shRNA-FAM38A基因对肺癌A549细胞系增殖、迁移和凋亡的影响

Effect of shRNA-FAM38A gene on proliferation,migration and apoptosis of lung cancer A549 cells

目的构建shRNA-FAM38A慢病毒载体,对肺腺癌A549细胞中FAM38A基因表达进行沉默干扰,研究其对肿瘤细胞增殖、迁移和凋亡的影响。方法采用慢病毒载体构建的方法,构建shRNA-FAM38A慢病毒载体,免疫组织化学染色检测FAM-38A的表达; RT-qPCR用来检测凋亡因子Bcl-2,Caspase-3和Caspase-9的表达量; CCK-8试剂盒检测肺癌细胞的增殖; Transwell小室检测肺癌细胞的迁移; AV-PI凋亡试剂盒检测肺癌细胞的凋亡。结果 (1)慢病毒转染效率较高,以肺癌细胞A549为靶点种子细胞,慢病毒转染率为90%。这说明慢病毒转染细胞具有较高的转染效率。(2)免疫组织化学方法检测到FAM-38A可以在肺癌组织中过度表达;(3)阴性对照组(shRNAFAM38A-Con),空质粒组,shRNAFAM38A1组和shRNAFAM38A 2组的FAM38A,凋亡因子Bcl-2,Caspase-3和Caspase-9的mRNA的相对表达量具有统计学差异(F=17. 967; P <0. 05)。shRNA FAM-38A1组中FAM38A,凋亡因子Bcl-2,Caspase-3和Caspase-9的mRNA的相对表达量与shRNAFAM38A 2组相比,表达量降低,表明shRNA FAM-38A1可以有效抑制FAM38A的表达,抑制Bcl-2的表达,促进Caspase-3和Caspase-9的表达,进而促进肿瘤细胞凋亡。然而shRNA FAM-38A2是个无效干扰序列,没有起到沉默FAM38A基因表达的作用。(4) Transwell实验,CCK-8实验和AV-PI实验的结果显示,shR-NA FAM-38A1可以抑制肺癌肿瘤细胞的增殖,抑制肿瘤细胞的迁移,并且可以促进肿瘤细胞的过度凋亡。结论 shRNA FAM38A1可以有效的抑制肺癌细胞的迁移和增殖,并且提高其凋亡率。

Objective To study its effect on proliferation,migration and apoptosis of tumor cells by constructing shRNA-FAM38A lentivirus vector to interfere with the expression of FAM38A gene in lung adenocarcinoma A549 cells.Methods It used constructing lentivirus vector method to construct shRNA-FAM38A lentivirus vector,and the expression of FAM-38A was detected by immunohistochemistry.RT-qPCR was used to detect the expression of apoptosis factor Bcl-2 Caspase-3 and Caspase-9.CCK-8 kit was used to detect the proliferation of lung cancer cells.Transwell chamber was used to detect the migration of lung cancer cells and AV-PI apoptosis kit was used to detect the apoptosis of lung cancer cells.Results (1)The efficiency of lentivirus transfection was high,and the lentivirus transfection efficiency was 90 when the lung cancer cell A549 was used as the target seed cell.It indicated that lentivirus transfection cells had higher transfection efficiency.(2)The expression of FAM-38A in lung cancer tissues was detected by immunohistochemistry.(3)In the negative control group,the empty plasmid group,the shRNAFAM38A1 group and the shRNAFAM38A2 group,the relative expression of mRNA of Bcl-2Caspase-3 and Caspase-9 were significantly different(P<0.05).The relative expression of FAM38A,Bcl-2Caspase-3 and Caspase-9 mRNA had the same trend.The shRNA FAM-38A1 group was lower than that the shRNAFAM38A2 group,and the relative expression of FAM38A,Bcl-2,Caspase-3 and Caspase-9 in the FAM-38A1 group was lower than that in the shRNAFAM38A2 group,and the relative expression of FAM38A,Bcl-2,Caspase-3 and Caspase-9 in the FAM-38A1 group was significantly lower than that in the shRNAFAM38A2 group.These Results suggested that shRNA FAM-38A1 could effectively inhibit the expression of FAM38A and Bcl-2,promote the expression of Caspase-3 and Caspase-9,and then promote the apoptosis of tumor cells.However,shRNA FAM-38A2 was an invalid interference sequence and did not silence the expression of FAM38A gene.(4)The Results of Transwell assay,CCK-8 test and AV-PI assay showed that shRNA FAM38A1 could inhibit the proliferation of lung cancer cells,inhibit the migration of tumor cells,and promote the excessive apoptosis of tumor cells.Conclusion shRNA-FAM38A can effectively inhibit the migration and proliferation of lung cancer cells and increase its apoptosis rate.