微小RNA-325对肾小管上皮细胞缺氧复氧损伤后凋亡的影响

Effect of microRNA-325 on renal tubular epithelial apoptosis after hypoxia-reoxygenation injury

目的探讨微小RNA-325(miRNA-325)在肾小管上皮细胞缺氧复氧(H/R)损伤模型中的作用及其机制。方法选取人肾小管上皮细胞HK-2,建立适当的肾小管上皮细胞H/R模型(缺氧24 h复氧2 h)。将HK-2细胞分为对照组、H/R组、H/R+抑制剂(inhibitor)组和H/R+抑制剂阴性对照(inNC),转染miRNA-325 inhibitor和inNC后进行缺氧复氧处理,然后进行后续实验。用流式细胞术检测各组细胞凋亡率。利用蛋白质印迹法(Western blot)检测HK2细胞在H/R后活化半胱氨酰天冬氨酸特异性蛋白酶-3(cleaved Caspase-3)、B细胞淋巴瘤-2(bcl-2)、bcl-2相关X蛋白(bax)和X连锁凋亡抑制蛋白(XIAP)等的表达水平。利用实时荧光定量聚合酶链式反应(RT-qPCR)检测HK2细胞中miRNA-325、Caspase-3和XIAP mRNA表达水平。使用双荧光素酶报告基因实验检测miRNA-325与XIAP 3’端非编码区(3’UTR)之间的关系。组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果H/R组细胞凋亡率高于对照组[(12.11±1.65)%比(3.88±0.24)%,t=8.311,P<0.05];H/R+Inhibitor组细胞凋亡率低于H/R组[(7.60±3.07)%比(12.11±1.65)%,t=4.557,P<0.05]。Western blot结果显示,实验组Cleaved Caspase-3[(0.50±0.02)、(0.63±0.01)、(0.61±0.02)比(0.41±0.01),F=212.095,P<0.05]和bax[(0.53±0.02)、(0.63±0.01)、(0.64±0.04)比(0.41±0.02),F=105.138,P<0.05]蛋白表达量显著高于对照组,bcl-2[(0.57±0.02)、(0.52±0.02)、(0.49±0.01)比(0.62±0.02),F=89.498,P<0.05]和XIAP[(0.29±0.01)、(0.24±0.03)、(0.24±0.01)比(0.33±0.03),F=27.575,P<0.05]表达量显著低于对照组;H/R+Inhibitor组Cleaved Caspase-3[(0.50±0.02)比(0.61±0.02),t=11.437,P<0.05]和bax[(0.53±0.02)比(0.64±0.04),t=7.253,P<0.05]的表达低于H/R组,bcl-2[(0.57±0.02)比(0.49±0.01),t=9.471,P<0.05]和XIAP[(0.29±0.01)比(0.24±0.01),t=4.231,P<0.05]的表达量高于H/R组,差异均有统计学意义。qRT-PCR结果显示,实验组miRNA-325[(7.27±0.30)、(4.66±0.21)、(7.11±0.25)比(1.05±0.13),F=1453.045,P<0.05]和Caspase-3 mRNA[(1.52±0.05)、(1.26±0.06)、(1.53±0.05)比(1.00±0.01),F=241.375,P<0.05]表达水平显著高于对照组,而XIAP mRNA[(0.38±0.01)、(0.76±0.02)、(0.39±0.02)比(1.01±0.02),F=2569.583,P<0.05]表达低于对照组;H/R+Inhibitor组miRNA-325[(4.66±0.21)比(7.27±0.30),t=24.236,P<0.05]和Caspase-3 mRNA[(1.26±0.06)比(1.52±0.05),t=11.451,P<0.05]低于H/R组,而XIAP mRNA[(0.76±0.02)比(0.38±0.01),t=45.678,P<0.05]高于H/R组,差异均有统计学意义。荧光素酶活性检测表明共转染miRNA-325的野生型组酶活性低于对照组[(0.69±0.03)比(1.01±0.02),t=22.809,P<0.05],而突变型组酶活性无明显变化[(1.04±0.11)比(0.99±0.06),t=0.983,P>0.05]。结论XIAP是miRNA-325的靶基因之一,抑制miRNA-325可以下调凋亡蛋白的表达,降低细胞凋亡率,从而保护肾小管上皮细胞。

Objective To study the pro-apoptotic effect of microRNA-325(miRNA-325)in a model of hypoxia reoxygenation(H/R)of renal tubular epithelial cells and the mechansim.Methods The renal tubular epithelial cell line HK2(purchased from China Center for Type Culture Collection)was selected and an appropriate H/R model of renal tubular epithelial cells(24 h hypoxia and 2 h reoxygenation)was established.HK2 cells were divided into control group,H/R group,H/R+inhibitor group and H/R+inhibitor negative control(inNC)group.The HK2 cells were transfected with miRNA-325 inhibitor or inNC,and then subjected to hypoxia and reoxygenation.Flow cytometry was used to detect cell apoptosis rate in each group.The expression levels of cleaved Caspase-3,B-cell lymphoma-2(bcl-2),bcl-2-associated X protein(bax),and X-linked inhibitor of apoptosis protein(XIAP)in HK2 cells after H/R were detected by immunoblotting(Western blotting).Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression levels of miRNA-325,Caspase-3,and XIAP mRNA in HK2 cells.The relationship between miRNA-325 and XIAP 3'untranslated regions(3'UTR)was examined using a dual luciferase reporter gene assay.The Student t-test was used between two groups.ANOVA test was adopted among multiple groups.Results The results of flow cytometry showed that the apoptosis rate in the H/R group was significantly higher than that in the control group[(12.11±1.65)%vs.(3.88±0.24)%,t=8.311,P<0.05];the apoptosis rate in the H/R+inhibitor group was significantly lowered in comparison with the H/R group[(12.11±1.65)%vs.(7.60±3.07)%,t=4.557,P<0.05].Western blotting results demonstrated that cleaved Caspase-3(0.50±0.02,0.63±0.01,0.61±0.02 vs.0.41±0.01,F=212.095,P<0.05)and bax(0.53±0.02,0.63±0.01,0.64±0.04 vs.0.41±0.02,F=105.138,P<0.05)proteins in experimental groups were significantly higher than in control group,and bcl-2(0.57±0.02,0.52±0.02,0.49±0.01 vs.0.62±0.02,F=89.498,P<0.05)and XIAP(0.29±0.01,0.24±0.03,0.24±0.01 vs.0.33±0.03,F=27.575,P<0.05)were significantly lowered.Cleaved Caspase-3(0.50±0.02 vs.0.61±0.02,t=11.437,P<0.05)and bax(0.53±0.02 vs.0.64±0.04,t=7.253,P<0.05)in H/R+inhibitor group were significantly decreased as compared with those the H/R group,and bcl-2(0.57±0.02 vs.0.49±0.01,t=9.471,P<0.05)and XIAP(0.29±0.01 vs.0.24±0.01,t=4.231,P<0.05)significantly increased compared to H/R group.The qRT-PCR results illustrated that miRNA-325(7.27±0.30,4.66±0.21,7.11±0.25 vs.1.05±0.13,F=1453.045,P<0.05)and Caspase-3 mRNA(1.52±0.05,1.26±0.06,1.53±0.05 vs.1.00±0.01,F=241.375,P<0.05)in experimental groups were significantly higher than those in the control group,and XIAP mRNA(0.38±0.01,0.76±0.02,0.39±0.02 vs.1.01±0.02,F=2569.583,P<0.05)was significantly was significantly lowered as compared with the control group.The H/R+inhibitor group decreasingly expressed miRNA-325(4.66±0.21 vs.7.27±0.30,t=24.236,P<0.05)and Caspase-3 mRNA(1.26±0.06 vs.1.52±0.05,t=11.451,P<0.05),and increasingly expressed XIAP mRNA(0.76±0.02 vs.0.38±0.01,t=45.678,P<0.05)as compared with the H/R group.The luciferase activity assay indicated that the luciferase activity in wild group co-transfected with miRNA-325 was significantly lower than that in the control group(0.69±0.03 vs.1.01±0.02,t=22.809,P<0.05),and the oppsite in mutant group(1.04±0.11 vs.0.99±0.06,t=0.983,P>0.05).Conclusion XIAP is one of the target genes of miRNA-325.Inhibition of miRNA-325 can down-regulate the expression of apoptotic proteins and reduce the apoptosis rate,thereby protecting renal tubular epithelial cells.