长链非编码RNA00963通过微小RNA-150-5p/白介素1受体关联激酶1轴对脂多糖诱导的大鼠肾小管上皮细胞凋亡影响的研究  

Effect of long non-coding RNA00963 on lipopolysaccharide-induced apoptosis of rat renal tubular epithelial cells via microRNA-150-5p/interleukin-1 receptor-associated kinase 1 axis

作　　者：崔雯萱 王彦[1] 于洋[1] 贾娟 赵海歌 CUI Wenxuan;WANG Yan;YU Yang;ZHAO Haige(Department of Laboratory Medicine,Affiliated Hospital of Hebei University,Baoding 071000,China)

机构地区：[1]河北大学附属医院检验科,保定071000 [2]河北大学附属医院肾内科

出　　处：《中国糖尿病杂志》2024年第12期941-950,共10页Chinese Journal of Diabetes

基　　金：河北省财政厅,河北省卫生健康委2024年政府资助临床医学优秀人才培养项目(ZF2024213);保定市科学技术局2023年保定市科技计划自筹经费项目(第二批)(2341ZF350);河北大学附属医院/临床医学院2022年度院内基金项目(2022QC33)。

摘　　要：目的探讨长链非编码RNA00963(LINC00963)通过微小RNA-150-5p(miR-150-5p)/白介素1受体关联激酶1(IRAK1)轴对脂多糖(LPS)诱导的大鼠肾小管上皮细胞凋亡的影响。方法体外培养大鼠肾小管上皮细胞NRK-52E,分为正常对照(Con)组、LPS组、LPS+敲低阴性对照组(LPS+si-NC组)、LPS+敲低LINC00963组(LPS+si-LINC00963)、LPS+si-LINC00963+抑制剂阴性对照组(LPS+si-LINC00963+inhibitor NC组)、LPS+si-LINC00963+miR-150-5p抑制剂组(LPS+si-LINC00963+miR-150-5p inhibitor组)、LPS+si-LINC00963+过表达阴性对照组(LPS+si-LINC00963+oe-NC组)、LPS+si-LINC00963+过表达IRAK1组(LPS+si-LINC00963+oe-IRAK1)组。MTT和Ed U法检测细胞增殖,流式细胞术检测细胞凋亡率,ELISA试剂盒检测TNF-α、IL-6和IL-10,商品化试剂盒检测丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物歧化酶(GSH-Px),q RT-PCR检测LINC00963、miR-150-5p、IRAK1 mRNA表达,Western blot法检测胱天蛋白酶-3(Caspase-3)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、IRAK1蛋白表达。双荧光素酶报告基因和RNA pull down实验验证miR-150-5p与LINC00963和IRAK1的关系。结果与Con组比较,LPS、LPS+si-NC组LINC00963和IRAK1 mRNA表达、TNF-α、IL-6、MDA、细胞凋亡率、Caspase-3、Bax、IRAK1蛋白表达升高(P<0.05),miR-150-5p表达、24 h A490值、48 h A490值、细胞增殖率、IL-10、SOD和GSH-Px活性、Bcl-2蛋白表达降低(P<0.05)。与LPS+si-NC组比较,LPS+si-LINC00963组LINC00963和IRAK1 mRNA表达、TNF-α、IL-6、MDA、细胞凋亡率、Caspase-3、Bax、IRAK1蛋白表达降低(P<0.05),miR-150-5p表达、24 h A490值、48 h A490值、细胞增殖率、IL-10、SOD和GSH-Px活性、Bcl-2蛋白表达升高(P<0.05)。抑制miR-150-5p表达或过表达IRAK1均可降低干扰LINC00963对LPS诱导NRK-52E细胞损伤的改善作用(P<0.05)。结论干扰LINC00963可调控miR-150-5p/IRAK1轴减轻LPS诱导的NRK-52E细胞氧化应激、炎症反应和凋亡。Objective To investigate the effect of long-chain non-coding RNA00963(LINC00963)on lipopolysaccharide(LPS)induced apoptosis in rat renal tubular epithelial cells via micro RNA-150-5p(miR-150-5p)/interleukin-1 receptor-associated kinase 1(IRAK1)axis.Methods Rat renal tubular epithelial cells NRK-52E were cultured in vitro.NRK-52E cells were divided into control group(Con),LPS group,LPS+si-NC group,LPS+si-LINC00963 group,LPS+si-LINC00963+inhibitor NC group,LPS+si-LINC00963+miR-150-5p inhibitor group,LPS+si-LINC00963+oe-NC group,and LPS+si-LINC00963+oe-IRAK1 group.MTT and EdU methods were used to detect cell proliferation.Flow cytometry was used to detect cell apoptosis rate.ELISA kits were used to detect TNF-α,IL-6,and IL-10 levels,and commercial reagent kits were used to analyze malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxide dismutase(GSH-Px)levels.The q RT-PCR was used to detect the expression levels of LINC00963,miR-150-5p,and IRAK1 genes in each group.Western blot was used to detect the expression of cysteine aspartic acid specific protease-3(Caspase-3),Bax,Bcl-2,and IRAK1 proteins in cells.Dual luciferase reporter gene and RNA pull down experiment were used to verify the relationship between miR-150-5p and LINC00963 and IRAK1.Results Compare with the Con group,the expression of LINC00963 and IRAK1 m RNA,the levels of TNF-α,IL-6,the content of MDA,apoptosis rate,the expression of Caspase-3,Bax,and IRAK1 proteins in NRK-52E cells were obviously increased(P<0.05),the expression of miR-150-5p,A_(490)(24 hours,48 hours)values,proliferation rate,the level of IL-10,the activities of SOD and GSH-Px,and the expression of Bcl-2 protein were obviously reduced(P<0.05)in the LPS group and LPS+si-NC group.Compare with the LPS+si-NC group,the expression of LINC00963and IRAK1 m RNA,the levels of TNF-α,IL-6,the content of MDA,apoptosis rate,the expression of Caspase-3,Bax,and IRAK1 proteins in NRK-52E cells were obviously reduced(P<0.05),the expression of miR-150-5p,A_(490)(24 hours,48 hours)values,prol

关 键 词：长链非编码RNA00963 微小RNA-150-5p/白介素1受体关联激酶1 脂多糖 肾小管上皮细胞 凋亡 

分 类 号：R692.9[医药卫生—泌尿科学] R587.2[医药卫生—外科学]