MiR-21靶向调控Mfn2对缺血再灌注损伤肾小管上皮NRK-52E细胞自噬及凋亡的影响

Effects of miR-21 Targeting Mfn2 on Autophagy and Apoptosis of Renal Tubular Epithelial NRK-52E Cells after Ischemia-reperfusion Injury

目的:探讨mi R-21对缺血再灌注损伤肾小管上皮细胞自噬及凋亡的影响及其与线粒体融合素2(mitochondria fusion protein mitofusin2,Mfn2)的靶向关系。方法:将大鼠近端肾小管上皮细胞株NRK-52E细胞按处理方式不同分组:I/R+control mimics组(转染control mimics后缺氧3 h/复氧3 h),I/R+mi R-21mimics组(转染mi R-21mimics后缺氧3 h/复氧3 h),I/R组(缺氧3 h/复氧3 h)及对照组(正常培养)。选取30只Sprague-Dawley(SD)大鼠,随机分为假手术组、缺血再灌注模型组(I/R组)。取大鼠肾组织进行HE染色,自动生化分析仪检测大鼠血清尿素氮(BUN)、肌酐(Cr),四甲基偶氮唑盐比色法(MTT)检测细胞增殖能力,TUNEL法检测细胞凋亡,实时荧光定量PCR检测细胞自噬和凋亡相关基因LC3-Ⅱ、LC3-Ⅰ、Beclin1、Bcl-2、Bax及Mfn2 m RNA表达,Western blot法检测细胞自噬和凋亡相关蛋白的表达,荧光素酶实验验证mi R-21与Mfn2的靶向关系。结果:Sham组大鼠血清BUN、Cr水平,大鼠肾组织细胞凋亡率高于I/R组(P<0.05)。I/R组大鼠肾组织肾小管结构紊乱,大量炎症细胞浸润。Sham组大鼠肾组织mi R-21水平高于I/R组(P<0.05)。48和72 h时,I/R+mi R-21 mimics组细胞活力明显低于I/R+control mimics组,I/R组及对照组(P<0.05),I/R组细胞活力低于对照组(P<0.05)。I/R+mi R-21mimics组凋亡率显著高于I/R+control mimics组,I/R组及对照组(P<0.05),I/R组凋亡率显著高于对照组(P<0.05)。与对照组比较,I/R组细胞Beclin1、LC3-Ⅱ/LC3-Ⅰ、Bax蛋白及基因m RNA表达量升高,Bcl-2蛋白及基因m RNA表达量降低(P<0.05);与I/R组比较,I/R+mi R-21mimics组细胞Beclin1、LC3-Ⅱ/LC3-Ⅰ、Bax蛋白及基因m RNA表达量升高,Bcl-2蛋白及基因m RNA表达量降低(P<0.05)。mi R-21与Mfn2具有靶向关系。结论:mi R-21可靶向Mfn2促进肾缺血再灌注损伤引起的凋亡及自噬。

Objective:To investigate the effect of miR-21 on autophagy and apoptosis of renal tubular epithelial cells after ischemia-reperfusion injury and its targeting relationship with mitochondrial fusion protein 2(Mfn2).Methods:NRK-52 E cells were divided into three groups:I/R+control mimics group(hypoxia 3 h/reoxygenation 3 h after transfection of control mimics),I/R+miR-21 mimics group(hypoxia 3 h/reoxygenation 3 h after transfection of miR-21 mimics),I/R group(hypoxia 3 h/reoxygenation 3 h)and control group(normal culture).Thirty Sprague Dawley(SD)rats were randomly divided into sham operation group and I/R group.Renal tissue was stained with HE.Serum urea nitrogen(BUN)and creatinine(CR)were detected by automatic biochemical analyzer.Cell proliferation was detected by MTT assay.Apoptosis was detected by TUNEL assay.Autophagy and apoptosis related genes LC3-Ⅱ,LC3-Ⅰ,Beclin1,Bcl-2,Bax and Mfn2 m RNA expression were detected by real-time fluorescent quantitative PCR.Western blot was used to detect the autophagy and apoptosis related proteins.The targeting relationship between miR-21 and Mfn2 was verified by luciferase assay.Results:The levels of serum BUN,Cr and the apoptosis rate of renal cells in Sham group were higher than those in I/R group(P<0.05).In I/R group,the structure of renal tubules was disordered and a large number of inflammatory cells infiltrated.The level of miR-21 in Sham group was higher than that in I/R group(P<0.05).At 48 and 72 h,the cell viability of I/R+miR-21 mimics group was significantly lower than that of I/R+control mimics group,I/R group and control group(P<0.05),and the cell viability of I/R group was lower than that of control group(P<0.05).The apoptosis rate of I/R+miR-21 mimics group was significantly higher than that of I/R+control mim ics group,I/R group and control group(P<0.05),and the apoptosis rate of I/R group was significantly higher than that of control group(P<0.05).Compared with the control group,the expressions of Beclin1,LC3-Ⅱ/LC3-Ⅰ,Bax protein and gene m RNA were increased and the expressions of Bcl-2 protein and gene m RNA were decreased in I/R group(P<0.05).Compared with I/R group,the expressions of Beclin1,LC3-Ⅱ/LC3-Ⅰ,Bax protein and gene m RNA were increased and the expressions of Bcl-2 protein and gene m RNA were decreased in I/R+miR-21 mimics group(P<0.05).Mi R-21 has a targeted relationship with Mfn2.Conclusion:miR-21 can target Mfn2 and promote apoptosis and autophagy induced by renal ischemia-reperfusion injury.