Ech1基因表达下调对小鼠肝癌细胞株Hca-F黏附能力的影响

Down-regulation of Echl decreases the adhesion ability of mouse hepatocarcinoma Hca-F cells

目的在蛋白质水平检测Ech1在小鼠肝癌高低淋巴道转移Hca—F细胞／Hca—P细胞中的表达及其对Hca-F细胞黏附能力的影响。方法采用荧光差异双向凝胶电泳（2DDIGE）和质谱技术定量分析鉴定Ech1在Hca—F细胞和Hca-P细胞中的表达；构建pGPU6／GFP／Neo--shRNA—Ech1质粒，稳定转染至Hca-F细胞中，并通过实时荧光定量PCR和Westemblot验证Ech1表达下调效果，检测Ech1基因表达下调后对细胞外基质和淋巴结的黏附能力。各组细胞间的差异采用单因素方差分析。结果Ech1在Hca-F细胞的表达明显高于其在Hca-P细胞表达;Ech1表达下调细胞株构建成功，Ech1基因表达下调后，降低Hca-F细胞对细胞外基质纤维连接蛋白和I型胶原成分的黏附能力，降低其对体内淋巴结的黏附能力。Hca-F组与pGPU6／GFP/Neo-shRNA-Ech1组黏附吸光度值的差异有统计学意义，层黏连蛋白和I型胶原吸光度值分别为1．42±0．26和1．14±0．07与1．01±0．27和0．90±0．09，P值均〈0．05。结论抑制Ech1基因和蛋白的表达，可以降低小鼠肝癌细胞株Hca—F细胞的黏附能力。

Objective To examine the differential expression pattern of Echl protein in mouse Hca-F and Hca-P hepatocarcinoma cell lines with high and low rates of lymphatic metastasis, respectively, and to investigate the relationships between Echl expression and adhesion of Hca-F cells. Methods Fluorescence two-dimensional difference in-gel electrophoresis （2D DIGE） and mass spectrometry were used to detect Echl expression. Echl gene silencing was achieved by stable transfection of Hca-F cells with a plasmid vector harboring short hairpin RNA （shRNA） targeting Echl, pGPU6/GFP/Neo-shRNA-Echl. Echl mRNA and protein expressions were detected by real-time quantitative polymerase chain reaction （qRT-PCR） and Western blotting analysis, respectively. Adhesive properties of cells were assessed by hematoxylin-eosin staining and fluorimetric detection of extracellular matrix （ECM） proteins. Results Endogenous Echl protein level was remarkably higher in the highly metastatic Hca-F cell line than in the Hca-P cell line （2.7- fold by 2D DIGE; 1.5-fold by Western blotting）, shRNA-induced silencing of Echl significantly reduced the adhesion ability of Hca-F cells, as evidenced by decreased absorbance values of fibronectin and collagen I （Hca-F cells vs. pGPU6/GFP/Neo-shRNA-Echl cells： 1.42 ± 0.26 vs. 1.01 ±0.27 and 1.14± 0.07 vs. 0.90 ± 0.09, respectively; P 〈 0.05）. Conclusion Down-regulation of Echl can inhibit the adhesive capacity of metastatic Hea-F cells.