siRNA 沉默 ERK5基因对 MC3 T3-E1成骨细胞增殖及BMP2／BMP7的影响

摘要

Objective To investigate the effect of extracellular signal-regulated kinase 5 ( ERK5 ) signaling pathway on cell proliferation and BMP2/BMP7 expression in MC3T3-E1 osteoblasts.Methods The technique of small interfering RNA ( siRNA) was used to silence the gene expression of ERK5.In order to select the optimal silencing effect of ERK5 siRNA, the concentrations of 20, 40, 60, and 80 nmol/L ERK5 siRNA were used to culture MC3T3-E1 osteoclasts, respectively.Cell proliferation at the 12th hour, 24th hour, 36th hour, and 48th hour after the transfection was detected using MTT assay.The expression of ERK5, BMP2, and BMP7 mRNA was detected using real-time PCR ( RT-PCR ) .The expression of BMP2 and BMP7 protein was detected using Western blotting.Results The expression of ERK5 mRNA reduced significantly with 60 nmol/L siRNA, and the silence rate was 76.8 ±2.2%(P<0.01).After silencing ERK5 gene, the proliferation of MC3T3-E1 osteoblasts was inhabited at the 12th hour, 24th hour, and 36th hour (P<0.05), but no significant difference at the 48th hour was observed (P>0.05).After 24h transfection with ERK5 siRNA, the expression of BMP2/BMP7 mRNA and protein decreased significantly in ERK5 siRNA group when compared with that in the blank group and siRNA control groups (P<0.05).Conclusion ERK5 siRNA can significantly inhibit cell proliferation and the expression of BMP2/BMP7 mRNA and protein in MC3T3-E1 osteoblasts.%目的：观察ERK5信号通路对MC3T3-E1成骨细胞的增殖，及功能分化蛋白BMP2/BMP7的影响。方法应用小RNA分子干扰技术（ siRNA）沉默ERK5基因，分别将浓度为20、40、60、80 nmol/L的ERK5 siRNA干预MC3T3-E1成骨细胞，筛选ERK5 siRNA转染的最佳浓度。噻唑蓝实验（ MTT）检测转染后12 h、24 h、36 h和48 h细胞的增殖情况，实时荧光定量PCR检测ERK5、BMP2和BMP7 mRNA的表达。 Westernblot 检测不同干预组BMP2及 BMP7蛋白的表达变化。结果当 ERK5 siRNA浓度为60 nmol/L时，MC3T3-E1成骨细胞的ERK5 mRNA的表达下降最为显著，沉默率为76．8±2．2％（P＜0．01）。ERK5 siRNA转染后12 h、24 h、36 h，成骨细胞的增殖受到明显抑制（ P＜0．05），但48 h转染组未发现明显变化（ P＞0．05）。ERK5 siRNA转染24 h后，实时荧光定量PCR结果显示：空白组与对照siRNA组成骨细胞的BMP2/BMP7 mRNA变化水平无显著性差异（P＞0．05），但ERK5 siRNA转染组的BMP2/BMP7 mRNA明显降低（P＜0．05）；Westernblot结果显示，空白组与对照siRNA组成骨细胞的BMP2/BMP7蛋白未发现明显变化（ P＞0．05），但转染组成骨细胞的BMP2/BMP7显著降低（ P＜0．05）。结论 ERK5 siRNA对MC3T3-E1成骨细胞的增殖以及骨形态蛋白BMP2和BMP7的表达有明显抑制作用。