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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n t检验，多组间比较采用单因素方差分析。n 结果:H/R组细胞凋亡率高于对照组[(12.11±1.65)%比(3.88±0.24)%，n t＝8.311，n 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)%，n t＝4.557，n 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)，n F＝212.095，n P<0.05]和bax[(0.53±0.02)、(0.63±0.01)、(0.64±0.04)比(0.41±0.02)，n F＝105.138，n 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)，n F＝89.498，n P<0.05]和XIAP[(0.29±0.01)、(0.24±0.03)、(0.24±0.01)比(0.33±0.03)，n F＝27.575，n 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)，n t＝11.437，n P<0.05]和bax[(0.53±0.02)比(0.64±0.04)，n t＝7.253，n P<0.05]的表达低于H/R组，bcl-2[(0.57±0.02)比(0.49±0.01)，n t＝9.471，n P<0.05]和XIAP[(0.29±0.01)比(0.24±0.01)，n t＝4.231，n 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)，n F＝1453.045，n P<0.05]和Caspase-3 mRNA[(1.52±0.05)、(1.26±0.06)、(1.53±0.05)比(1.00±0.01)，n F＝241.375，n 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)，n F＝2569.583，n 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)，n t＝24.236，n P<0.05]和Caspase-3 mRNA[(1.26±0.06)比(1.52±0.05)，n t＝11.451，n P<0.05]低于H/R组，而XIAP mRNA[(0.76±0.02)比(0.38±0.01)，n t＝45.678，n 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)，n t＝22.809，n P0.05]。n 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 n t-test was used between two groups. ANOVA test was adopted among multiple groups.n 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)%, n t=8.311, n 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)%,n t=4.557, n 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,n F=212.095, n P<0.05) and bax (0.53±0.02, 0.63±0.01, 0.64±0.04 vs. 0.41±0.02,n F=105.138, n 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,n F=89.498, n P<0.05) and XIAP (0.29±0.01, 0.24±0.03, 0.24±0.01 vs. 0.33±0.03,n F=27.575, n P<0.05) were significantly lowered. Cleaved Caspase-3 (0.50±0.02 vs. 0.61±0.02,n t=11.437, n P<0.05) and bax (0.53±0.02 vs. 0.64±0.04,n t=7.253, n 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,n t=9.471, n P<0.05) and XIAP (0.29±0.01 vs. 0.24±0.01,n t=4.231, n 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,n F=1 453.045, n P<0.05) and Caspase-3 mRNA (1.52±0.05, 1.26±0.06, 1.53±0.05 vs. 1.00±0.01,n F=241.375, n 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,n F=2 569.583, n 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,n t=24.236, n P<0.05) and Caspase-3 mRNA (1.26±0.06 vs. 1.52±0.05,n t=11.451, n P<0.05), and increasingly expressed XIAP mRNA (0.76±0.02 vs. 0.38±0.01,n t=45.678, n 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,n t=22.809, n P0.05).n 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.