摘要
目的探讨miR-199a对高糖刺激人肾小管上皮细胞间质转化(EMT)的影响。方法体外培养人肾小管上皮细胞,按不同糖浓度培养分为低糖组(LG)、高渗组(HM)和高糖组(HG),按是否转染miR-199a抑制剂(miR-199ai)分为miR-199a抑制组(miR-199ai组)、阴性对照组(miR-iNC组)和脂质体组(Mock组),按照是否转染沉默缺氧诱导因子-1α(si-HIF-1α)及miR-199ai分为si-HIF-1α组、miR-199ai组、miR-199ai+si-HIF-1α组及阴性对照组(NC组),采用实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹法(Westernblot)检测miR-199a、HIF-1α、纤连蛋白(FN)、α-平滑肌肌动蛋白(α-SMA)在不同组细胞中的表达情况。采用双荧光素酶靶标实验验证miR-199a和HIF-1α基因的靶向关系。结果HG组miR-199amRNA表达水平明显低于LG组及HM组(均P<0.01),HIF-1α、FN及α-SMA的mRNA及蛋白表达水平明显高于LG组及HM组(均P<0.01)。miR-199ai组HIF-1α、FN、α-SMA的mRNA及蛋白表达水平均明显高于miR-iNC组和Mock组(均P<0.01)。通过TargetScan和miRanda进行靶基因预测,发现miR-199a可靶向结合HIF-1α的3'-UTR。双荧光素酶报告结果显示,抑制miR-199a可导致荧光素酶活性明显升高(P<0.01),而HIF-1α的3°-UTR突变后,荧光素酶活性无明显变化(P>0.05)。转染si-HIF-1α后,si-HIF-1α组HIF-1α、FN、α-SMA的mRNA及蛋白表达水平均明显低于NC组(均P<0.05);转染miR-199ai后,miR-199ai组HIF-1α、FN、α-SMA的mRNA及蛋白表达水平均明显高于NC组(均P<0.05);而si-HIF-1α与miR-199ai共转染时,可以恢复si-HIF-1α或miR-199ai单独转染导致的HIF-1α、FN、α-SMA的mRNA及蛋白表达变化。结论miR-199a可通过靶向调控HIF-1α减轻高糖诱导的人肾小管上皮细胞EMT。
Objective To investigate the effect of miR-199a on interstitial transdifferentiation(EMT)of human renal tubular epithelial cells stimulated by high glucose.Methods Human renal tubular epithelial cells were cultured in vitro and divided into low glycemic group(LG),high osmotic group(HM)and high glycemic group(HG)according to different glucose concentrations.They were divided into miR-199a inhibition group(miR-199ai group),negative control group(miR-INC group)and liposome group(Mock group)according to whether they were transfected with miR-199a inhibitors(miR199ai).They were divided into si-hypoxia-inducible factor-lα(si-HIF-1α)group,miR-199ai group,miR-199ai+si-HIF-1αgroup and negative control group(NC group)according to whether they converted to si-HIF-lαand miR-199ai.The expressions of miR-199a,HIF-1α,fibronectin(FN)andα-smooth muscle actin(α-SMA)in different groups of cells were detected by real-time quantitative fluorescent polymerase chain reaction(qRT-PCR)and Western blot.Dual luciferase target assay was used to verify the targeting relationship between miR-199a and HIF-1αgene.Results The mRNA expression level of miR-199a in the HG group was significantly lower than that in the LG group and the HM group(all P<0.01),and the mRNA and protein expression levels of HIF-1α,FN andα-SMA in Hg group were significantly higher than those in LG group and HM group(all P<0.01).The mRNA and protein expression levels of HIF-1α,FN andα-SMA in the miR-199ai group were significantly higher than those in the miR-iNC group and the Mock group(all P<0.O1).Target gene prediction by TargetScan and miRanda showed that miR-199a could target the 3'-untranslated region(3'-UTR)that binds HIF-1α.Dual luciferase reported results:Inhibition of miR-199a resulted in a significant increase in luciferase activity(P<0.01),but there was no significant change in luciferase activity after 3'-UTR mutation of HIF-1α(P>O.05).After transfection with si-HIF-1α,the mRNA and protein expression levels of HIF-Iα,FN andα-SMA in si-HIF-lαgroup were significantly lower than those in NC group(all P<0.05).After transfection with miR-199ai,the mRNA and protein expression levels of HIF-lα,FN andα-SMA in miR-199ai group were significantly higher than those in NC group(all P<0.05).However,when co-transfected with si-HIF-1αand miR-199ai,the expression levels of mRNA and protein in miR-199AI group were significantly higher than those in NC group(all P<0.05).The mRNA and protein expression changes of HIF-lα,FN andα-SMA induced by transfection of si-HIF-lαor miR-199ai alone can be recovered.Conclusions miR-199a can alleviate hyperglycemia-induced EMT in human renal tubular epithelial cells through targeted regulation of HIF-1α.
作者
蒋科威
乐颖
张莹
赵颖
薛萌
Jiang Kewei;Le Ying;Zhang Ying;Zhao Ying;Xue Meng(Department of Geriatrics,Shenzhen People's Hospital,the Second Affiliated Hospital of Jinan University,the First Affiliated Hospital of Southern University of Science and Technology,Shenzhen 518020,China;Department of Endocrinology and Metabolism,Shenzhen People's Hospital,the Second Affiliated Hospital of Jinan University,the First Afiliated Hospital of Southern University of Science and Technology,Shenzhen 518020,China)
出处
《中国医师杂志》
CAS
2024年第10期1477-1482,共6页
Journal of Chinese Physician
基金
国家自然科学基金(81900378)
广东省医学科研基金(2021112622548309)。
关键词
缺氧诱导因子1
Α亚基
糖尿病肾病
miR-199a
肾小管上皮细胞
皮-间质转化
Hypoxia-inducible factor 1,alpha subunit
Diabetic nephropathies
miR-199a
Renal tubular epithelial cell
Epithelial-mesenchymal transition