摘要
目的 探讨微小RNA-146a-5p(miR-146a-5p)通过调控高迁移率族蛋白A2(HMGA2)对高糖诱导的肾小管上皮细胞损伤的影响。方法 体外培养人肾小管上皮细胞HK-2,按不同处理方法将细胞分为正常对照组(Con组)、高糖组(HG组)、HG+miR-146a-5p组、HG+si-HMGA2组、HG+miR-146a-5p+pcDNA-HMGA2组、miR-NC组、miR-146a-5p组、anti-miR-NC组和anti-miR-146a-5p组。采用实时荧光定量聚合酶链反应(qRT-PCR)检测各组细胞中miR-146a-5p、HMGA2 mRNA表达水平;采用双荧光素酶报告实验验证miR-146a-5p与HMGA2的靶向关系;采用蛋白质印迹法(Western blot)检测各组细胞中活化半胱氨酸天冬氨酸蛋白酶3(cleaved-caspase3)、活化半胱氨酸天冬氨酸蛋白酶9(cleaved-caspase9)、HMGA2蛋白表达水平;采用流式细胞术测定各组细胞凋亡率;采用酶联免疫吸附试验(ELISA)检测各组细胞中炎症因子[白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)]水平;检测各组细胞中氧化应激指标[过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、丙二醛(MDA)]水平。结果 HMGA2的3′-非翻译区(3′-UTR)序列中存在与miR-146a-5p互补的核苷酸序列;miR-146a-5p组HMGA2蛋白表达水平低于miR-NC组,anti-miR-146a-5p组HMGA2蛋白表达水平高于anti-miR-NC组,差异有统计学意义(P<0.05)。HG组总细胞凋亡率、早期细胞凋亡率、晚期细胞凋亡率和cleaved-caspase3蛋白、cleaved-caspase9蛋白、HMGA2 mRNA、HMGA2蛋白、TNF-α、IL-6、IL-8、MDA水平高于Con组,miR-146a-5p、CAT、SOD水平低于Con组,差异有统计学意义(P<0.05);HG+miR-146a-5p组总细胞凋亡率、早期细胞凋亡率、晚期细胞凋亡率和cleaved-caspase3蛋白、cleaved-caspase9蛋白、HMGA2 mRNA、HMGA2蛋白、TNF-α、IL-6、IL-8、MDA水平低于HG组,miR-146a-5p、CAT、SOD水平高于HG组,差异有统计学意义(P<0.05);HG+si-HMGA2组总细胞凋亡率、早期细胞凋亡率、晚期细胞凋亡率和cleaved-caspase3蛋白、cleaved-caspase9蛋白、HMGA2 mRNA、HMGA2蛋白、TNF-α、IL-6、IL-8、MDA水平低于HG组,CAT、SOD水平高于HG组,差异有统计学意义(P<0.05);HG+miR-146a-5p+pcDNA-HMGA2组总细胞凋亡率、早期细胞凋亡率、晚期细胞凋亡率和cleaved-caspase3蛋白、cleaved-caspase9蛋白、HMGA2 mRNA、HMGA2蛋白、TNF-α、IL-6、IL-8、MDA水平高于HG+miR-146a-5p组,CAT、SOD水平低于HG+miR-146a-5p组,差异有统计学意义(P<0.05)。结论 miR-146a-5p可能通过靶向HMGA2,发挥对高糖诱导的肾小管上皮细胞损伤的调控作用。
Objective To investigate the effect of microRNA-146a-5p(miR-146a-5p)on high glucose-induced injury of renal tubular epithelial cells by regulating high mobility group A2(HMGA2).Methods Human renal tubular epithelial cells(HK-2)were cultured in vitro and divided into different groups according to various treatment methods:normal control group(Con group),high glucose group(HG group),HG+miR-146a-5p group,HG+si-HMGA2 group,HG+miR-146a-5p+pcDNA-HMGA2 group,miR-NC group,miR-146a-5p group,anti-miR-NC group,and anti-miR-146a-5p group.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-146a-5p and HMGA2 mRNA in each group.A dual-luciferase reporter assay was employed to verify the targeting relationship between miR-146a-5p and HMGA2.Western blot was utilized to detect the expression levels of cleaved-caspase3,cleaved-caspase9,and HMGA2 protein in each group.Flow cytometry was applied to determine the apoptosis rate of cells in each group.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory factors[interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),interleukin-8(IL-8)]in each group.The levels of oxidative stress indicators[catalase(CAT),superoxide dismutase(SOD),malondialdehyde(MDA)]in each group were also measured.Results A nucleotide sequence complementary to miR-146a-5p was found in the 3′-untranslated region(3′-UTR)of HMGA2.The expression level of HMGA2 protein in the miR-146a-5p group was lower than that in the miR-NC group,while was higher in the anti-miR-146a-5p group than that in the anti-miR-NC group(P<0.05).The total apoptosis rate,early apoptosis rate,late apoptosis rate,and the levels of cleaved-caspase3 protein,cleaved-caspase9 protein,HMGA2 mRNA,HMGA2 protein,TNF-α,IL-6,IL-8,and MDA in the HG group were higher than those in the Con group,whereas the levels of miR-146a-5p,CAT,and SOD were lower(P<0.05).In the HG+miR-146a-5p group,these indicators showed opposite trends compared to the HG group(P<0.05).Similarly,in the HG+si-HMGA2 group,the total apoptosis rate,early apoptosis rate,late apoptosis rate,and the levels of cleaved-caspase3 protein,cleaved-caspase9 protein,HMGA2 mRNA,HMGA2 protein,TNF-α,IL-6,IL-8,and MDA were lower than those in the HG group,while the levels of CAT and SOD were higher(P<0.05).In the HG+miR-146a-5p+pcDNA-HMGA2 group,the trends of these indicators were consistent with those between the HG group and the HG+miR-146a-5p group,showing statistically significant differences when compared to the HG+miR-146a-5p group(P<0.05).Conclusion MiR-146a-5p may play a regulatory role in high glucose-induced injury of renal tubular epithelial cells by targeting HMGA2.
作者
王晨鸽
杜海梅
尹东升
WANG Chenge;DU Haimei;YIN Dongsheng(Department of Endocrinology,Shangluo Central Hospital of Shaanxi Province,Shangluo,Shaanxi,726000;Department of General Practice,the Affiliated Hospital of Yan'an University,Yan′an,Shaanxi,716000)
出处
《实用临床医药杂志》
2025年第11期14-19,25,共7页
Journal of Clinical Medicine in Practice
基金
陕西省自然科学基金项目(2020FR26003)。
