摘要
目的研究沉默miR-141表达对肾小管上皮细胞损伤的影响,并探讨其可能的分子机制。方法体外培养肾小管上皮细胞HK-2并分为:正常(5.5 mmol/L D-葡萄糖)组、高渗组、高糖(30 mmol/L D-葡萄糖)组、阴性对照+高糖组(转染NC inhibitor质粒)和si-miR-141+高糖组(转染miR-141 inhibitor)。miR-141和Sirt1 mRNA相对表达量用实时荧光定量聚合酶链反应(qRT-PCR)进行检测。DCFH-DA荧光染色、CCK-8法和流式细胞术分别检测细胞中活性氧(ROS)生成量、细胞存活率和凋亡率。Sirt1/Nrf2信号通路相关蛋白的表达量用Western blot法检测。荧光素酶报告基因实验验证miR-141和Sirt1 mRNA之间的靶向关系。结果①与正常组相比,转染si-miR-141后,细胞miR-141相对表达量降低(1.00±0.03 vs 0.52±0.06),差异有统计学意义(F=278.104,P<0.05);②与正常组[DCFH-DA荧光强度(7.18±0.59)%]相比,高糖组[DCFH-DA荧光强度(84.95±3.21)%]细胞ROS水平明显升高,而与高糖组相比,si-miR-141+高糖组[DCFH-DA荧光强度(45.10±4.29)%]细胞ROS水平明显降低,差异有统计学意义(均P<0.05);③与正常组(5.13%±0.78)%和高渗组(5.96±0.81)%相比,高糖组(32.76±2.95)%细胞凋亡率显著增加,而si-miR-141+高糖组(17.54%±2.79)%细胞凋亡率则较高糖组和阴性对照+高糖组(33.40%±3.14)%显著降低,差异有统计学意义(F=221.419,P<0.05);④与正常组(100±3.98)%和高渗组(95.68±5.14)%相比,高糖组(67.24±5.18)%HK-2细胞存活率显著降低;同时,与高糖组(67.24±5.18)%和阴性对照+高糖组(65.33±3.10)%相比,si-miR-141+高糖组(83.55±5.10)%细胞存活率显著增加,差异有统计学意义(F=93.008,P<0.05);⑤与正常组和高渗组相比,高糖组Cleaved caspase 3蛋白表达量升高,同时Sirt1、Nrf2和HO-1蛋白表达量下调;然而,与高糖组相比,si-miR-141+高糖组中Cleaved caspase 3蛋白表达量降低,同时Sirt1、Nrf2和HO-1蛋白表达量升高,差异有统计学意义(均P<0.05)。结论下调miR-141表达能通�
Objective To investigate the effect of miR-141 down-regulation on the damage of renal tubular epithelial cell,and further to explore its mechanism.Methods The renal tubular epithelial cell line HK-2 cells were divided into normal(5.5 mmol/L D-glucose)group,hypertonic group,high glucose(30 mmol/L D-glucose)group,negative control+high glucose group(transfected with NC inhibitor vector)and si-miR-141+high glucose group(transfected with miR-141 inhibitor vector).Real time quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative expression of miR-141.The production of reactive oxygen species(ROS),cell viability and apoptosis were detected by DCFH-DA fluorescence staining,CCK-8 method and flow cytometry.The expression of Sirt1/Nrf2 signaling pathway related proteins was detected by Western blot.Luciferase reporter gene assay verified the targeting relationship between miR-141 and Sirt1 mRNA.Results①Compared with the normal group,after transfection with si-miR-141,the relative expression of miR-141 decreased(1.00±0.03 vs 0.52±0.06),the difference was statistically significant(F=278.104,P<0.05);②Compared with the normal group[DCFH-DA fluorescence intensity(7.18±0.59)%],the high glucose group[DCFH-DA fluorescence intensity(84.95±3.21)%]cell ROS level was significantly increased,and compared with the high glucose group[DCFH-DA fluorescence intensity(84.95±3.21)%]Compared with the si-miR-141+high glucose group[DCFH-DA fluorescence intensity(45.10±4.29)%]cell ROS levels were significantly reduced,the difference was statistically significant(all P<0.05);③compared with the normal group(5.13%±0.78)%Compared with the hypertonic group(5.96±0.81)%,the high glucose group(32.76±2.95)%cell apoptosis rate was significantly increased,while the si-miR-141+high glucose group(17.54%±2.79)%apoptosis rate was significantly lower in the higher glucose group and the negative control+high glucose group(33.40%±3.14)%,the difference was statistically significant(F=221.419,P<0.05);④compared with the normal
作者
姚建平
宁建文
钟晓敬
金月萍
邱蔚
Yao Jianping;Ning Jianwen;Zhong Xiaojing;Jin Yueping;Qiu Wei(Department of Endocrinology,Huzhou Central Hospital,Huzhou Normal University Affiliated Central Hospital,Medical College of Zhejiang University,Huzhou 313000,China)
出处
《中华内分泌外科杂志》
CAS
2021年第3期225-229,共5页
Chinese Journal of Endocrine Surgery
基金
浙江省科技计划项目(2016C37119)
浙江省湖州市科技局公益性技术应用研究项目(2018GYB24)。
