified the correctness of our prediction. Overexpressed miR-433-3 p inhibited the expressions of B
16、CKDHB at both m RNA (P0.01) and protein (P0.05) levels in ovine preadipocyte. The expression of miR-433-3 p 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-433-3 p negatively regulated the expression of BCKDHB and its c
