摘要
目的建立诱导型蛋白酶活化受体4(PAR4)表达抑制的人肠癌细胞SW620/PAR4D细胞模型,探讨PAR4对肠癌细胞增殖迁移的影响。方法建立带有四环素诱导表达调控系统的SW620/Tet-On人肠癌细胞,并构建针对PAR4的诱导型人工小RNA(miRNA)表达慢病毒载体p LVX-Tight-Puro-PAR4-miR,获得诱导型PAR4表达抑制的SW620/PAR4D细胞模型。用Western blot法对经强力霉素(DOX)药物诱导后SW620细胞中PAR4的表达抑制情况进行验证,采用CCK-8法检测抑制PAR4对SW620细胞增殖的影响,划痕实验检测SW620细胞的迁移情况。结果成功建立了PAR4低表达的SW620/PAR4D细胞模型。与对照组细胞相比,DOX诱导PAR4表达抑制前后,SW620/PAR4D细胞的生长增殖情况无明显变化,但运动迁移能力明显下降。结论下调PAR4水平不影响SW620细胞的增殖但抑制其迁移。
Objective To establish the human colorectal cancer cell model SW620/PAR4D with inducible suppression of proteinase activated receptor 4 （PAR4） expression, and investigate the role PAR4 plays in the proliferation and migration of cancer cells. Methods A human colorectal cancer cell line with tetracycline-inducible expression regulatory system, namely SW620/Tet-on, was＇established; inducible expression lentiviral vector with artificial microRNA targeting PAR4, pLVX-Tight- Puro-PAR4-miR, was constructed and transfected into SW620/Tet-on to make an inducible PAR4-suppressed cell model SW620/PAPAD. Western blotting was used to confirm the suppression of PAR4 expression after the doxycycline （DOX） treatment. CCK-8 assay was used to evaluate the impact of suppressed PAR4 expression on cell proliferation, and wound-healing assay was used to analyze the migration of the cells. Results The SW620/PAR4D cell model was established successfully. Suppression of PAR4 expression by DOX treatment had no significant impact on the growth/proliferation of SW620/PAR4D cells, but markedly inhibited the cell migration. Conclusion Suppression of PAR4 expression has no significant effect on the proliferation of SW620 cells, but can inhibit the migration of the cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2016年第5期609-614,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(30271450
30672365
81172516)
浙江省自然科学基金(LQ15H160008)
作者简介
陈丽红（1987-），女，浙江上虞人，助理实验师Tel：0571-87315203；E-mail：yongandhong@126．com
叶景佳，E-mail：yej001@zju．edu．cn
通讯作者，曹江，E-mail：caoj@zju．edu．cn
