ANXA11表达稳定下调的肝癌Hca-P细胞株的建立

Construction of mouse hepatoma Hca-P with stable knockdown of ANXA11 gene

关键词:  ANXA11  淋巴道转移  Hca-P  RNA干扰

Key words:  ANXA11  Lymphatic metastasis  Hca-P  RNA interference

摘要:

目的　构建针对膜联蛋白A11(Annexin A11，ANXA11) 的shRNA(small hairpin RNA)，稳定转染小鼠低淋巴道转移力肝癌Hca-P细胞，筛选ANXA11稳定下调的单克隆细胞株，为后续研究奠定基础。　方法　合成2条ANXA11的shRNA序列(shRNA-1和-2),与真核表达载体pGPU6/GFP/Neo连接，构建pGPU6/GFP/Neo-shRNA-ANXA11表达载体并转染Hca-P细胞，通过G418筛选及有限稀释法获得单克隆细胞株，Western blot检测Hca-P细胞ANXA11蛋白表达，并与空载体转染及对照组比较。　结果　成功构建了pGPU6/GFP/Neo-shRNA- ANXA11重组表达质粒，并获得pGPU6/GFP/Neo- shRNA- ANXA11-Hca-P单克隆细胞株；shRNA-1重组质粒的抑制效果在mRNA和蛋白水平对ANXA11抑制率分别为80.2%和77.7%，P<0.01。　结论　通过RNA干扰和基因转染技术成功建立了ANXA11表达稳定下调的Hca-P细胞株，得到了其单克隆细胞株，为进一步研究ANXA11在恶性肿瘤淋巴道转移中的作用及机制打下了基础。

Abstract:

[Abstract] Objective 〗To design small hairpin RNA (shRNA) targeting Annexin A11 (ANXA11), to transfect mouse hepatoma Hca-P with Annexin A11 (ANXA11), to obtain the Hca-P cell line with Anxa11 stable knock-down and to establish material basis for future study. 　Methods　Two Anxa11 shRNAs, shRNA-1 and shRNA-2, were synthesized, ligated to pGPU6/GFP/Neo expression plasmid and transfected into Hca-P cell. The monoclonal pGPU6/GFP/Neo-ANXA11- Hca-P cell with stable knockdown of ANXA11 was obtained by G418 screening and limited dilution. ANXA11 mRNA and protein expression levels were measured by RT-PCR and Western blot,and compared with empty vector transfected Hca-P and Hca-P control. 　Results　The recombinant expression vector pGPU6/GFP/Neo-shRNA-ANXA11 was successfully constructed. The monoclonal pGPU6/GFP/Neo-ANXA11-Hca-P cell lines were obtained. Compared to normal Hca-P cell, the ANXA11 mRNA and protein level in Hca-P cells transfected with shRNA-1 wese down-regulated by about 80.2% and 77.7% with statistic significance (P<0.01). 　Conclusion　Mouse monoclonal hepatoma Hca-P cell with stable knock-down of ANXA11 was successfully constructed by RNAi and gene transfection, which provides a solid base for studying the effect and mechanism of ANXA11 in hepatoma lymphatic metastasis.