shRNA干扰BAMBI对非小细胞肺癌生长、侵袭和迁移的调控作用

Effect of shRNA interfering BAMBI on growth,invasion and migration of non-small cell lung cancer

目的:探究shRNA干扰骨形成蛋白和激活素的穿膜抑制剂(BAMBI)对非小细胞肺癌A549细胞生长、侵袭和迁移的影响及其潜在的分子机制。方法:构建shRNA BAMBI质粒和shRNA scramble载体。将构建好的质粒转染A549细胞后,实时定量PCR(qRT-PCR)和蛋白质印迹检测转染效果;细胞计数试剂盒8(CCK-8)检测细胞增殖;流式细胞术检测细胞凋亡情况;划痕实验检测细胞迁移能力;体外侵袭实验检测细胞侵袭能力;蛋白质印迹检测Ki67、Bax、cl-caspase-3、血管内皮生长因子(VEGF)和基质金属蛋白酶9(MMP-9)的表达;蛋白质印迹检测TGF-β、Smad2和磷酸化Smad2的表达。构建移植瘤裸鼠,检测肿瘤体积、细胞凋亡和VEGF的表达水平。结果:转染后shRNA BAMBI组细胞中BAMBI的mRNA和蛋白水平均明显低于Scramble组。与Scramble组相比,shRNA BAMBI组细胞增殖倍数显著降低;凋亡细胞比率显著增加;划痕愈合率显著降低;侵袭细胞数目显著减少;Ki67、VEGF、MMP-9的蛋白表达水平显著降低,Bax的表达水平显著增加;TGF-β的表达水平和p-Smad2/Smad2的比值均显著升高。动物实验结果显示,与Scramble组相比,shRNA BAMBI组肿瘤体积明显减少,细胞凋亡比率显著增加,VEGF的表达水平显著降低。结论: shRNA干扰BAMBI抑制非小细胞肺癌增殖、侵袭和迁移并诱导细胞凋亡,其作用机制可能与TGF-β/Smad2信号通路的激活相关。

Objective: To investigate the effect of shRNA interference with BAMBI on the growth,invasion and migration of non-small cell lung cancer A549 cells and its potential molecular mechanism. Methods: shRNA BAMBI plasmid and shRNA scramble vector were constructed.After the constructed plasmid was transfected into A549 cells,the transfection effect was detected by qRT-PCR and Western blot.Cell proliferation was detected by cell counting kit 8 (CCK-8).Apoptosis was detected by flow cytometry.Cell migration was detected by scratch test.Cell invasion was detected by in vitro invasion test.The expression level of Ki67,VEGF,MMP-9,TGF-β,Smad2 and phosphorylated Smad2 were detected by Western blot. Results: After transfection,the levels of BAMBI mRNA and protein in shRNA BAMBI group were significantly lower than those in Scramble group.Compared with Scramble group,shRNA BAMBI group had significantly decreased cell proliferation,increased apoptotic cell ratio,significantly decreased scratch healing rate,significantly decreased number of invasive cells,significantly decreased expression of Ki67,VEGF,MMP-9,and significantly increased expression of TGF-β and the values of p-Smad2/Smad2 increased significantly.Animal model results showed that compared with Scramble group,tumor size was significantly reduced,apoptosis rate was significantly increased and VEGF expression level was significantly decreased in shRNA BAMBI group. Conclusion: shRNA interference with BAMBI inhibits proliferation,invasion,migration and induces apoptosis of non-small cell lung cancer,which may be related to the activation of TGF-β/Smad2 signaling pathway.