长链非编码RNA前列腺雄激素调节转录因子1对帕金森模型细胞损伤的实验研究

Damage of Parkinson's model cells induced by long non-coding RNA prostatic androgen-regulated transcription 1

目的探讨长链非编码RNA(lncRNA)前列腺雄激素调节转录因子1(PART1)对1-甲基-4-苯基-吡啶离子(1-methyl-4-phenyl,MPP+)诱导的帕金森模型细胞氧化应激和凋亡的影响及分子机制。方法将SK-N-SH细胞分为对照(Con)组(细胞未经任何处理)、MPP+组、空白组、转染组(pcDNA-PART1过表达载体质粒转染)、阴性组、干扰组[微小RNA(miR)-448抑制剂转染]、转染对照组、转染实验组(pcDNA-PART1与miR-448模拟物转染),后7组细胞用100μmol/L的MPP+处理。实时荧光定量PCR检测lncRNA PART1和miR-448表达;ELISA检测丙二醛(MDA)和谷胱甘肽(GSH)含量;流式细胞术检测细胞凋亡;蛋白印迹检测裂解的半胱氨酸天冬氨酸蛋白酶(cleaved-Caspase)3和cleaved-Caspase9蛋白表达;双荧光素酶报告实验检测lncRNA PART1和miR-448的靶向关系。结果与Con组比较,MPP+组细胞PART1表达降低(0.31±0.03 vs 1.00±0.00,P=0.000),miR-448表达升高(2.86±0.21 vs 1.00±0.00,P=0.000),且MPP+组细胞MDA含量升高,GSH含量降低,细胞凋亡率升高(P<0.05)。与空白组比较,转染组PART1表达升高,MDA含量降低,GSH含量升高,细胞凋亡率降低,cleaved-Caspase3和cleaved-Caspase9蛋白表达降低(P<0.05)。与阴性组比较,干扰组细胞miR-448表达降低,MDA含量降低,GSH含量升高,细胞凋亡率降低,cleaved-Caspase3和cleaved-Caspase9蛋白表达降低(P<0.01)。与转染对照组比较,转染实验组miR-448表达升高,MDA含量升高,GSH含量降低,细胞凋亡率升高,cleaved-Caspase3和cleaved-Caspase9蛋白表达升高(P<0.01)。PART1可靶向调控miR-448的表达。结论lncRNA PART1过表达通过靶向下调miR-448抑制MPP+诱导的帕金森模型细胞氧化应激和凋亡。

Objective To study the effect of lncRNA PART1 on MPP^(+)-induced oxidative stress and apoptosis of Parkinson’s model cells and its molecular mechanism.Methods SK-N-SH cells were divided into control group, MPP^(+)group, blank group, transfection group, negative group, interference group, transfection control group, transfection experimental group.The Parkinson’s model cells in the last 7 groups were treated in 100 μmol/L MPP^(+).The expressions of lncRNA PART1 and miR-448 in Parkinson’s model cells were detected by RT-qPCR.The serum MDA and GSH levels were measured by ELISA.The apoptosis of Parkinson’s model cells was assayed by flow cytometry.The expressions of cleaved Caspase-3 and 9 protein were detected by Western blot.The targeting relationship between lncRNA-PART1 and miR-448 was studied in dual leuciferase reporter experiment.Results The expression level of PART1 was lower while that of miR-448 was higher in MPP^(+)group than in control group(0.31±0.03 vs 1.00±0.00,P=0.000;2.86±0.21 vs 1.00±0.00,P=0.000).The serum level of MDA was higher while that of GSH was lower and the cell apoptosis rate was higher in MPP^(+)group than in control group(P<0.05).The expression level of PART1 was higher while that of cleaved Caspase-3 and 9 protein and the apoptosis rate of Parkinson’s model cells were lower, the serum level of MDA was lower while that of GSH was higher in transcription group than in blank group(P<0.05).The expression levels of miR-448 and cleaved Caspase-3 and 9 protein and apoptosis rate of Parkinson’s model cells were lower, the serum level of MDA was lower while that of GSH was higher in interference group than in negative group(P<0.01).The expression level of miR-448 and cleaved Caspase-3 and 9 protein, and the apoptosis rate of Parkinson’s model cells were higher, the serum level of MDA was higher while that of GSH was lower in transcription group than in transfection group(P<0.01).PART1 could target and regulate the expression of miR-448.Conclusion Overexpression of lncRNA-PART1 inhibits MPP^(+)-induced oxidative stress and apoptosis of Parkinson’s model cells by down-regulatiing the targeted miR-448.