脂多糖促进肠道L细胞miR-425-5p表达的机制研究  

Mechanism of lipopolysaccharide promoting the expression of miR-425-5p in intestinal L cells in vitro

作　　者：韦丽瑞 赵雪楠 陈东东 丁成智[2] 郭丰[1] 刘彦玲 王娇[1] Wei Lirui;Zhao Xuenan;Chen Dongdong;Ding Chengzhi;Guo Feng;Liu Yanling;Wang Jiao(Department of Endocrinology and Metabolic Disease,First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Department of Thoracic Oncology,Henan Provincial Chest Hospital,Zhengzhou 450008,China)

机构地区：[1]郑州大学第一附属医院内分泌与代谢病科,郑州450052 [2]河南省胸科医院胸部肿瘤科,郑州450008

出　　处：《中华糖尿病杂志》2022年第8期817-822,共6页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金(81500646);河南省科技项目(192102310064,212102310204)。

摘　　要：目的:探讨脂多糖(LPS)促进肠道L细胞miR-425-5p表达的机制。方法:将肠道L细胞系GLUTag细胞分为对照(NC)组、LPS组(LPS 200 ng/ml培养24 h)、小干扰RNA(siRNA)NC组(LPS 200 ng/ml培养24 h后转染siRNA NC)和NF-κB siRNA组[LPS 200 ng/ml培养24 h后转染核因子-κB(NF-κB)siRNA],每组3个复孔。采用Western blotting检测细胞核及细胞质中NF-κB p65蛋白表达水平,实时荧光定量聚合酶链反应检测miR-425-5p表达水平,酶联免疫吸附测定法检测胰高糖素样肽-1(GLP-1)的分泌水平。采用Promo软件分析预测miR-425-5p启动子上NF-κB的潜在结合位点,双荧光素酶报告实验检测miR-425-5p启动子区域结合位点的相对活性,染色质免疫沉淀反应验证NF-κB与miR-425-5p之间的相互作用。两组之间比较采用独立样本t检验,多组之间比较采用单因素方差分析。结果:与对照组相比,LPS组的GLP-1分泌水平显著下降(P<0.01),miR-425-5p表达水平显著升高(P<0.01)、细胞质和细胞核中NF-κB p65蛋白表达水平显著升高(P<0.05)。与siRNA NC组相比,NF-κB siRNA组细胞核及细胞质中NF-κB p65的蛋白表达水平、miR-425-5p表达水平均明显下降(P<0.01)。LPS诱导下,含有两个结合位点(2-Luc/3-Luc)的miR-425-5p启动子活性明显高于含有单独的结合位点(3-Luc),差异具有统计学意义(P<0.01);NF-κB与miR-425-5p基因启动子序列结合位点2的结合能力最强(P<0.01)。结论:LPS通过活化NF-κB,促进其与miR-425-5p基因启动子序列的结合位点2结合,进而促进肠道L细胞miR-425-5p转录,最终调节肠道L细胞GLP-1分泌。Objective To investigate the mechanism of lipopolysaccharide(LPS)promoting the expression of miR-425-5p in intestinal L cells.Methods Intestinal L cell line GLUTag cells were divided into normal control(NC)group,LPS group(LPS 200 ng/ml cultured for 24 hours),small interfering RNA(siRNA)NC group(LPS 200 ng/ml cultured for 24 hours and then transfected with siRNA NC)and nuclear factor-κB(NF-κB)siRNA group(LPS 200 ng/ml cultured for 24 hours and then transfected with NF-κB siRNA),with 3 replicate wells in each group.The protein levels of NF-κB p65 in the nucleus and cytoplasm were detected by Western blotting.The expression level of miR-425-5p was detected by real-time fluorescence quantification polymerase chain reaction.The secretion level of glucagon-like peptide-1(GLP-1)was determined by enzyme-linked immunosorbent assay.The potential binding sites of NF-κB on the miR-425-5p promoter were analyzed and predicted using Promo software.The relative activity of the binding site in the promoter region of miR-425-5p was detected by dual luciferase reporting assay.The interaction between NF-κB and miR-425-5p was verified by chromatin immunoprecipitation reaction.The independent sample t test was used for comparison between the two groups,and one-way analysis of variance was used for comparison between multiple groups.Results Compared with the control group,the secretion level of GLP-1 in the LPS group was significantly decreased(P<0.01),the expression level of miR-425-5p was increased(P<0.01),and the protein expression level of NF-κB p65 in cytoplasm and nucleus was increased(P<0.05).Compared with the siRNA NC group,the protein level of NF-κB p65 in the nucleus and cytoplasm and the expression level of miR-425-5p in the NF-κB siRNA group were significantly decreased(all P<0.01).Under LPS induction,the activity of the miR-425-5p promoter with two binding sites(2-Luc/3-Luc)was significantly higher than that with a single binding site(3-Luc)(P<0.01).The binding ability of NF-κB to miR-425-5p promoter sequence at

关 键 词：脂多糖类 NF-ΚB miR-425-5p 胰高糖素样肽-1 

分 类 号：R333[医药卫生—人体生理学]