沉默环状RNA_0084043对高糖诱导的人肾小管上皮细胞损伤的影响

Effect of circ_0084043 silence upon human renal tubular epithelial cell injury induced by high glucose

目的探讨沉默环状RNA_0084043(circular RNA_0084043,circ_0084043)对高糖诱导的人肾小管上皮细胞炎性反应、氧化应激及凋亡的影响及其对微小RNA-142-5p(micro RNA-145-5p,miR-142-5p)的靶向调控作用。方法肾小管上皮细胞HK-2按照随机数字表法随机分为Con组、HG组、HG+si-NC组、HG+si-circ_0084043组、HG+miR-NC组、HG+miR-142-5p组、HG+si-circ_0084043+anti-miR-NC组、HG+si-circ_0084043+anti-miR-142-5p组;qRT-PCR法检测circ_0084043与miR-142-5p的表达量;ELISA法检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素8(interleukin8,IL-8)的水平;试剂盒检测丙二醛(malondialedhyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)的水平;流式细胞术检测细胞凋亡率;双荧光素酶报告基因实验检测circ_0084043与miR-142-5p的靶向关系;Western blot法检测B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bcl-2-associated X protein,Bax)蛋白表达量。结果与Con组比较,HG组circ_0084043的表达水平升高[(1.00±0.04)比(3.57±0.49)](P<0.05),miR-142-5p的表达水平降低[(1.00±0.05)比(0.34±0.06)](P<0.05),TNF-α、IL-8、MDA的水平升高[(18.43±2.06)mg/mL比(49.55±2.65)mg/mL;(11.74±0.83)mg/mL比(27.62±2.25)mg/mL;(0.23±0.08)nmol/mg比(2.86±0.37)nmol/mg](P<0.05),凋亡率[(6.74±0.95)%比(23.15±2.49)%]与Bax蛋白水平升高(P<0.05),SOD的活性降低[(40.05±3.07)U/mg比(17.35±1.54)U/mg](P<0.05),Bcl-2蛋白水平降低(P<0.05);与HG组、HG+si-NC组比较,HG+si-circ_0084043组TNF-α、IL-8、MDA的水平降低[(49.55±2.65)/(48.42±4.04)mg/mL比(34.90±4.95)mg/mL;(27.62±2.25)/(27.76±3.53)mg/mL比(19.14±1.02)mg/mL;(2.86±0.37)/(2.81±0.43)nmol/mg比(0.89±0.12)nmol/mg](P<0.05),凋亡率[(23.15±2.49)/(23.73±3.28)%比(14.47±0.93)%]与Bax蛋白水平降低(P<0.05),SOD的活性[(17.35±1.54)/(17.40±0.63)U/mg比(28.54±2.56)U/mg]升高(P<0.05),Bcl-2蛋白水平升高(P<0.05);circ_0084043可靶向调控miR-142-5p;与HG+miR-NC组比较,HG+miR-142-5p组TNF-α、IL-8、MDA的水平降低[(48.78±4.56)mg/mL比(35.18±3.93)mg/mL;(27.98±1.64)mg/mL比(18.31±1.11)mg/mL;(2.81±0.32)nmol/mg比(0.96±0.08)nmol/mg](P<0.05),凋亡率[(22.27±2.68)%比(16.01±0.48)%]与Bax蛋白水平降低(P<0.05),SOD的活性升高[(17.40±1.09)U/mg比(25.22±1.67)U/mg](P<0.05),Bcl-2蛋白水平升高(P<0.05);与HG+si-circ_0084043+anti-miR-NC组比较,HG+si-circ_0084043+anti-miR-142-5p组TNF-α、IL-8、MDA的水平升高[(34.74±1.51)mg/mL比(46.31±2.65)mg/mL;(19.03±0.66)mg/mL比(23.07±1.19)mg/mL;(0.89±0.06)nmol/mg比(1.43±0.11)nmol/mg](P<0.05),凋亡率[(14.69±0.49)%比(18.37±0.89)%]与Bax蛋白水平升高(P<0.05),SOD的活性降低[(28.38±1.03)U/mg比(19.56±0.83)U/mg](P<0.05),Bcl-2蛋白水平降低(P<0.05)。结论沉默circ_0084043可通过负向调控miR-142-5p而抑制细胞炎性反应、氧化应激及凋亡,从而减轻高糖诱导的人肾小管上皮细胞损伤。

Objective To explore the effect of silencing circular RNA_0084043(circ_0084043)on the inflammatory responses,oxidative stress and apoptosis of human renal tubular epithelial cells induced by high glucose and its targeted regulation of miR-142-5p.Methods Renal tubular epithelial cells HK-2 were divided into the groups of Con,HG,HG+si-NC,HG+si-circ_0084043,HG+miR-NC,HG+miR-142-5p,HG+si-circ_0084043+anti-miR-NC and HG+si-circ_0084043+anti-miR-142-5p.Quantitative realtime polymerase chain reaction(qRT-PCR)was utilized for detecting the expression levels of circ_0084043 and miR-142-5p according to the random number table Enzyme-linked immunosorbent assay(ELISA)was utilized for detecting the levels of tumor necrosis factor-alpha(TNF-α)and interleukin-8(IL-8).The levels of malondialedhyde(MDA)and superoxide dismutase(SOD)were detected.Flow cytometry was employed for detecting apoptotic rate.Dual luciferase reporter gene was utilized for detecting the targeting relationship between circ_0084043 and miR-142-5p and Western blot for detecting the expressions of Bcl-2 and Bax protein.Results As compared with Con group,the expression level of circ_0084043 spiked in HG group[(1.00±0.04)vs(3.57±0.49)](P<0.05)while the expression level of miR-142-5p declined[(1.00±0.05)vs(0.34±0.06)](P<0.05);the levels of TNF-α,IL-8 and MDA[(18.43±2.06)vs(49.55±2.65)mg/mL;(11.74±0.83)vs(27.62±2.25)mg/mL;(0.23±0.08)vs(2.86±0.37)nmol/mg](P<0.05),apoptotic rate[(6.74±0.95)%vs(23.15±2.49)%]and protein level of Bax increased(P<0.05)while activity of SOD decreased[(40.05±3.07)vs(17.35±1.54)U/mg](P<0.05)and protein level of Bcl2 dropped(P<0.05).Compared with HG/HG+si-NC group,the levels of TNF-α,IL-8 and MDA declined in HG+si-circ_0084043 group[(49.55±2.65)/(48.42±4.04)vs(34.90±4.95)mg/mL;(27.62±2.25)/(27.76±3.53)vs(19.14±1.02)mg/mL;(2.86±0.37)/(2.81±0.43)vs(0.89±0.12)nmol/mg](P<0.05)and apoptotic rate[(23.15±2.49)/(23.73±3.28)%vs(14.47±0.93)%]and protein level of Bax declined(P<0.05)while the activity of SOD[(17.35±1.54)/(17.40±0.63)vs(28.54±2.56)U/mg](P<0.05)and protein level of Bcl-2 rose(P<0.05).And circ_0084043 could target miR-142-5p.As compared with HG+miR-NC group,the levels of TNF-α,IL-8 and MDA declined in HG+miR-142-5p group[(48.78±4.56)vs(35.18±3.93)mg/mL;(27.98±1.64)vs(18.31±1.11)mg/mL;(2.81±0.32)vs(0.96±0.08)nmol/mg](P<0.05)and apoptotic rate[(22.27±2.68)%vs(16.01±0.48)%]and protein level of Bax dropped(P<0.05)while activity of SOD[(17.40±1.09)vs(25.22±1.67)U/mg](P<0.05)and protein level of Bcl-2 rose(P<0.05).As compared with HG+si-circ_0084043+antimiR-NC group,the levels of TNF-α,IL-8 and MDA rose in HG+si-circ_0084043+anti-miR-142-5p group[(34.74±1.51)vs(46.31±2.65)mg/mL;(19.03±0.66)vs(23.07±1.19)mg/mL;(0.89±0.06)vs(1.43±0.11)nmol/mg](P<0.05)and apoptotic rate[(14.69±0.49)%vs(18.37±0.89)%]and protein level of Bax spiked(P<0.05)while the activity of SOD[(28.38±1.03)vs(19.56±0.83)U/mg](P<0.05)and protein level of Bcl-2 declined(P<0.05).Conclusion Silencing circ_0084043 could inhibit cell inflammatory responses,oxidative stress and apoptosis through negatively regulating miR-142-5p,thereby blunting the damage of human renal tubular epithelial cells induced by high glucose.