氯离子通道1过表达对小鼠肝癌细胞株Hca-P生物学行为的影响

Effect of CLIC1 overexpression on tumor biological behavior in mouse hepatocarcinoma cell line Hca-P in vitro

背景与目的:氯离子通道l(chloride intracellular channel l,CLICl)是CLIC家族中的一员,研究表明CLIC1与肿瘤转移相关。本研究旨在探讨CLICl过表达在体外对小鼠肝癌低淋巴道转移细胞株Hca-P生长、体外迁移和侵袭能力的影响。方法:构建CLIC1过表达真核载体pcDNA3.1(+)-CLIC1表达质粒,将重组的pcDNA3.1(+)-CLIC1基因和空载体pcDNA3.1(+)转染Hca-P母系细胞,通过G418筛选获得稳定表达CLIC1基因的pcDNA3.1(+)-CLIC1-Hca-P细胞株和转染空载体的pcDNA3.1(+)-Hca-P细胞株,RT-PCR和ELISA鉴定CLIC1过表达水平,CCK-8法检测细胞活力和分裂增殖能力,Transwell实验检测细胞的体外迁移能力和侵袭能力。结果:成功建立了稳定表达CLIC1基因的细胞株pcDNA3.1(+)-CLIC1-Hca-P,与空载体对照组相比,pcDNA3.1(+)-CLIC1质粒转染入Hca-P细胞后CLIC1基因表达水平显著升高。pcDNA3.1(+)-CLIC1-Hca-P细胞增殖明显高于Hca-P母系细胞组和空载体对照组,差异有统计学意义(P<0.05),且细胞增殖主要集中在72～96 h;Transwell检测各组肿瘤细胞迁移能力结果显示,pcDNA3.1(+)-CLIC1-Hca-P、pcDNA3.1(+)-Hca-P和Hca-P细胞株迁移到膜下和小室下室的平均细胞数分别为205.43±22.87、132.72±20.45和121.35±19.64。pcDNA3.1(+)-CLIC1-Hca-P与Hca-P、pcDNA3.1(+)-Hca-P细胞株比较差异有统计学意义(P<0.05);Transwell检测各组肿瘤细胞侵袭能力结果显示,pcDNA3.1(+)-CLIC1-Hca-P细胞穿过基膜的细胞数为(76.2±4.62)个,明显多于pcDNA3.1(+)-Hca-P的穿膜细胞数(48.34±3.45)个和Hca-P细胞的穿膜细胞数(49.8±5.51)个,差异有统计学意义(P<0.05)。结论:CLIC1过表达可以显著促进Hca-P细胞增殖、增强其侵袭能力。CLIC1有望成为临床治疗肝癌的基因治疗靶点之一。

Background and purpose： Our previous study have identified many lymphatic metastais- associated genes/proteins using gene chip and quantitative proteomics technology from a couple of the same parents＇ mouse hepatocarcinoma cell lines with high and low lymphatic metastatic potential. In this study, we select chloride intracellular channel 1 （CLIC1） which is highly expressed in cells with high metastatic potential to understand the effect of CLIC1 overexpression on tumor biological behavior in mouse hepatocarcinoma cell line Hca-P in vitro. Methods： The plasmids pcDNA3. I（＋）-CLIC 1 was constructed. Then the Hca-P cells were transfected with the pcDNA3.1（＋） and pcDNA3.1（＋）-CLIC1. The stably transfected cells were obtained by growing them in G418 for 4 weeks. The levels of expression of CLIC1 mRNA and protein were detected by semi-quantitative polymerase chain reaction （RT-PCR） and enzyme-linked immunosorbant assay （ELISA） analysis, respectively. The cell proliferation was detected by CCK- 8 method; the invasive and migrative abilities were detected by Transwell assay. Results： Restrictive endonuclease assay and sequence analysis verified the successful construction of the recombinant vector pcDNA3.1 （＋）-CLIC 1. The cell line Hca-P that steadily expressing CLIC1 gene was constructed. RT-PCR assay and ELISA showed that the expression level of CLIC 1 mRNA and protein in the Hca-P cells were increased significantly after pcDNA3.1 （＋）- CLIC1 transfection （P〈0.05）. The cell proliferation, the average number of invasive and migrative cells were increased in Hca-P cells with CLIC1 transfection as compared with that of the Hca-P without CLIC1 transfection （P〈0.05）. Conclusion： CLIC1 promotes the proliferation, invasive and migration in Hca-P cells with CLIC1 transfection in vitro obviously. The results suggest that CLIC 1 may become a target of gene for treatment of hepatocarcinoma.