人ETV4蛋白截短体的构建与其在结直肠中的功能分析

Construction and identification of human ETV4 deletion mutants

目的:研究ETV4转录因子各保守结构在转录调节结直肠癌细胞增殖以及迁移过程中的作用以及机制。方法:利用PCR介导的克隆技术,在前期构建的ETV4载体基础上,构建含不同ETV4结构域的蛋白截短体ETV4(1~339)(包含酸性结构域)与ETV4(340~484)(含DNA结合结构域)。利用CCK-8试剂盒检测不同截短体对结直肠癌细胞增殖的影响,利用细胞划痕实验检测不同截短体对结直肠癌细胞迁移的影响。进一步利用定量PCR技术检测表达不同截短体过表达细胞中上皮间质转换(epithelial-mesenchymal transition,EMT)标志物的变化情况。结果:CCK-8实验结果显示截短体ETV4(1~339)无明显促进结直肠癌细胞增殖的作用,而截短体ETV4(340~484)和全长蛋白都可明显促进结直肠癌细胞增殖(P<0.01)。细胞划痕实验显示截短体ETV4(340~484)和全长蛋白可促进结直肠癌细胞迁移而截短体ETV4(1~339)却无此作用。进一步对EMT标志物检测发现ETV4(340~484)和全长蛋白可导致E-cadherin表达下降,N-cadherin、Vimentin和Twist表达上调(P<0.01),而ETV4(1~339)中EMT标志物无明显变化。结论:本实验首次证明,ETV4促进结直肠癌细胞增殖和迁移主要是通过ETV4中的ETS结构域起作用,并且可能通过介导EMT标志物来调控结直肠癌细胞转移。

Objective：To construct ETS Variant 4（ETV4）truncated mutants and to investigate their role in colorectal cancer cell growth and migration. Methods：Based on our previous constructed ETV4 vector,ETV4 deletion mutants that containing different conserved domains were constructed：ETV4（1-339）and ETV4（340-484）. Each mutant function was analyzed in colorectal cancer cell growth by CCK-8 kit. Each mutant function in colorectal cancer cell migration was detected with wound healing assay. Moreover,each mutant function in EMT was further measured by q RT-PCR. Results：CCK-8 assay showed that mutant ETV4（1-339）didn＇t promote cancer cell growth whereas mutant ETV4（340-484） and wide type ETV4 evidently promoted cancer cell growth. Wound healing assay revealed that mutant ETV4（340-484）and wide type ETV4 promoted colorectal cancer cell migration. Further q RTPCR analysis showed that mutant ETV4（340-484）and wide type ETV4 were able to alter the expression of ETM markers whereas mutant ETV4（1-339）could not. Conclusion：ETS domain of ETV4 is indispensable in ETV4-induced colorectal cancer cell proliferation and migration,which is probably related with EMT.