miR-320a调控水通道蛋白4对阿尔茨海默病细胞模型的影响

The effect of aquaporin 4 regulated by microRNA-320a on a cell model of Alzheimer's disease

目的探讨miR-320a调控水通道蛋白4(AQP4)对阿尔茨海默病细胞模型的影响。方法采用神经生长因子(NGF)诱导大鼠肾上腺嗜铬细胞瘤细胞株(PC12)为神经元细胞,观察PC12组和神经元细胞组的细胞形态。采用β淀粉样蛋白(Aβ)处理诱导的神经元细胞,构建AD细胞模型。根据处理因素的不同将培养细胞分为对照组(不做特殊处理)、miR-320a模拟剂组(加入miR-320a模拟剂50 nmol/L)、miR-320a抑制剂组(加入miR-320a抑制剂50 nmol/L)、Aβ处理组(加入Aβ)、Aβ+miR-320a模拟剂组(加入Aβ+miR-320a模拟剂50 nmol/L)和Aβ+miR-320a抑制剂组(加入Aβ+miR-320a抑制剂50 nmol/L)。采用CCK8法检测细胞活力。利用RT-PCR检测目标基因AQP4、miR-320a、Bax、B淋巴细胞瘤-2基因(Bcl-2)mRNA的相对表达。采用免疫印迹法检测目标基因AQP4、Bax、Bcl-2蛋白的相对表达。结果PC12经NGF诱导后,与PC12组比较,神经元细胞组泛素羧基末端水解酶-L1(Uch-L1)基因表达明显下调,神经丝蛋白(NFP)、微管结合蛋白2(MAP2)、AQP4基因表达明显上调,MAP2和AQP4蛋白相对表达明显增高。造模后,与对照组比较,Aβ处理组神经元细胞凋亡增加,细胞活力明显减低,miR-320a、AQP4、Bcl-2的mRNA及AQP4、Bcl-2蛋白表达明显减少,Bax的mRNA及蛋白表达明显增加。与Aβ处理组比较,Aβ+miR-320a模拟剂组,细胞活力明显增加,miR-320a、AQP4、Bcl-2的mRNA及AQP4、Bcl-2蛋白表达明显增加、Bax的mRNA及蛋白表达明显降低。与Aβ+miR-320a模拟剂组比较,Aβ+miR-320a抑制剂组细胞活力明显降低,miR-320a、AQP4、Bcl-2的mRNA及AQP4、Bcl-2蛋白表达明显降低,Bax的mRNA及蛋白表达明显升高。结论miR-320a可以上调AD细胞模型中AQP4的表达,减少细胞凋亡,增加细胞存活率。

Objective To explore the effect of aquaporin 4(AQP4)regulated by miR-320a on a cell model of Alzheimer′s disease.Methods A rat adrenal pheochromocytoma cell line(PC12)was induced into neurons using nerve growth factor(NGF).The morphology of the PC12 cells and the neurons was observed,and ubiquitin carboxy terminal hydrolase L1(Uch-L1)and neurofilament protein(NFP)were detected.Levels of microtubule-associated protein(MAP2)and AQP4 target genes were related to the mRNA expression of NFP to determine the neuron-inducing effect.The neurons were then randomly divided into a control group(given no treatment),an miR-320a mimic transfection group(cultured by adding 50nmol/L miR-320a as a mimic agent),an miR-320a inhibitor group(cultured by adding 50nmol/L miR-320a as an inhibitor),an Aβtreatment group(cultured by adding Aβ),an Aβ+miR-320a mimic group(cultured by adding both 50nmol/L miR-320a and Aβ),and an Aβ+miR-320a inhibitor group(also cultured by adding Aβ,but with 50nmol/L miR-320a as an inhibitor).Cell activity was measured by the CCK8 method.Reverse-transcription polymerase chain reactions were used to detect the relative expression of the target gene miR-320a,AQP4,B-cell bcl2-associated X(Bax),and B-cell bcl-2(Bcl-2)mRNA.Western blotting was employed to detect the relative expression of AQP4,Bax and Bcl-2 proteins.Results After PC12 was induced by 50μg/L NGF,the expression of Uch-L1 genes in the induced neuron was significantly down-regulated compared with the PC12.The expressions of NFP,MAP2 and AQP4 genes were significantly up-regulated,and the relative expressions of MAP2 and AQP4 proteins increased significantly.Compared with the control group,the apoptosis and cell activity of neurons in the treatment group increased,the mRNA and protein expressions of miR-320a,AQP4,bcl-2,AQP4 and Bcl-2 decreased significantly,and the mRNA and protein expressions of Bax increased significantly.Compared with the Aβ-treated group,the cell activity of the Aβ+Mir-320a mimic group increased significantly,the mRNA and protein expressions of miR-320a,AQP4 and Bcl-2 increased significantly,and the mRNA and protein expressions of Bax decreased significantly.Compared with the Aβ+miR-320a mimic group,the cell activity of the Aβ+miR-320a inhibitor group decreased significantly,the mRNA and protein expressions of miR-320a,AQP4 and Bcl-2 decreased significantly,and the mRNA and protein expressions of Bax increased significantly.Conclusion miR-320a can up-regulate AQP4 expression in a cell model of Alzheimer′s disease,reduce apoptosis and increase the cell survival rate.