核转录因子NF-κB调节人肾小管上皮细胞SIGIRR表达机制的研究  

Regulation of SIGIRR expression mechanism in human renal tubular epithelial cells by nuclear transcription factor NF-κB

作　　者：蒋克国 朱莉 赵文曼 石瑞 王德光[1] Jiang Keguo;Zhu Li;Zhao Wenman;Shi Rui;Wang Deguang(Dept of Nephrology,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601;Dept of Nephrology,The Third Affiliated Hospital of Anhui Medical University,Hefei 230001)

机构地区：[1]安徽医科大学第二附属医院肾脏内科,合肥230601 [2]安徽医科大学第三附属医院肾脏内科,合肥230001

出　　处：《安徽医科大学学报》2023年第3期358-365,共8页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金(编号:2008085MH244);安徽医科大学第二附属医院国家自然科学基金面上孵育计划项目(编号:2020GMFY04);安徽医科大学第二附属医院临床研究培育计划项目(编号:2020LCZD01);安徽医科大学校科学研究基金(编号:2019xkj140)。

摘　　要：目的探讨人肾小管上皮细胞(HKC)内核转录因子NF-κB(NF-κB)反馈调控单免疫球蛋白白介素1受体相关蛋白(SIGIRR)表达的分子机制。方法分子克隆法构建pLNCX2-G418-SIGIRR过表达载体,经PT67细胞包装后感染HKC细胞,构建SIGIRR过表达细胞和对照细胞。使用IL-1β诱导,Western blot验证过表达SIGIRR可抑制NF-κB活化。使用NF-κB阻断剂和干涉NF-κB活性后,免疫荧光法验证活化的NF-κB可调控SIGIRR表达。在线工具预测SIGIRR启动子区存在NF-κB结合位点。分子克隆法从人基因组DNA内获取含有结合位点的SIGIRR启动子序列,连接至荧光素酶载体pGL3-Luc,构建pGL3-Luc-SIGIRR,并突变结合位点。使用荧光素酶报告基因实验和染色质免疫沉淀技术(ChIP)共同验证活化的NF-κB可结合在SIGIRR启动子区调控SIGIRR基因的表达。结果构建的pLNCX2-G418-SIGIRR逆转录病毒载体经酶切、测序验证后对比SIGIRR基因编码序列完全一致,重组体和对照载体经病毒包装后转入HKC细胞后成功构建HKC/SIGIRR实验组和HKC/Co对照组细胞系,在mRNA和蛋白水平的SIGIRR表达差异有统计学意义(P<0.05,P<0.001)。过表达SIGIRR细胞组相比对照组细胞可以减少IL-1β诱导的NF-κB的活化(P<0.001),抑制NF-κB活化和干涉NF-κB表达后,SIGIRR的表达出现下调。提取人基因组DNA后,分子克隆法获得SIGIRR目的启动子序列连接至载体,成功构建pGL3-Luc-SIGIRR荧光素酶载体并定点突变载体,经酶切和测序验证与目的序列完全一致。通过荧光素酶报告基因实验和CHIP实验证实NF-κB可结合SIGIRR启动子区域,调控SIGIRR表达。结论HKC细胞中SIGIRR可以影响NF-κB的活化,活化的NF-κB可以结合到SIGIRR的启动子区域,调控SIGIRR的基因表达变化,形成一个反馈体系,控制NF-κB的过度活化。Objective To investigate the molecular mechanisms underlying the feedback regulation of single immunoglobin interleukin-1 related receptor(SIGIRR)expression by the nuclear factor kappa-κB(NF-κB)in human renal tubular epithelial cells(HKC).Methods The pLNCX2-G418-SIGIRR overexpression vector was constructed by molecular cloning,and the SIGIRR overexpression cells and control cells were constructed by infecting HKC cells after packaging with PT67 cells.Using IL-1βinduction,Western blot verified that overexpression of SIGIRR inhibited NF-κB activation.After using NF-κB blocker and interfering with NF-κB activity,immunofluorescence assay verified that activated NF-κB regulated SIGIRR expression.Online tools predicted the presence of NF-κB binding sites in the SIGIRR promoter region.The SIGIRR promoter sequence containing the binding site was obtained from within human genomic DNA by molecular cloning,ligated to the luciferase vector pGL3-Luc,constructed pGL3-Luc-SIGIRR,and mutated the binding site.The luciferase reporter gene assay and chromatin immunoprecipitation technique(ChIP)were used to jointly verify that activated NF-κB could bind to the SIGIRR promoter region to regulate SIGIRR gene expression.Results The results showed that the constructed pLNCX2-G418-SIGIRR retroviral vector was verified by enzymatic digestion and sequencing to be identical to the coding sequence of the SIGIRR gene for comparison,the recombinant and control vectors were transferred into HKC cells after viral packaging,and the HKC/SIGIRR experimental and HKC/Co control cell lines were successfully constructed at the mRNA and protein levels of SIGIRR expression differences were statistically significant(P<0.05,P<0.001).Overexpression of SIGIRR cell groups reduced IL-1β-induced NF-κB activation compared to control cells(P<0.001).SIGIRR expression was downregulated after inhibition of NF-κB activation and interference with NF-κB expression.After extracting human genomic DNA,the SIGIRR target promoter sequence was obtained by molec

关 键 词：人肾小管上皮细胞 TLR样受体 核转录因子 单免疫球蛋白白介素1受体相关蛋白 

分 类 号：R692.6[医药卫生—泌尿科学]