POT1对黑色素瘤细胞生物学行为及DNA修复相关分子表达的研究

Study on the effect of POT1 on biological behavior of melanoma cells and the expression of DNA repair-related molecules

摘要

目的 探究端粒保护蛋白1（protection of telomeres 1,POT1）对黑色素瘤细胞生物学行为及DNA修复相关分子表达的影响。方法 使用小干扰RNA（small interfering RNA,siRNA）技术敲低黑色素瘤A2058和A375细胞中POT1的表达。通过细胞计数试剂盒8（cell counting kit-8,CCK8）实验、细胞划痕实验和transwell实验检测细胞增殖、迁移和侵袭能力。采用实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction,qRT-PCR）和蛋白质免疫印迹法验证敲低效率并检测线粒体凋亡相关分子包括B细胞淋巴瘤2（B-cell lymphoma 2,Bcl2）、Bcl2相关X蛋白（Bcl2-associated X protein,Bax）、半胱天冬酶3（cysteine-aspartic acid protease 3,Caspase3）及DNA损伤修复相关分子包括DNA连接酶3（DNA ligase 3,LIG3）、磷酸化组蛋白H2A.X（phosphorylated histone H2A.X,γH2A.X）、剪切型聚ADP核糖聚合酶1[cleaved poly（ADP-ribose） polymerase 1,c-PARP1]的表达水平。结果 与对照组相比,A2058细胞和A375细胞敲低组POT1 mRNA和蛋白表达水平显著下降（P<0.05）。CCK8结果显示2种细胞的敲低组在24 h、48 h、72 h和96 h的光密度（optical density,OD）值均低于对照组,组间、时点间、组间·时点间交互作用差异有统计学意义（P<0.05）。细胞划痕实验显示2种细胞敲低组的划痕愈合率显著低于对照组（P<0.05）。Transwell实验显示2种细胞的敲低组穿过基底膜的细胞数较对照组均显著降低（P<0.05）。在敲低POT1后,2种细胞Bcl2 mRNA表达均显著下调（P<0.05）,Bax mRNA表达显著上调（P<0.05）,蛋白免疫印迹结果与之一致（P<0.05）,且敲低组Caspase3蛋白表达量高于对照组（P<0.05）。2种细胞敲低组LIG3、γH2A.X、c-PARP1的蛋白表达均显著上调（P<0.05）。结论 敲低POT1可使黑色素瘤细胞的增殖、迁移和侵袭能力下降,激活线粒体凋亡途径并上调DNA修复相关分子的表达。

Abstract

Objective To investigate the effects of protection of telomeres 1 （POT1） on the biological behavior of melanoma cells and the expression of DNA repair-related molecules. Methods Small interfering RNA （siRNA） technology was used to knock down POT1 expression in melanoma A2058 and A375 cells. Cell proliferation, migration, and invasion were assessed using the cell counting kit-8 （CCK-8） assay, wound healing assay, and Transwell assay. The knockdown efficiency was verified, and the expression levels of mitochondrial apoptosis-related molecules including B-cell lymphoma 2 （Bcl2）, Bcl2-associated X protein （Bax）, and cysteine-aspartic acid protease 3 （Caspase3）, as well as DNA damage repair-related molecules including DNA ligase 3 （LIG3）, phosphorylated histone H2A.X （γH2A.X）, and cleaved poly（ADP-ribose） polymerase 1 （c-PARP1）, were detected by quantitative real-time polymerase chain reaction （qRT-PCR） and Western blotting. Results Compared with the control group, the expression levels of POT1 mRNA and protein in the A2058 and A375 cell knockdown groups were significantly decreased （P<0.05）. The CCK8 assay showed that the optical density （OD） values of the knockdown groups of both cell lines at 24 h, 48 h, 72 h and 96 h were significantly lower than those of the control groups, and significant differences were found for the between-group effect,time effect,or group-by-time interaction effect （P<0.05）. The wound healing assay indicated that the cell migration rate in the knockdown groups of both cell lines was significantly lower than that of the control group （P<0.05）. Transwell assay demonstrated that the number of cells passing through the basement membrane in the knockdown groups of both cell types was significantly lower than that in the control group （P<0.05）. After POT1 knockdown, the mRNA expression levels of Bcl2 in A2058 and A375 cells were significantly downregulated （P<0.05）, while the Bax mRNA levels were significantly upregulated in both cell lines （P<0.05）, which was consistent with the Western blotting results （P<0.05）. Meanwhile, the expression levels of Caspase3 protein in the knockdown groups were significantly higher than those in the control group （P<0.05）. In addition, the protein expression levels of LIG3, γH2A.X and c-PARP1 in the knockdown groups of both cell lines were significantly upregulated （P<0.05）. Conclusion Knockdown of POT1 can reduce the proliferation, migration and invasion abilities of melanoma cells, activate the mitochondrial apoptotic pathway and affect the expression of DNA repair-related molecules.

关键词

黑色素瘤 / POT1 / 细胞增殖 / 细胞凋亡

Key words

melanoma / POT1 / cell proliferation / apoptosis