内皮素3基因对结直肠癌LoVo细胞迁移和侵袭能力的影响

Over-expression of endothelin 3 suppresses migration and invasion in colorectal cancer LoVo cells

目的探讨内皮素3(endothelin 3,EDN3)基因对结直肠癌细胞的增殖、迁移和侵袭能力的影响及其相关机制。方法实时荧光定量PCR检测EDN3 mRNA在人结直肠癌组织及对应癌旁组织中的表达情况,半定量PCR检测EDN3及内皮素受体B(endothelin receptor B,EDNRB)mRNA在结直肠癌细胞(CaCO_2、LoVo、HT-29)中的表达情况。将包装过载体pCDH-CMV-MCS-EF1-Puro-EDN3的病毒感染至结直肠癌细胞LoVo,MTS、克隆形成法、划痕法、Transwell检测EDN3对细胞增殖、克隆、迁移和侵袭能力的改变。Western blot检测LoVo中p-ERK1/2和t-ERK1/2蛋白改变。结果 EDN3在人结直肠癌组织和细胞中表达降低。表达EDN3基因的重组载体成功感染至LoVo细胞。MTS和克隆实验结果显示,实验组和对照组细胞增殖和克隆形成差异无统计学意义(P>0.05),划痕实验和Transwell实验提示实验组细胞迁移和侵袭能力明显低于对照组(P<0.01)。Western blot检测结果显示磷酸化的ERK1/2蛋白表达下降(P<0.01)。结论 EDN3在结直肠癌组织和细胞中表达降低,过表达EDN3能抑制结直肠癌LoVo细胞的迁移和侵袭能力,其机制可能与ERK1/2信号通路有关。

Objective To determine the effect of endothelin 3（EDN3） on the proliferation,migration and invasion in colorectal cancer cell line LoVo and investigate the underlying mechanism. Methods Real-time fluorescent quantitative PCR was used to detect the expression level of EDN3 in colorectal cancer tissues and para-cancerous tissues（n = 15）. Semi-quantitative PCR was adopted to detect the mRNA expression of EDN3 and endothelin receptor B（EDNRB） in the colorectal cancer cell lines CaCO_2,LoVo and HT-29. The recombinant plasmid pCDH-CMV-MCS-EF1-Puro-EDN3 was packaged into lentivirus and then infected into the LoVo cells. MTS assay,colony formation assay,wound healing assay,Transwell migration and invasion assay were conducted to analyze the changes of cells in the proliferation,colony formation,migration and invasion. Western blotting was used to detect the protein expression changes of p-ERK1/2 and t-ERK1/2 in the LoVo cells. Results The expression of EDN3 were down-regulated in colorectal cancer tissues and cell lines. The recombinant lentiviral vectors which express EDN3 gene were successfully infected into LoVo cells. MTS assay and colony formation assay showed that there were no changes in the proliferation and colony formation between the LoVo cells with the lentiviral infection and those with blank vector（P〈0. 05）. Wound healing test and Transwell assay revealed that the migration and invasion ability were apparently reduced in the experimental cells compared to the control cells（P〈0. 01）. Western blot assay showed that the protein level of p-ERK1/2 was decreased after the infection（P〈0. 01）. Conclusion The expression of EDN3 gene is generally down-regulated in colorectal cancer tissues and cell lines. EDN3 overexpression inhibits the migration and invasion in colorectal cancer LoVo cells,which may be through ERK1/2signaling pathway.