摘要
目的研究微小RNA(miR)-29a在急性肾损伤患者血清中的表达情况,探究miR-29a是否通过调控膦酸酯酶张力蛋白同源物(PTEN)表达影响肾小管细胞凋亡。方法收集113例急性肾损伤(AKI)患者以及110例健康对照者血清,通过实时荧光定量PCR检测血清中miR-29a的表达情况。体外培养人肾小管上皮细胞(HK-2),通过转染miR-29a NC和miR-29a mimic到HK-2细胞中,用TGF-β1诱导HK-2细胞凋亡,采用流式细胞仪检测细胞凋亡,用生物信息学预测miR-29a的潜在靶基因,荧光素酶报告基因实验验证miR-29a与PTEN的互补配对关系,Western blot检测潜在靶基因PTEN的表达情况。再通过转染过表达PTEN的质粒,用流式细胞仪检测HK-2细胞的凋亡情况。结果与健康对照者血清相比,AKI患者血清中的miR-29a表达水平降低(P<0.05);流式细胞仪结果显示转染miR-29a mimic后,HK-2细胞的凋亡明显少于转染miR-29a NC组(P<0.05);荧光素酶报告基因实验结果显示,miR-29a与PTEN具有互补配对关系;转染过表达PTEN后,肾小管上皮细胞的凋亡率增加(P<0.05)。结论在AKI患者血清中,miR-29a表达水平降低,过表达miR-29a通过抑制PTEN蛋白的表达从而抑制肾小管上皮细胞凋亡。
ObjectiveTo investigate the expression of miR-29a in serum of acute kidney injury patients, and whether miR-29a can affect the apoptosis of renal tubular cells by regulating the expressions of phosphatase and tensin homologue deleted on chromosome ten （PTEN）.
MethodsBlood samples were collected from 113 patients with acute kidney injury （AKI） and 110 healthy controls. The expressions of miR-29a in serum were detected by real-fluorescence quantitative polymerase chain reaction （RT-q-PCR）. HK-2 cells were cultured in vitro, miR-29a NC or miR-29a mimic was transfected into HK-2 cells, and apoptosis of HK-2 cells was induced by transforming growth factor-β1 （TGF-β1）. Flow cytometry was used to investigate the apoptosis of HK-2 cells. Bioinformatics was used to predict potential target genes of miR-29a. Luciferase reporter gene test was used to verify the complementary pairing relationship between miR-29a and PTEN. The expression of PTEN was detected by Western blot. After transfection of plasmids overexpressing PTEN, the apoptosis of HK-2 cells was detected by flow cytometry.
ResultsCompared to the healthy control group, the serum expression of miR-29a was decreased in AKI patients （P〈0.05）. Flow cytometry results showed that transfection of miR-29a mimic was significantly decreased HK-2 cells apoptosis compared to miR-29 NC group （P〈0.05）. The Luciferase reporter assay results showed that miR-29a and PTEN had complementary relationship. After transfected with overexpression of PTEN, the HK-2 cells apoptosis rate was significantly increased （P〈0.05）.
ConclusionsIn the serum of AKI patients, the expression of miR-29a was decreased, overexpression of miR-29a inhibited the apoptosis of renal tubular epithelial cells by inhibiting the expression of PTEN protein.
出处
《中国医师杂志》
CAS
2018年第2期195-199,共5页
Journal of Chinese Physician
基金
山西省科技攻关项目(20140313013-5)~~
