RhoE对cd44基因启动子的转录活性及大肠癌细胞系恶性生物学行为的影响

The Affect of RhoE on cd44 Promoter and the Malignant Behaviors of Colorectal Cancer Cell

目的研究RhoE对cd44基因启动子的转录调控,并探讨RhoE对裸鼠体内成瘤性的影响。方法采用PCR技术从人胚肾HEK293细胞中扩增出cd44启动子,并将其插入荧光素酶报告基因载体pGL3-Basic中,经测序确定所扩增的DNA序列,并将其转染入SW480及LoVo细胞中监测其活性。将pcDNA3.1-RhoE和pcDNA3.1分别与pGL3-CD44 promoter共转染大肠癌LoVo细胞和SW480细胞,双荧光素报告基因检测活性。另外将pcDNA3.1-RhoE和对照组分别稳定转染LoVo细胞和SW480细胞,并将筛选的稳定表达的细胞分别接种裸鼠,观察肿瘤的生长;用免疫组化的方法检测RhoE对肿瘤组织细胞形态及CD44分子表达的影响。结果测序结果表明,扩增的cd44启动子序列正确,双报告基因实验检测荧光素酶活力表明构建的报告基因具有启动子活性。含cd44启动子序列的报告基因在pcDNA3.1-RhoE阳性的LoVo细胞中表达受到抑制;HE染色显示pcDNA3.1-RhoE转染组较对照组肿瘤细胞明显变小,且大小和形态呈现一致性,已没有瘤巨细胞,相应的肿瘤细胞核的体积也变小。结论 RhoE通过抑制cd44基因启动子的活性部分逆转肿瘤的恶性生物学行为。为研究RhoE表达与cd44基因的关系及CD44蛋白表达与结直肠癌发生、发展及浸润转移的关系提供了良好的实验基础。

Objective To study the effect of RhoE on the transcriptional regulation of cd44 and in vivo tumorigenicity of nude mice.Methods cd44 promoter was amplified from human embryonic kidney HEK293 cells with PCR and insert into Dual-Luciferase○R Reporter plasmid pGL3-Basic.After confirmed with sequence analysis,the generated recombinant was transfected into SW480 and LoVo cells to monitor their activity.Colon cancer SW480 and LoVo cells were cotransfected with pGL3-CD44 promoter along with pcDNA3.1-RhoE and pcDNA3.1 respectively.SW480 and LoVo cells were stably transfected with pcDNA3.1-RhoE and the control group and were inoculated into nude mice to observe tumor growth.Immunohistochemistry assay was applied to observe the morphology of tumor cells and the expression of CD44 molecules.Results The cd44 promoter sequence was amplified correctly,Dual-Luciferase○R Reporter Assay showed that the constructed reporter gene has promoter activity.The expression of cd44 promoter sequence containing reporter gene in pcDNA3.1-RhoE expression positive LoVo cells was inhibited;HE staining demonstrated that the pcDNA3.1-RhoE transfected tumor cells was significantly smaller than that in the control group,and consistent size and shape tumor cells were observed but no tumor giant cells,the corresponding volume of the tumor nuclei were also small.Conclusion RhoE could partially reverse the malignant biological behavior of tumors by inhibiting the transcriptional regulation of cd44 promoter.