PARP1对黑素瘤细胞克隆形成的影响及其作用机制

The effect of PARP1 on the colony formation of melanoma cells and its possible mechanism

目的:探究聚二磷酸腺苷核糖多聚酶1[poly (ADP-ribose) polymerase 1,PARP1]对黑素瘤细胞克隆形成的影响及可能的作用机制。方法:在黑素瘤A2058细胞系中利用小干扰RNA干涉PARP1表达水平,利用平板克隆形成实验观察干涉PARP1对黑素瘤细胞克隆形成的影响。提取PARP1干涉片段及对照片段转染细胞的总RNA进行全基因组表达谱芯片检测。Realtime RT-PCR对部分差异性基因进行验证。应用DAVID数据库对差异性基因进行GO及KEGG通路富集分析。Realtime RT-PCR对富集分析结果中可疑靶基因进行验证。结果:干涉PARP1表达可显著抑制黑素瘤细胞克隆形成。全基因组芯片结果显示干涉PARP1表达可引起128个基因表达上调,77个基因表达下调。通过Realtime RT-PCR对部分差异表达基因进行验证,结果表明与芯片筛选结果一致。GO和KEGG富集分析结果显示PARP1调控的差异性表达基因在生物学功能及参与的信号通路中存在部分交集,即MAPK信号通路及其正向调控机制。双特异性磷酸酶5(DUSP5)作为MAPK通路中的抑制分子,Realtime RT-PCR证实干涉PARP1可促进其表达水平升高。结论:干涉PARP1可能通过促进DUSP5表达从而抑制MAPK信号通路活性,进而发挥抑制黑素瘤细胞克隆形成的作用。

Objective:To detect the effect of PARP1 on the colony formation of melanoma cells and its possible mechanism.Methods:The expression level of PARP1 was knocked down by small interfering RNA(siRNA) in the melanoma A2058 cell line.The colony formation assay was performed to determine the effect of PARP1 to A2058 cells.Total RNA of cells transfected with PARP1 siRNA and control siRNA was isolated for whole-genome expression spectrum chip detection.Realtime RT-PCR was used to verify some differentially expressed genes.DAVID database was used to carry out GO and KEGG pathway enrichment analysis of differentially expressed genes.Realtime RT-PCR was used to verify the suspicious target gene in the enrichment analysis results.Results:The colonies formation of melanoma cells were significantly inhibited in the group transfected with PARP1 siRNA.Whole-genome microarray results showed that knockdown of PARP1 expression could induce up-regulation of 128 genes and down-regulation of 77 genes.Some differentially expressed genes were verified by Realtime RT-PCR and the results were consistent with chip screening.The results of GO and KEGG enrichment analysis showed that the differentially expressed genes had partial intersections in the biological function and the signal pathways.That were MAPK signaling pathway and its positive regulation.DUSP5 as an inhibitory molecule in MAPK pathway,was upregulated after PARP1 knockdown.Conclusion:PARP1 knockdown may inhibit MAPK signaling pathway activity by promoting the expression of DUSP5 and thereby play a role in inhibiting the colony formation of melanoma cells.