CyclinD1反义核酸诱导肺腺癌细胞A549的凋亡

Anti-sense nucleic acid of CyclinD1 induces apoptosis of lung adenocarcinoma cancer cell A549

为了探讨CyclinD1反义核酸在肺癌基因治疗中的作用,本研究构建了含有CyclinD1反义核酸的表达载体(命名为:pcDNA3.1-CyclinD1),并将该载体转染肺腺癌细胞A549,进行G418筛选后,获得稳定表达CyclinD1反义核酸的A549细胞(命名为:anti-CyclinD1-A549)。通过MTT法检测细胞增殖,流式细胞术检测细胞的凋亡情况,结果显示稳定转染CyclinD1反义核酸后,A549细胞增殖显著被抑制,细胞凋亡增加。为探讨CyclinD1反义核酸诱导细胞凋亡的机理,应用Westernblot的方法检测了细胞内视网膜母细胞瘤蛋白质(retinoblastoma protein,pRb)、adenovirus E2 factor-1(E2F-1)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、基质金属蛋白酶(matrix metalloproteinase,MMP)-2和MMP-9的表达,其结果显示,稳定转染CyclinD1反义核酸后,A549细胞内pRb、E2F-1、VEGF、MMP-2和MMP-9蛋白表达均显著降低。以上结果表明,Cy-clinD1反义核酸可引起肺癌细胞的凋亡,其机制可能与细胞内pRb、E2F-1、VEGF、MMP-2和MMP-9的表达降低相关。

To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy,the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1.The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors.After being screened by G418,the stable expression positive clones were obtained.MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis,respectively.The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth,compared with negative control and empty vector groups.To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis,the expression levels of retinoblastoma protein（pRb）,adenovirus E2 factor-1（E2F-1）,vascular endothelial growth factor（VEGF）,matrix metalloproteinase（MMP）-2 and MMP-9 were detected by Western blot,and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group,compared with those in negative control and empty vector groups.In a word,anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells,and the depressions of pRb,E2F-1,VEGF,MMP-2 and MMP-9 expressions may be the possible mechanism.