摘要
目的探讨microRNA-709（miR-709）在顺铂引起肾小管上皮细胞损伤模型中的作用及机制。方法体外培养小鼠肾小管上皮细胞，予不同浓度（0、1、5、10、20μmol／L）顺铂刺激24h，以10μmol／L顺铂刺激不同时间（0、2、6、12、24h）。采用实时荧光定量PCR（RT—PCR）技术检测细胞miR-709的表达变化。实验组分为上调对照组、miR-709过表达组、下调对照组、miR-709下调组，其中miR-709下调组和下调对照组加顺铂刺激。应用AnnexinV—FITC／PI双染法检测细胞凋亡，分光光度法检测含半胱氨酸的天冬氨酸水解酶（Caspase-3）活性，Westernblot法和RT—PCR检测上皮细胞标志蛋白E-cadherin及其mRNA的变化。JC-1（线粒体膜电位）荧光探针检测线粒体膜电位，流式细胞术检测线粒体超氧化物（mltoSOX）生成，PCR检测线粒体基因（mtDNA）拷贝数。结果miR-709在5μmol／L顺铂刺激小管上皮细胞后开始明显升高（F=22．17，P〈0．05），10μmol／L顺铂刺激12h开始明显升高（F=33．462，P〈0．05）；过表达miR-709后细胞凋亡数目、Caspase-3活性较上调对照组增加，E-cadhenn表达较上调对照组下降，差异均有统计学意义（凋亡：1．54±0．20比1．00±0．23；Caspase-3活性：1．27±0．08比0．97±0．08；E-cadherin：0．47±0．15比1．00±0．10；t=-3．086、-5．882、5．671，P均〈0．05）；线粒体功能指标线粒体膜电位、mtDNA拷贝数较对照组降低，mitoSOX生成较对照组增加，差异均有统计学意义（JC-1：0．80±0．04比1．05±0．08；mtDNA：0．58±0．15比1．00±0．75；mitoSOX：1．31±0．16比1．00±0．05；t=4．687、4．943、-3．694，P均〈0．05）。而顺铂刺激后，miR-709下调组细胞凋亡数目、Caspase-3活性较下调对照组降低，E—cadhefin表达较下调对照组增高，差异均有统计学意义（凋亡：1．35±0．10比1．86±0．44；Caspase-3活性：1．04±0．12比1．30±0．09；E-cadhefin：0．86±0．08比0．54±0．05；F=18．489、20．932、33．323，P均〈0．05）；线粒体膜电位、mtDNA拷贝数较对照组增加，mitoSOX生成较下调对照组降低，差异均有统计学意义（Jc-1：0．94±0．06比0．75±0．05；mtDNA：0．68±0．09比0．27±0．12；mitoSOX：0．91±0．09比1．22±0．08；F=21．726、59。330、23．813，P均〈0．05）。结论miR-709可能通过负性调控线粒体功能参与顺铂引起的肾小管上皮细胞损伤。
Objective To investigate the role of microRNA -709 （miR -709）in murine renal tubular epithe- lial cells with Cisplatin treatment and its underlying mechanism. Methods The ceils were cultured with Cisplatin in the concentrations （0,1,5,10,20 μmol/L） for 24 h in vitro,and underwent 10 μmol/L Cisplatin stimulation at diffe- rent time points （ 0,2,6,12,24 h）. Real - time fluorescence quantitative polymerase chain reaction （ RT - PCR） was used to investigate the level of miR - 709. mPTC were divided into a negative control group, miR - 709 mimic group, in- hibitor negative control （INNC） group, miR - 709 inhibitor group, INNC group and miR - 709 inhibitor group were trea- ted with Cisplatin treatment. Annexin V - FITC/PI double staining was applied to detect apoptosis. Caspase - 3 activity was detected by spectrophotometry. The mRNA and protein levels of E - cadherin were detected by RT - PCR and Western blot. Mitochondrial membrane potential and mitochondrial superoxide （mitoSOX） generation were detected by flow cytometry. Mitochondrial DNA（mtDNA） copy number was verified by PCR. Results The level of miR -709 in- creased in Cisplatin 5 μmol/L stimulation group（ F = 22. 17 ,P 〈 0.05 ） and increased further 12 h later after cultured in 10 μmol/L Cisplatin compared with that of the control group （ F = 33. 462, P 〈 0. 05 ）. After overexpression of miR - 709, apoptotic rate and Caspase - 3 activity of the mPTC increased, while the expression of E - cadherin declined when compared with that in the negative control group （ apoptotic rate ： 1.54 ± 0.20 vs 1.00± 0. 23 ; Caspase - 3 activity：1.27±0.08vs0.97 ±0.08;E-cadherin：0.47±0.15 vs 1.00±0.10;t= -3.086, -5.882,5.671,allP〈 0.05 ）. In addition, the levels of JC - 1 and mtDNA decreased while mitoSOX increased compared with that of the con-trol group （JC - 1：0.80 ±0.04 vs 1.05 ±0. 08;mtDNA：0.58 ±0. 15 vs 1.00 ±0.75;mitoSOX： 1.31 ±0. 16 vs 1.00 ± 0.05 ; t = 4. 687,4. 943, - 3. 694, all P 〈 0.05 ）. Downregulation of miR - 709 attenuated the tubular cell injury and the mitochondrial dysfunction was induced by Cisplatin （ apoptotic rate ： 1.35 ± 0.10 vs 1.86± 0.44 ; Caspase - 3 activity ： 1.04 ±0.12 vs 1.30 ± 0.09 ; E - cadherin ：0.86 ± 0.08 vs 0. 54 ±0.05 ; JC - 1：0.94± 0.06 vs 0. 75 ± 0.05 ; mtDNA ：0.68 ± 0.09 vs 0. 27 ± 0.12 ; mitoSOX ： 0.91 ± 0.09 vs 1.22 ± 0.08 ; F = 18. 489,20. 932,33. 323,21. 726, 59.330,23. 813, all P 〈 0.05 ）. Conclusion Mitochondrial function negatively regulated by miR - 709 may be involved in the renal tubular epithelial cell injury induced by Cisplatin.
出处
《中华实用儿科临床杂志》
CAS
CSCD
北大核心
2015年第9期711-714,共4页
Chinese Journal of Applied Clinical Pediatrics
基金
国家自然科学基金（81170635）
