摘要
目的 探讨长链非编码RNA Opa相互作用蛋白5-反义转录物1(lncRNA OIP5-AS1)对高糖诱导的人肾小管上皮细胞增殖、凋亡和氧化应激损伤的影响及分子机制。方法 体外培养人肾皮质近曲小管上皮细胞HK-2,分为正常葡萄糖组(NG组)、高糖组(HG组)、HG+si-NC组、HG+si-OIP5-AS1组、HG+miR-NC组、HG+miR-25-3p组、HG+si-OIP5-AS1+inhibitor-NC组、HG+si-OIP5-AS1+miR-25-3p inhibitor组。转染48 h后,实时荧光定量PCR(qPCR)检测细胞中lncRNA OIP5-AS1、miR-25-3p和性别决定区Y框蛋白4(SOX4)m RNA水平;CCK-8法检测细胞活力;检测细胞培养上清液中乳酸脱氢酶(LDH)活性;流式细胞术分析细胞凋亡情况;检测细胞中丙二醛(MDA)水平和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性;DCFH-DA荧光探针检测细胞内活性氧(ROS)的产生;Western blot实验检测细胞中SOX4、B细胞淋巴瘤因子2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶-3(Caspase-3)和裂解的Caspase-3(Cleaved-Caspase-3)蛋白表达。双荧光素酶报告基因实验确认miR-25-3p与lncRNA OIP5-AS1和SOX4的靶向关系。结果 与NG组相比,HG组细胞中lncRNA OIP5-AS1和SOX4 mRNA和蛋白表达水平升高,miR-25-3p水平降低(P<0.05);敲低lncRNA OIP5-AS1可显著下调SOX m RNA和蛋白水平,上调miR-25-3p水平,增加HK-2细胞活力和SOD、CAT活性以及Bcl-2蛋白水平,降低细胞凋亡率、LDH活性、MDA、ROS水平、Bax蛋白水平及Cleaved-Caspase-3/Caspase-3比值(P<0.05);上调miR-25-3p表达与敲低lncRNA OIP5-AS1的作用一致;在敲低lncRNA OIP5-AS1的基础上,下调miR-25-3p可明显减弱lncRNA OIP5-AS1敲低对高糖诱导的HK-2细胞氧化应激损伤的保护作用(P<0.05)。双荧光素酶报告基因实验显示lncRNA OIP5-AS1和miR-25-3p,以及miR-25-3p和SOX4之间存在结合位点。结论 lncRNA OIP5-AS1可能通过miR-25-3p/SOX4轴促进高糖诱导的HK-2细胞损伤。
Objective To investigate the impact and molecular mechanism of long non-coding RNA Opa-interacting protein 5-antisense transcript 1(lncRNA OIP5-AS1) on the proliferation,apoptosis and oxidative stress damage of human renal tubular epithelial cells induced by high glucose.Methods Human renal cortical proximal tubule epithelial cells HK-2 were cultured in vitro.HK-2 cells were transfected with lncRNA OIP5-AS1 small interfering RNA(si-OIP5-AS 1),miR-25-3p mimic,miR-25-3p inhibitor and their negative controls si-NC,miR-NC and inhibitor-NC.Cells were divided into the normal glucose group(NG group),the high glucose group(HG group),the HG+si-NC group,the HG+si-OIP5-AS1group,the HG+miR-NC group,the HG+miR-25-3p group,the HG+si-OIP5-AS1+inhibitor-NC group and the HG+siOIP5-AS1+miR-25-3p inhibitor group.Forty-eight hours after transfection,real-time quantitative PCR(qPCR) was performed to detect levels of lncRNA OIP5-AS 1,miR-25-3p and sex-determining region Y-box protein 4(SOX4) mRNA in cells.CCK-8 assay was performed to detect cell viability and lactate dehydrogenase(LDH) activity in the cell culture supernatant.Flow cytometry was performed to analyze apoptosis,malondialdehyde(MDA),superoxide dismutase(SOD) and catalase(CAT) activities in cells.DCFH-DA fluorescent probe was implemented to detect intracellular reactive oxygen species(ROS) production.Western blot experiment was performed to detect the protein expression of SOX4,B-cell lymphoma factor 2(Bcl-2),Bcl-2-associated X protein(Bax),caspase-3(Caspase-3) and cleaved-Caspase-3(CleavedCaspase-3) in cells.Dual luciferase reporter assay confirmed the targeting relationship between lncRNA OIP5-AS1 and miR-25-3p,and between miR-25-3p and SOX4.Results Compared with the NG group,the expression levels of lncRNA OIP5-AS1 and SOX4 were significantly increased in the HG group,and the level of miR-25-3p was significantly decreased(P <0.05).Knockdown of lncRNA OIP5-AS1 was able to significantly down-regulate SOX mRNA and protein levels,and up-regulate miR-25-3p level,increase HK-2 cell viability,SOD,CAT activities and Bcl-2 protein level,and reduce apoptosis rate,LDH activity,MDA,ROS levels,Bax protein level and Cleaved-Caspase-3/Caspase-3 ratio(P<0.05).The up-regulating miR-25-3p expression was consistent with that of knocking down lncRNA OIP5-AS1.On the basis of knockdown of lncRNA OIP5-AS1,down-regulation of miR-25-3p significantly attenuated the protective effect of lncRNA OIP5-AS1 knockdown on high glucose-induced oxidative stress damage in HK-2 cells(P<0.05).Dual luciferase reporter assay showed binding sites between lncRNA OIP5-AS1 and miR-25-3p,as well as between miR-25-3p and SOX4.Conclusion The lncRNA OIP5-AS1 may promote high glucose-induced HK-2 cell damage through the miR-25-3p/SOX4 axis.
作者
杨娟
张厚芬
吴松
陈莹
罗华荣
YANG Juan;ZHANG Houfen;WU Song;CHEN Ying;LUO Huarong(Department of Nephrology Endocrinology,302 Hospital of China Guihang Group,Anshun 561000,China)
出处
《天津医药》
CAS
北大核心
2023年第2期131-138,共8页
Tianjin Medical Journal
基金
安顺市科技计划项目(安市科社[2021]45号)。
