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

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。

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

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.