缺血性脑卒中患者miR-146a表达及对钙调蛋白的影响

Expression of miR-146a and Its Effect on Calmodulin in Patients with Ischemic Stroke

目的探讨缺血性脑卒中患者miR-146a表达及对钙调蛋白的影响.方法选取急性缺血性脑卒中患者110例为缺血性脑卒中组,另收集健康志愿者110例为对照组,实时荧光定量PCR检测血清miR-146a表达水平;通过查询PicTar、Targetscan生物信息学数据库,预测miR-146a的靶基因为钙调蛋白编码基因CALM2;应用脂质体将mimics-miR-146a、inhibitor-miR-146a及相对应的阴性对照物分别转染至PC12细胞中进行培养,应用RT-PCR和Western blot检测Bcl-2同源拮抗蛋白/致死蛋白(Bax)、半胱天冬氨酸蛋白酶-9(Caspase-9)、B淋巴细胞瘤/白血病-2(Bcl-2)、钙调蛋白编码基因2(CALM2)的mRNA和蛋白表达水平.结果缺血性脑卒中组患者血清miR-146a表达水平明显低于对照组受试者(P<0.01);mimics-miR-146a转染组PC12细胞增殖能力明显高于对照组和mimics阳性对照组,PC12细胞凋亡率明显低于对照组和mimics阴性对照组(P<0.05);inhibitor-miR-146a转染组PC12细胞增殖能力明显低于对照组和inhibitor阴性对照组,PC12细胞凋亡率明显高于对照组和mimics阴性对照组(P<0.05).mimics-miR-146a转染组Bax、Caspase-9 m的RNA和蛋白表达水平明显低于对照组,Bcl-2的mRNA和蛋白表达水平明显高于对照组(P<0.05);inhibitor-miR-146a转染组Bax、Caspase-9的mRNA和蛋白表达水平明显高于对照组,Bcl-2的mRNA和蛋白表达水平明显低于对照组(P<0.05).mimics-miR-146a转染组CALM2的mRNA和蛋白表达水平明显低于对照组,inhibitor-miR-146a转染组CALM2的mRNA和蛋白表达水平明显高于对照组(P<0.05).结论缺血性脑卒中患者miR-146a表达下调,可能通过上调CALM2的表达而抑制神经细胞增殖,进而促进细胞凋亡,加重患者病情.

Objective To investigate the expression of miR-146a and its effect on calmodulin in patients with ischemic stroke.Method 110 patients with acute ischemic stroke were selected as the ischemic stroke group and 110 healthy volunteers as the control group.The expression level of serum miR-146a was detected by real-time fluorescence quantitative PCR;The bioinformatics databases of PiCtar and Targetscan were queried to predict the targeted binding relationship between miR-146a and calmodulin coding gene CALM2;Mimics-miR-146a,inhibitor-miR-146a and the corresponding negative control were transfected into PC12 cells for culture.Bcl-2 homologous antagonist/lethal protein(Bax),Caspase-9,B lymphocytoma/leukemia-2 and calmodulin coding gene 2(CALM2)were detected by RT-PCR and Western blot mRNA and protein expression levels.Results The expression level of serum miR-146a in ischemic stroke group was significantly lower than that in control group(P<0.01);The proliferation ability of PC12 cells in the mirics-miR-146a transfection group was significantly higher than that in the control group and the mirics positive control group,and the apoptosis rate of PC12 cells was significantly lower than that in the control group and the mimics negative control group(P<0.05);The proli-feration ability of PC12 cells in inhibitor-miR-146a transfection group was significantly lower than that in control group and inhibitor negative control group,and the apoptosis rate of PC12 cells was significantly higher than that in control group and mimics negative control group(P<0.05).The expression levels of Bax,Caspase-9 mRNA and protein in the mics-miR-146a transfection group were significantly lower than those in the control group,and the protein expression levels and Bcl-2 mRNA in the mics-miR-146a transfection group were significantly higher than those in the control group(P<0.05);The protein expression levels and Bax,Caspase-9 mRNA in inhibitor-miR-146a transfection group were significantly higher than those in the control group,and the protein expression levels of Bcl-2 mRNA and protein were significantly lower than those in the control group(P<0.05).The expression levels of CALM2 mRNA and protein in the mics-miR-146a transfection group were significantly lower than those in the control group,and the protein expression levels and CALM2 mRNA in the inhibitor-miR-146a transfection group were significantly higher than those in the control group(P<0.05).Conclusion The down-regulation of miR-146a expression in patients with ischemic stroke may inhibit nerve cell proliferation,promote apoptosis and aggravate the patient’s condition by up-regulating CALM2 expression.