抑制微小RNA-21可减轻高糖诱导的肾小球系膜细胞自噬抑制  

Abated microRNA- 21 attenuates high glucose- induced autophagy inhibition in rat mesangialcells by PTEN/Akt/mTOR pathway

作　　者：卢新星[2] 范秋灵[1] 徐莉[1] 曹旭[1] 苏彦[1] 张东成[1] 王九宁[1] 

机构地区：[1]中国医科大学附属第一医院肾内科,沈阳110001 [2]辽宁省人民医院肾内科

出　　处：《中华肾脏病杂志》2017年第1期48-54,共7页Chinese Journal of Nephrology

摘　　要：目的探讨高糖是否通过改变肾小球系膜细胞中miRNA-21的表达，调控PTEN和P13uAk/mTOR信号通路，进而调节自噬和糖尿病肾脏损伤，及抑制miRNA-21的作用对该过程的影响。方法大鼠肾小球系膜细胞（HBZY-1）应用脂质体2000转染试剂分别转染miRNA．21抑制物和阴性对照，转染稳定后分别在正常糖及高糖条件下培养48h，分为正常糖组（5．5mmol／L葡萄糖）、正常糖阴性对照组（转染阴性对照＋5．5mmol／L葡萄糖）、正常糖miRNA-21抑制物组（转染miRNA-21抑制物＋5．5mmol／L葡萄糖）、高糖组（25．0mmol／L葡萄糖）、高糖阴性对照组（转染阴性对照＋25．0mmol／L葡萄糖）、高糖miRNA-21抑制物组（转染miRNA-21抑制物＋25．0mmol／L葡萄糖）。采用MTT法检测细胞的存活及增殖能力；总蛋白／细胞数评估细胞肥大；Western印迹和实时定量PCR检测miRNA-21、PTEN-P13K／AkffmTOR信号通路活性、I型胶原及自噬标志物（p62和LC3Ⅱ）。透射电镜观察自噬体的形成及数量。结果与正常对照组比较，高糖组系膜细胞明显肥大、增殖，胞内miRNA-21表达上调，PTEN蛋白及mRNA的表达显著下调，P．Akt、p-mTOR、I型胶原、p62表达均增加，LC3Ⅱ表达和自噬体数量降低（均P〈0．01）。与高糖组比较，高糖miRNA-21抑制物组细胞肥大程度和增殖率均降低，p-Akt、p-mTOR、I型胶原、p62的表达均降低，PTEN、LC3II表达和自噬体数量均升高（均P〈0．01）。结论高糖上调系膜细胞内miRNA-21的表达，下调PTEN的表达，激活AkffmTOR通路，抑制自噬。抑制miRNA-21作用可上调PTEN的表达，进而抑制Akt/mTOR信号通路的活化，改善高糖诱导的系膜细胞异常的肥大、增殖，增强自噬，及减少细胞外基质蛋白聚积。Objective To investigate the effects of abated microRNA-21 （miRNA- 21） on phosphatase and tensin homologue on chromosome ten protein （PTEN） and PI3K/Akt/mTOR pathway, as well as their further influence on the autophagy in high glucose （HG, 25.0 mmol/L） induced rat glomerular mesangial cells. Methods MiRNA-21 inhibitor and negative control were transfected by liposome 2000 into rat glomerular mesangial cells （HBZY- 1）. The cells were divided into normal glucose （5.5 mmol/L） group, normal glucose ＋ negative control group, normal glucose ＋ miRNA- 21 inhibitor group, HG group, HG＋ negative control group and HG＋miRNA- 21 inhibitor group. Cell proliferation and hypertrophy were assayed by MTF and the ratio of total protein to cell number respectively. The miRNA-21 expression was detected using real time PCR. The expressions of PTEN/ Akt/mTOR signaling signatures, autophagy-associated protein （p62 and LC3 II） and collagen I was detected by Western blotting and real time PCR. Autophagosomes were observed using electron microscopy. Results Compared with those in normal glucose group, in HG group cells had hypertrophy and proliferation, up-regulated miRNA-21 expression, and down-regulated PTEN protein and mRNA expressions （all P 〈 0.01）. Also there were and up-regulated p-Akt, p-mTOR, p62 and collagen I expression, and lower LC3 Ⅱ expression and autophagosomes （all P 〈 0.01）. Further, compared with those in HG group, cells hypertrophy and proliferation in HG＋miRNA- 21 inhibitor group were reduced, expressions of p-Akt, p-mTOR, p62 and collagen [ were down-regulated, while expressions of PTEN and LC3 Ⅱ and autophagosomes were up-regulated （all P 〈 0.01）. Conclusions MiRNA- 21 inhibitor up- regulates PTEN expression, which inhibits the activation of Akt/mTOR signaling pathway, ameliorates cell hypertrophy, proliferation and enhances autophagy to reduce extracellular matrix accumulation.

关 键 词：糖尿病肾病 自噬 微RNAS PTEN磷酸水解酶 肾小球系膜细胞 

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