抑制miR-181a对鱼藤酮诱导的SH-SY5Y多巴胺能细胞损伤的保护作用

Inhibition of mi R-181a expression protects dopaminergic SH-SY5Y cells against the rotenone-induced cell injury

目的:通过抑制mi R-181a表达研究其对鱼藤酮诱导SH-SY5Y的细胞损伤、内质网应激以及未折叠蛋白反应(unfolded protein response,UPR)的影响,为mi R-181a在帕金森病治疗方面提供初步的实验依据。方法:不同浓度的鱼藤酮诱导SH-SY5Y作用12、24、48、72 h;mi R-181a类似物和mi R-181a抑制剂及0.4μmol/L鱼藤酮诱导SH-SY5Y作用24 h;采用CCK-8法检测细胞存活率;Annexin V/PI双染检测细胞凋亡;双荧光素酶报告基因检测mi R-181a与葡萄糖调节蛋白78(glucose regulated protein 78 k D,GRP78)的靶标关系;q RT-PCR检测mi R-181a和GRP78 m RNA表达水平;Western Blot检测GRP78、肌醇需酶1α(inositol-requiring enzyme 1,IRE1α)、X-盒结合蛋白1(X box-binding protein 1,XBP1)和CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein-homologous protein,CHOP)的蛋白表达水平。结果:(1)CCK-8结果显示:随着鱼藤酮浓度(>0.1μmol/L)增加,SH-SY5Y存活率降低,并存在浓度和时间依赖性;其中0.4μmol/L作用24 h后存活率已降为0.6(P<0.05),加入mi R-181a抑制剂后细胞存活率升高达0.8(P<0.05);(2)Annexin V/PI双染结果显示:抑制mi R-181a表达,细胞的凋亡降低至0.23(P<0.05);(3)双荧光素酶报告基因检测显示:mi R-181a与GRP78存在直接的靶标关系;(4)q RT-PCR结果显示:0.4μmol/L鱼藤酮作用24 h后细胞mi R-181a呈高表达(P<0.05),抑制mi R-181a表达可上调GRP78 m RNA水平(P<0.05);(5)Western Blot结果显示:抑制mi R-181a表达可上调GRP78和下调p IRE1α、XBP1和CHOP的蛋白表达(P<0.05)。结论:抑制mi R-181a表达可减轻鱼藤酮诱导的SH-SY5Y细胞损伤,其机制可能通过上调GRP78进而调节内质网应激以及抑制UPR相关信号通路,从而发挥对神经元细胞的保护作用。

Objective： To investigate the effect of inhibition mi R-181 a on the cell injury,endoplasmic reticulum stress and the unfolded protein response（ UPR） in SH-SY5 Y cells induced by rotenone and to provide preliminary experimental basis for treatment of Parkinson＇s disease（ PD） with mi R-181 a. Methods： SH-SY5 Y cells were treated with various concentrations of rotenone for 12,24,48 h and 72 h; SH-SY5 Y cells were treated with mi R-181 a mimics and inhibitor for 24 h in the presence with 0. 4 μmol / L rotenone. Cell survival of SH-SY5 Y cells was detected by CCK-8 assay. Cell apoptosis was tested with Annexin V / propidium iodide（ PI） double staining method. The target relationship between mi R-181 a and GRP78 was analyzed using dual-luciferase reporter assay. The expression levels of mi R-181 a and GRP78 m RNA were examined by q RT-PCR. The protein expression levels of glucose regulated protein 78 k D（ GRP78）,inositol-requiring en-zyme 1α（ p IRE1α）,CCAAT / enhancer-binding protein-homologous protein（ CHOP） and X box-binding protein 1（XBP1） were detected by Western Blot. Results：（1） CCK-8 results showed that the cell survival rate of rotenone-induced SH-SY5 Y was gradually decreased with the increasing rotenone concentration（ 〉0. 1 μmol / L） in time- and dosedependent manner,and cell survival rate was decreased at 0. 6 with 0. 4 μmol / L rotenone for 24 h（ P 〈0. 05）. Treatment of mi R-181 a inhibitor significantly promoted cell survival rate to almost 0. 8.（ 2） Annexin V / PI double staining showed that inhibiting expression of mi R-181 a decreased cell apoptosis to 0. 23（ P 〈0. 05）.（3） Dual-luciferase reporter assay showed that the mi R-181 a and GRP78 had a directly targeting relationship.（4） q RT-PCR analysis showed that the expression of mi R-181 a was significantly upregulated by treatment with 0. 4 μmol / L rotenone for 24 h and transfection with mi R-181 a inhibitor significantly increased GRP78 m RNA expression.（5） Western Blot showed that mi R-181 inhibitor transfection significantly increased the protein expression of GRP78 and markedly decreased the protein expression of p IRE1α,XBP1 and CHOP（ P 〈0. 05）. Conclusion： Inhibition of mi R-181 a expression can alleviate the cell injury induced by rotenone in SH-SY5 Y cells and the potential underlying mechanism may be associated with promoting GRP78 expression which regulates endoplasmic reticulum stress and prevents UPR signaling pathway to protect neurons.