miRNA-133b-3p对PC12细胞P2rx4的表达调控及细胞钙活动的实验观察

Effect of miRNA-133b-3p on P2rx4 Expression and Calcium Mobilization in PC12 Cells

目的:从离体细胞水平观察miRNA-133b-3p调控P2rx4表达对神经元钙活动的影响。方法:用转染试剂将miRNA-133b-3p mimic和miRNA-133b-3p inhibitor转入PC12细胞中。转染48 h后,用荧光定量PCR的方法检测PC12细胞中miRNA-133b-3p的含量,P2rx4 mRNA的表达变化;用免疫印迹的方法检测P2rx4受体蛋白的表达变化;用胞内钙成像技术检测ATP孵育后细胞中钙离子浓度的变化。结果:转染48 h后,与阴性对照组相比,加入miRNA-133b-3p mimic后PC12细胞中miRNA-133b-3p的含量明显增加,P2rx4 mRNA和P2rx4蛋白的表达减少,ATP孵育后细胞中钙离子浓度的升高明显减弱。转染inhibitor后,细胞内miRNA-133b-3p的含量明显减少,P2rx4 mRNA和P2rx4蛋白的表达增加,ATP孵育后胞内钙离子浓度的升高明显增强。结论:miRNA-133b-3p通过负向调控P2rx4的表达来影响细胞钙活动。

Objective：To observe the effect of miRNA-133b-3 p on the calcium mobilization of neurons by regulating the expression of P2rx4.Method：Transfection reagents and miRNA-133b-3p mimic or inhibitor were added into the culture medium of PC12 cell.After 48 hours of transfection,the expression of miRNA-133b-3 p and P2rx4 mRNA were measured by real-time PCR.Western blot method was used for quantitative analysis on P2rx4 protein.The mobilization of intracellular calcium was measured with calcium imaging technique.Result：After 48 hours of transfection,compared with the negative control group,after adding miRNA-133b-3 p mimic,the expression of miRNA-133b-3 p in PC12 cells was significantly increased, the expressions of P2rx4 mRNA and P2rx4 protein were significantly decreased,and calcium ion concentration increase induced by ATP was decreased obviously.After adding miRNA-133b-3 p inhibitor,the expression of miRNA-133b-3 p was decreased significantly,and the expressions of P2rx4 mRNA and P2rx4 protein were significantly increased,the calcium ion concentration increase induced by ATP was enhanced obviously.Conclusion：miRNA-133b-3 p downregulation of P2rx4 affectes intracellular calcium activity and is involved in chronic pain.