LKB1基因与乳腺癌细胞侵袭性相关因子的研究

Relationship between LKB1 gene and invasion-related factors of breast cancer cells

目的观察 LKB1基因对乳腺癌细胞的侵袭性影响及机制。方法筛选 LKB1基因转染的 MDA-MB-435乳腺癌细胞,随机取样一株高表达细胞株和一株低表达细胞株,同时与未转染细胞株和空质粒转染细胞株比较。采用逆转录聚合酶链反应(RT-PCR)和明胶-酶谱法观察基质金属蛋白酶(MMP)-2、MMP-9和促进微血管生成的因子 VEGF、bFGF 的基因表达情况,并应用 Western 印迹对其蛋白定量分析。应用 Tramswell 试验对其进行膜穿透能力检测。结果 LKB1基因转染的乳腺癌细胞的 mRNA 和蛋白水平上 MMP-2、MMP-9和 VEGF、bFGF 基因表达的相对吸光度(A)值皆低于未转染细胞和空质粒细胞,且 LKB1基因高表达细胞株较低表达细胞株更低。同时发现 Tramswell 试验中 LKB1转染的细胞侵袭能力下降(18.1%±1.0%、22.4%±1.8%vs 47.6%±1.3%、45.6%±1.2%,均 P<0.01)。结论 LKB1基因作为一种抑癌基因对乳腺癌细胞的侵袭性可能起重要作用。

Objective To investigate the relationship between LKB1 gene and invasion of breast cancer cells. Methods Human breast cancer cells of the line MDA-MB-435 were cultured and transfected with plasmids with or without LKB1 gene. The clone of the transfected MDA-MB-435 cells with high expression of LKB1 was called MDA-MB-435/LKB1 （ H）, and that with low expression of LKB1 was called MDA-MB-435/LKB1 （L）. The MDA-MB-435 cells transfected with blank vector was called MDA-MB-435/ vec. MDA-MB-435 cells without transfection were used as controls. RT-PCR was used to detect the mRNA expression of the invasion-related factors of breast cancer： matrix metalloproteinase （ MMP）-2, MMP-9, vascular endothelial growth factor （VEGF）, and basic fibroblast growth factor （bFGF）, and Western blotting was used to detect the protein expression of these factors. Gelatin zymography was used to expression of the secretive MMP-2 and MMP-9. Transwell test was used to examine the membrane penetration of the different MDA-MB-435 cells. Results The invasion rate was （47.6 ± 1.3） % in the MDA-MB-435 cells, （45.6 ±1.2）% in the MDA-MB-435/vec cells, both significantly higher than those of the MDA-MB-435/ LKB1 （H） and MDA-MB-435/LKB1 （L） cells [ （ 18.1 ± 1.0） % and （22.4± 1.8） % respectively, all P 〈0.01] , with significant difference between the latter 2 groups （P 〈0.05）. The mRNA expression and protein expression of MMP-2, MMP-9, VEGF, and b-FGF were all significantly lower in the MDA-MB-435/ LKB1 cells. Gelatin zymography showed that the secretive MMP-2 and MMP-9 were expressed at the protein level significantly lower in the MDA-MB-435/LKB1 cells. Conclusion LKB1 gene inhibits the invasion of breast cancer cells.