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目的 :探讨骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)是否可以通过BMPs/SMAD信号通路抑制人肺腺癌细胞A549的增殖。方法 :采用慢病毒感染的方式将外源性BMP9转入人肺腺癌A549细胞中,并应用半定量RT-PCR及蛋白质印迹法检测病毒感染后A549细胞中BMP9表达的改变。采用MTT法和FCM法检测BMP9过表达对A549细胞增殖和周期的影响。蛋白质印迹法检测BMPs/SMAD信号通路中总SMAD1/5/8和磷酸化SMAD1/5/8蛋白表达水平的改变。半定量RT-PCR及蛋白质印迹法检测BMP9过表达对分化抑制因子3(inhibitor of dif erentiation 3,ID3)m RNA和蛋白表达的影响。将构建有BMP9的慢病毒和构建有SMAD4-sh RNA的慢病毒共感染A549细胞,以阻断BMPs/SMAD通路的激活,采用蛋白质印迹法检测磷酸化SMAD1/5/8和ID3表达的改变。结果 :与空白对照组和阴性对照组相比,感染BMP9慢病毒后A549细胞中BMP9 m RNA和蛋白表达水平明显升高(P值均<0.05)。MTT法和FCM法检测结果显示,病毒感染3 d后,BMP9过表达组吸光度(D)值均较空白对照组的和阴性对照组明显降低(P值均<0.05);细胞周期阻滞在G2/M期,G2/M期细胞所占的比例均较空白对照组和阴性对照组明显提高(P值均<0.05)。蛋白质印迹法检测结果提示,BMP9过表达可使磷酸化SMAD1/5/8和ID3蛋白的表达水平明显上调,激活BMPs/SMAD通路。当干扰BMPs/SMAD信号通路关键分子SMAD4的表达后,可抑制BMP9激活的BMPs/SMAD通路,下调磷酸化SMAD1/5/8和ID3蛋白的表达水平(P值均<0.05)。
Objective: To investigate whether bone morphogenetic protein 9 (BMP9) can inhibit the proliferation of human lung adenocarcinoma A549 cells via BMPs / SMAD signaling pathway. Methods: Exogenous BMP9 was transfected into human lung adenocarcinoma A549 cells by lentivirus infection. The expression of BMP9 in A549 cells was detected by semi-quantitative RT-PCR and Western blotting. The effects of BMP9 overexpression on the proliferation and cell cycle of A549 cells were detected by MTT assay and FCM assay. Western blotting was used to detect the expression of total SMAD1 / 5/8 and phosphorylated SMAD1 / 5/8 protein in BMPs / SMAD signaling pathway. Semiquantitative RT-PCR and Western blotting were used to detect the effect of BMP9 overexpression on mRNA and protein expression of inhibitor of dif erentiation 3 (ID3). A549 cells were co-infected with lentivirus constructed with BMP9 and lentivirus constructed with SMAD4-sh RNA to block the activation of BMPs / SMAD pathway. The changes of phosphorylated SMAD1 / 5/8 and ID3 expression were detected by Western blot. Results: Compared with the blank control group and negative control group, the expression of BMP9 mRNA and protein in A549 cells was significantly increased after infected with BMP9 lentivirus (all P <0.05). The results of MTT assay and FCM assay showed that the absorbance (D) of BMP9 overexpression group was significantly lower than that of blank control group and negative control group (all P <0.05) 3 days after virus infection; the cell cycle arrest was at G2 / M phase, G2 / M phase cells were significantly increased compared with the blank control group and negative control group (P all <0.05). The results of Western blotting showed that BMP9 overexpression significantly upregulated phosphorylated SMAD1 / 5/8 and ID3 proteins and activated BMPs / SMAD pathway. After interfering with the expression of SMAD4, a key molecule of BMPs / SMAD signaling pathway, it inhibited BMP9-activated BMPs / SMAD pathway and down-regulated the expression of phosphorylated SMAD1 / 5/8 and ID3 protein (all P <0.05).