RNA干扰血管平滑肌细胞高血压相关基因FXYD5表达及其功能研究

目的：通过RNA干扰（RNAi）技术特异性沉默大鼠胸主动脉平滑肌细胞（CRL-1444）高血压相关基因FXYD5的表达,探索该基因在高血压发病机制中的作用.方法：设计并合成4个针对大鼠FXYD5基因的特异性siRNA片段（siRNA-1、siRNA-2、siRNA-3、siRNA-4）,以含非特异性编码序列的siRNA-con为对照.将上述4个片段及阴性对照通过阳离子脂质体法转染进大鼠胸主动脉平滑肌细胞株（CRL-1444）内,采用CCK-8法检测FXYD5基因特异性沉默后对平滑肌细胞增殖的影响,采用划痕法（Woundhealing法）检测FXYD5基因特异性沉默后对细胞迁移力的影响,采用超微量Na＋-K＋-ATPase测试盒检测FXYD5基因特异性沉默后对细胞膜Na＋-K＋-ATPase活性的影响.结果：荧光实时定量PCR结果显示siRNA-1使FXYD5的mRNA表达与空白对照组相比下降86.71％（P＜ 0.01）；siRNA-2使FXYD5的mRNA表达与空白对照组相比下降90.17％（P＜ 0.01）；siRNA-con对照组与空白对照组FXYD5的mRNA表达差异无统计学意义（P＞ 0.05）.CCK-8法检测结果显示siRNA-1、siRNA-2两组与对照组间细胞增殖能力差异无统计学意义（P＞0.05）.划痕法检测细胞迁移力结果显示siRNA-1和siRNA-2两组平滑肌细胞迁移力低于siRNA-con对照组和空白对照组,差异有统计学意义（P＜ 0.01）.Na＋-K＋-ATPas活性检测结果显示siRNA-1和siRNA-2两组平滑肌细胞Na＋-K＋-ATPas活性低于siRNA-con对照组（P＜ 0.05）和空白对照组（P＜0.01）.结论：RNAi技术能特异性沉默大鼠胸主动脉平滑肌细胞的FXYD5基因的表达水平；特异性沉默FXYD5基因表达后血管平滑肌细胞的增殖能力无明显变化,而细胞迁移力和细胞膜的Na＋-K＋-ATPase活性明显降低.

Expression and its functional study on hypertension-related gene FXYD5 in smooth muscle cell by RNA interference

Objective：To explore the function of FXYD5 gene in the pathogenesis of hypertension by specifically silencing the FXYD5 expression in vascular smooth muscle cells （VSMCs）.Methods：Design and synthesize four siRNAs targeting FXYD5 （siRNA-1,siRNA-2,siRNA-3,siRNA-4）; transfect the rat thoracic artic smooth muscle cell line （CRL-1444） by using four synthesized siRNA and siRNA with nonspecific coding sequence （siRNA-con） as control group; Estimated the transfection efficiency through the laser scanning confocal microscope and analyze the mRNA expression of FXYD5 by real-time PCR and further analyze the cellular proliferation,mobility as well as Na＋-K＋-ATPase activity of CRL-1444 after specifically silencing the FXYD5 expression.Results：The results of real-time PCR showed FXYD5 mRNA was suppressed by 86.71％ （P＜0.01）and 90.17％ （P＜0.01） respectively by siRNA-1 and siRNA-2,while no distinctive change was found in siRNAcon treated VSMCs （P＞0.05）; The cellular proliferation among four groups was similar（P＞0.05）; but the mobility of siRNA-1 and siRNA-2 transfected groups was weaker than that in siRNA-con transfected group and blank control group （P＜0.01）; the activity of cell membrane Na＋-K＋-ATPase in siRNA-1 and siRNA-2 transfected groups was lower than that in siRNA-con transfected group and blank control group （P＜0.05）.Conclusion：RNA interference technology can selectively silence FXYD5 gene expression in rat aortic smooth muscle cells; and inhibit the migration of smooth muscle cells and cell membrane Na＋-K＋-ATPase activity.The result reveals the potential functions of FXYD5 in the pathogenesis of hypertension and leads to the inference that FXYD5 may be a new associative gene of hypertension.