摘要
The incidence rate of kidney diseases in China has always remained high.At present,the clinical treatment mainly focuses on symptomatic treatment to delay the progression of the disease,and there is a lack of economical and effective treatment methods.MicroRNA plays an important regulatory role in the occurrence and development of diseases.This study aims to explore the role and regulatory mechanism of miR⁃142a⁃3p in adriamycin(ADR)⁃induced renal tubular epithelial cell(TCMK⁃1)injury,with a focus on its potential as a therapeutic target for ADR nephropathy.First,cell viability was assessed using the CCK⁃8 kit,and a mouse renal tubular epithelial cell model induced by ADR was established.Subsequently,alterations in miR⁃142a⁃3p and its target gene ATG16L1 mRNA levels were quantified using RT⁃qPCR.Western blotting was used to detect the protein levels of autophagy marker proteins and pyroptosis marker proteins.Monodansylcadaverin(MDC)staining was performed and the autophagy of cells was detected by flow cytometry.The results showed that the relative expression of miR⁃142a⁃3p in TCMK⁃1 cells induced by ADR was increased and the relative expression of its target gene ATG16L1 was decreased(P<0.0001).Western blotting results showed that the levels of p62(P<0.001)and pyroptosis⁃related proteins(P<0.001)were increased,while the protein levels of autophagy⁃related proteins were decreased(P<0.05).The flow cytometry results showed that there was no difference in the mean fluorescence intensity of autoph⁃agosomes between the ADR group and the autophagosome inhibitor group(3⁃MA group)(P>0.05),indicating that after ADR induction,cell autophagy was inhibited and pyroptosis was enhanced.When the expression of miR⁃142a⁃3p was inhibited by transfecting miR⁃142a⁃3p inhibitor,the relative expression level of the target gene ATG16L1 was restored(P<0.001).Western blotting showed that the protein level of p62(P<0.01)and pyroptosis⁃related proteins(P<0.01)were decreased,and the protein level of autophagy⁃related proteins was restored(P<0.001).Flow cytometry results further indicated that cell autophagy was restored(P<0.0001).In conclusion,ADR targets ATG16L1 through miR⁃142a⁃3p to reduce the autophagy level of TCMK⁃1,and simultaneously activates GSDMD⁃mediated pyroptosis.
肾病在我国的发病率一直居高不下,目前,临床主要通过对症治疗来延缓疾病的进展,缺乏经济有效的治疗手段。微RNA在疾病的发生发展中发挥着重要的调控作用,本文旨在探究微RNA-142a-3p(miR-142a-3p)在阿霉素(adriamycin,ADR)诱导肾小管上皮细胞(TCMK-1)损伤过程中的作用、调控机制及其能否成为治疗ADR肾病的潜在靶点。首先,用CCK-8试剂盒检测细胞活性并构建ADR诱导的小鼠肾小管上皮细胞模型,利用RT-qPCR检测细胞miR-142a-3p及其靶基因ATG16L 1的表达情况;蛋白质免疫印迹(Western blotting,WB)检测自噬标志蛋白质和焦亡标志蛋白质表达水平;使用单丹磺酰尸胺(monodansylcadaverin,MDC)染色并通过流式细胞术检测细胞自噬情况。结果显示,TCMK-1细胞经ADR诱导后miR-142a-3p相对表达量升高,其靶基因ATG16L 1相对表达量降低(P<0.0001);WB显示,p62蛋白表达增加(P<0.001),自噬相关蛋白质表达减少(P<0.05),焦亡相关蛋白质表达增加(P<0.001);流式结果显示,ADR组与自噬体抑制剂组(3-MA组)间自噬体平均荧光强度无差别(P>0.05),表明ADR诱导后细胞自噬被抑制和焦亡增强。当转染miR-142a-3p inhibitor抑制miR-142a-3p表达时,靶基因ATG16L 1相对表达量恢复(P<0.001);WB显示,p62蛋白减少(P<0.01),自噬相关蛋白至表达恢复(P<0.001),焦亡相关蛋白质表达降低(P<0.01);流式结果显示,细胞自噬得到恢复(P<0.0001)。综上所述,ADR通过miR-142a-3p靶向ATG16L 1降低TCMK-1自噬水平,同时激活GSDMD介导的细胞焦亡。
出处
《中国生物化学与分子生物学报》
北大核心
2025年第7期1031-1039,共9页
Chinese Journal of Biochemistry and Molecular Biology
基金
内蒙古自治区自然科学基金(No.2020MS08007,No.2020MS08015)
内蒙古自治区留学人员创新创业启动支持计划人选项目(2021,No.00121273)资助。
