摘要
目的:探索LncRNA TUG1在糖尿病肾病(DN)大鼠肾纤维化中的作用及其潜在分子机制。方法:应用链脲佐菌素构建DN大鼠模型,LncRNA表达谱芯片筛选DN大鼠肾脏组织中LncRNA的差异表达。应用慢病毒试验过表达LncRNA TUG1,苏木精-伊红(HE)染色检测过表达LncRNA TUG1对DN大鼠肾脏组织形态学的改变;实时荧光定量逆转录-聚合酶链反应(RT-qPCR)检测过表达LncRNA TUG1对miR-21及纤维连接蛋白(FN)、人Ⅳ型胶原(Col-4)和转化生长因子β1(TGFβ1)mRNA表达水平的影响;Western blot检测FN、Col-4、TGFβ1、磷酸酯酶与张力蛋白同源物(PTEN)、磷酸化磷脂肌醇3激酶(p-PI3K)及磷酸化蛋白激酶(p-Akt)蛋白表达水平。应用生物信息软件预测miR-21与LncRNA TUG1的作用关系,双荧光素酶报告基因实验验证miR-21与LncRNA TUG1的相互作用关系。构建高糖诱导的肾小球系膜HBZY-1细胞模型,应用慢病毒试验方法过表达LncRNA TUG1或应用miR-21 mimic处理细胞,采用CCK-8试验检测他们对细胞增殖的影响,使用RT-qPCR实验检测其对HBZY-1细胞FN、Col-4、TGFβ1 mRNA表达水平的影响。结果:DN大鼠肾脏组织中LncRNA TUG1的表达水平显著降低。过表达LncRNA TUG1抑制DN大鼠FN、Col-4、TGFβ1 mRNA以及蛋白表达水平,肾组织病理形态学明显改善。双荧光素酶报告基因实验结果显示,LncRNA TUG1通过直接靶向方式与miR-21结合。过表达LncRNA TUG1能够抑制miR-21表达,上调PTEN并可能抑制磷脂酰肌醇3-激酶/蛋白激酶(PI3K/Akt)信号通路。过表达LncRNA TUG1抑制HBZY-1细胞增殖,抑制FN、Col-4、TGFβ1 mRNA在细胞中的表达水平;应用miR-21 mimic处理能够逆转LncRNA TUG1过表达产生的增殖抑制和纤维化抑制效应。结论:LncRNA TUG1靶向调节miR-21/PTEN轴抑制DN大鼠肾纤维化。
Objective:To explore the effects of LncRNA TUG1 on renal fibrosis in diabetic nephropathy(DN)rats and its potential molecular mechanism.Methods:The DN rat model was constructed by streptozotocin.LncRNA expression profiling was used to explore differentially expressed LncRNA in kidney tissues of DN rats.Lentivirus assay was applied to overexpress LncRNA TUG1 in DN rats.Hematoxylin-eosin(HE)staining was used to detect the histomorphological changes of overexpressed LncRNA TUG1 on DN rat kidney tissues.The effect of LncRNA TUG1 overexpression on mRNA expression levels of miR-21,fibronectin(FN),collagen typeⅣ(Col-4)and transforming growth factorβ1(TGFβ1)were detected by real time quantitative polymerase chain reaction(RT-qPCR).The protein expression levels of FN,Col-4,TGFβ1,phosphatase and tensin homolog(PTEN),phosphatidylin-ositol-3-kinase(p-PI3K)and Phosphorylated-Akt(p-Akt)proteins was assayed by Western blot assay.Bioinformatics software was applied to predict the interaction of miR-21 and LncRNA TUG1,and luciferin reporter assay method was used to verify the interaction between miR-21 and LncRNA TUG1.A high glucose-induced glomerular thylakoid HBZY-1 cell model was constructed.In HBZY-1 cells,LncRNA TUG1 was overexpressed using a lentivirus assay,cells were treated with miR-21 mimic,and then CCK-8 assay was used to detect their effect on cell proliferation.Their effect on the expression levels of FN,Col-4,and TGFβ1 mRNA was subsequently detected using RT-qPCR.Results:The expression level of LncRNA TUG1 was significantly reduced in renal tissue of DN rats.Overexpression of LncRNA TUG1 inhibited the mRNA and protein expression levels of FN,Col-4 and TGFβ1 in DN rats,and the pathological morphology of renal tissue was significantly improved.The results of dual luciferase reporter assay showed that LncRNA TUG1 bound to miR-21 through a direct targeting manner.Overexpression of LncRNA TUG1 inhibited the expression of miR-21,further upregulated PTEN and may inhibit the phosphatidylinositol 3-kinase/protein kina
作者
陈生晓
甘艳
邝才花
张蕾
符大天
CHEN Shengxiao;GAN Yan;KUANG Caihua;ZHANG Lei;FU Datian(Department of Nephrology and Rheumatology,No.928 Hospital,Joint Logistics Support Force of the Chinese People s Liberation Army,Haikou 571159,China;Department of Pharmacy,Hainan Women and Children s Medical Center,Haikou 571159,China;Department of Pharmacy,the First Affiliated Hospital of Hainan Medical College,Haikou 570102,China)
出处
《东南大学学报（医学版）》
CAS
2023年第2期218-227,共10页
Journal of Southeast University(Medical Science Edition)
基金
海南省卫生健康行业科研项目(20A200167)。
