敲减circ_0001879对高糖作用的肾小管上皮细胞HK-2损伤的影响机制研究  

The effects and mechanism of circ_0001879 knockdown on the injury of renal tubular epithelial cell HK-2 induced by high glucose

作　　者：荆立宝 白丽 Jing Libao;Bai Li(Department of Nephropathy Hemodialysis,the Second People's Hospital of Qingdao Chengyang District,Qingdao Women and Children's Hospital,Qingdao 266111,China;Department of Nephrology,Chongming Branch of Xinhua Hospital,Shanghai 202150,China)

机构地区：[1]青岛市妇女儿童医院城阳院区、青岛市城阳区第二人民医院肾病血液透析科,山东青岛266111 [2]新华医院崇明分院肾内科,上海202150

出　　处：《国际免疫学杂志》2023年第1期16-24,共9页International Journal of Immunology

摘　　要：目的探讨circ_0001879对高糖作用的肾小管上皮细胞HK-2凋亡和氧化应激的影响及作用机制。方法体外培养HK-2细胞,转染后用30 mmol/L葡萄糖干预24 h。未转染的HK-2细胞分为对照组(NG组)和高糖组(HG组);转染后的HK-2细胞分为HG+si-NC组、HG+si-circ_0001879组、HG+miR-136组、HG+miR-NC组、HG+si-circ_0001879+anti-miR-NC组和HG+si-circ_0001879+anti-miR-136组。流式细胞术检测细胞凋亡,Western blot检测兔抗人B淋巴细胞瘤-2(B lymphocytoma-2,Bcl-2)和兔抗人Bcl-2相关X蛋白(Bcl-2-associated X,Bax)表达,硫代巴比妥酸法检测丙二醛(malondialdehyde,MDA)含量,黄嘌呤氧化酶法检测超氧化物歧化酶(superoxide dismutase,SOD)活性。反转录实时荧光定量PCR(reverse transcription-real time quantitative PCR,RT-qPCR)检测circ_0001879和miR-136表达,双荧光素酶报告基因实验验证circ_0001879与miR-136靶向关系。结果HK-2细胞经高糖处理后,与NG组相比,HG组、HG+si-NC组和HG+si-circ_0001879组circ_0001879含量、HK-2细胞凋亡率、Bax表达及MDA含量明显升高,而miR-136含量、相关蛋白Bcl-2表达及SOD活性明显降低,差异具有统计学意义(F值分别为1036.48、181.50、191.80、380.29、1396.44、195.18、108.17,P值均<0.001);敲减circ_0001879后,与HG组、HG+si-NC组比较,HG+si-circ_0001879组circ_0001879含量、HK-2细胞凋亡率、Bax表达、MDA含量明显降低,而miR-136含量、Bcl-2表达、SOD活性明显升高,差异具有统计学意义{[(4.40±0.12)、(4.48±0.14)]比(1.44±0.08),[(24.89±1.43)%、(24.93±1.56)%]比(12.53±0.61)%,[(0.76±0.05)、(0.76±0.06)]比(0.27±0.02),[(621.27±23.75)nmol/mL、(626.98±15.24)nmol/mL]比(309.48±15.24)nmol/mL,[(0.12±0.01)、(0.12±0.01)]比(0.80±0.04),[(0.12±0.01)、(0.13±0.01)]比(0.54±0.05),[(94.00±5.59)U/L、(92.64±7.92)U/L]比(215.69±10.88)U/L,P<0.05}。Circular RNA Interactome靶基因在线预测软件显示,miR-136的核苷酸序列与circ_0001879存在结合位点。双荧光素酶报告基因研究�Objective To investigate the effect and mechanism of circ_0001879 on the apoptosis and oxidative stress of renal tubular epithelial cells HK-2 treated with high glucose.Methods HK-2 cells were cultured in vitro.After transfection,HK-2 cells were intervened with 30 mmol/L glucose for 24 h.The un-transfected HK-2 cells were divided into control group(NG group)and high glucose group(HG group),and the transfected HK-2 cells were divided into HG+si-NC group,HG+si-circ_0001879 group,HG+miR-136 group,HG+miR-NC group,HG+si-circ_0001879+anti-miR-NC group and HG+si-circ_0001879+anti-miR-136 group.Cell apoptosis was detected by flow cytometry.The protein expression of B lymphocytoma-2(Bcl-2)and Bcl-2-associated X(Bax)was detected by Western blot.The malondialdehyde(MDA)content and superoxide dismutase(SOD)activity were detected by the thiobarbituric acid and xanthine oxidase method respectively.The expression levels of circ_0001879 and miR-136 were detected by reverse transcription-real time quantitative PCR(RT-qPCR).The dual-luciferase reporter gene experiment was performed to verify the targeting relationship between circ_0001879 and miR-136.Results After HK-2 cells were treated with high glucose,compared with NG group,the circ_0001879 content,apoptosis rate,Bax expression and MDA content were significantly increased,whereas miR-136 and Bcl-2 expression and SOD activity were significantly decreased in HG group,HG+si-NC group and HG+si-circ_0001879 group(F values were 1036.48,181.50,191.80,380.29,1396.44,195.18 and 108.17 respectively,all P values<0.001).After circ_0001879 knockdown,compared with HG group and HG+si-NC group,the circ_0001879 content,HK-2 cell apoptosis rate,Bax expression and MDA content were significantly decreased,whereas miR-136 content,Bcl-2 expression and SOD activity were significantly increased in HG+si-circ_0001879 group{[(4.40±0.12),(4.48±0.14)]vs(1.44±0.08),[(24.89±1.43)%,(24.93±1.56)%]vs(12.53±0.61)%,[(0.76±0.05),(0.76±0.06)]vs(0.27±0.02),[(621.27±23.75)nmol/mL,(626.98±15.24)nmol/mL]vs

关 键 词：肾小管上皮细胞 circ_0001879 miR-136 细胞凋亡 氧化应激 

分 类 号：R692.6[医药卫生—泌尿科学]