甲基化修饰抑癌基因MSX1抑制黑色素瘤细胞迁移和侵袭的研究

Inhibitory migration and invasion of melanoma cells by methylation modified tumor suppressor gene MSX1

目的探讨MSX1在黑色素瘤细胞系和组织中表达和甲基化及对黑色素瘤细胞A375迁移/侵袭的影响和机制。方法qRT-PCR和免疫组织化学法分别检测MSX1在黑色素瘤细胞系和组织中的表达;甲基化PCR分析MSX1在黑色素瘤组织中甲基化;分别转染pcDNA3.1(+)-Flag-MSX1质粒(实验组)和pcDNA3.1(+)-Flag-Vector质粒(对照组)至A375细胞;Transwell迁移和侵袭实验检测MSX1对细胞迁移和侵袭的影响;RT-PCR验证MSX1对上皮间质转化及下游相关标志物的影响,免疫蛋白印迹实验验证MSX1对WNT/β-catenin信号通路的影响。结果与色素痣组织和细胞相比,MSX1 mRNA和蛋白在黑色素瘤细胞和组织中表达下调(P<0.001);MSX1甲基化水平表达上调,其甲基化程度明显高于癌旁组织和正常色素痣组织。过表达MSX1后抑制细胞迁移和侵袭(P<0.001),而上皮间质转化及下游相关标志物表达受到抑制。过表达MSX1能抑制WNT/β-catenin信号通路及其下游细胞因子的表达。结论MSX1在黑色素瘤组织中表达下调,能抑制Wnt/β-catenin信号通路参与黑色素瘤的进展。

Objective The objective of this study was to investigate the expression and methylation of MSX1 in melanoma cells and tissues,and its effect on migration/invasion of melanoma A375 cells.Methods qRT-PCR and immunohistochemical were used to examine the expression of MSX1 in A375 cells and tissues;methylation PCR was used to examine the methylation of MSX1 in melanoma tissues;pcDNA3.1(+)-Flag-MSX1(experimental group)and pcDNA3.1(+)-Flag-Vector(control group)were transfected into A375 cells,transwell migration and invasion assay were used to detect the effect of MSX1 on cell migration and invasion;RT-PCR was used to verify the effect of MSX1 on epithelial-mesenchymal transition and its downstream related markers;Westernblot was used to verify the effect of MSX1 on Wnt/β-catenin signaling pathway.Results MSX1 mRNA were down-regulated in human malignant melanoma cell lines and tissues(P<0.001).The methylation of MSX1 was up-regulated,which was significantly higher than that in adjacent tissues and normal nevus tissues.Overexpression of MSX1 inhibited cell migration and invasion in A375 cells(P<0.001),while epithelial to mesenchymal transition and the expression of downstream markers were also inhibited.MSX1 overexpressed could inhibit the expression of Wnt/β-catenin signaling pathway and its downstream markers in A375 cells.Conclusion MSX1 was down-regulated in melanoma tissues,indicating that MSX1 inhibits Wnt/β-catenin signaling pathway and participates in the progression of melanoma.