LKB1抑制人巨细胞肺癌细胞的增殖和迁移侵袭、mTOR磷酸化与VEGF和MMP9表达

LKB1 inhibits the proliferation and invasion of human lung giant cell cancer cell line through down-regulating the phosphorylation of mTOR and the expression of VEGF and MMP9

目的分析肝激酶B1(liver kinase B1,LKB1)对人巨细胞肺癌(the people giant cell lung cancer,PGCL3)细胞增殖、迁移、侵袭的影响及其机制。方法构建真核表达载体p CMV-LKB1,转染人巨细胞肺癌细胞。转染后48h,用免疫印迹法检测各组细胞LKB1蛋白表达水平,明确转染PGCL3效率。转染后每隔12h、连续72h检测LKB1对细胞增殖能力的影响;Transwell小室检测转染后各组细胞迁移、侵袭力。提取各组细胞蛋白检测基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)、基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)、雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达水平。结果 p CMV-LKB1经PCR、双酶切及DNA测序鉴定后,证实目的基因片段插入方向正确,核酸序列与NCBI公布的LKB1的核酸序列一致;免疫印迹分析显示,转染p CMV-LKB1的PGCL3细胞中LKB1表达水平明显增高,表明p CMV-LKB1构建成功。增殖曲线分析和5-乙炔基-2,脱氧嘧啶核苷(5-ethynyl-2,-deoxyuridine,Ed U)检测显示,转染p CMV-LKB1能显著抑制PGCL3细胞的增殖;Transwell小室检测显示,过表达LKB1可显著抑制PGCL3细胞的迁移与侵袭;免疫印迹分析显示,过表达LKB1的PGCL3细胞其LKB1下游分子中总m TOR表达量不变,磷酸化m TOR(p-m TOR)水平降低,m TOR下游分子VEGF表达量也显著降低,迁移侵袭相关蛋白MMP2表达无变化,但MMP9水平显著降低。结论 LKB1可能通过下调PGCL3细胞p-m TOR水平,降低VEGF表达,从而抑制巨细胞肺癌细胞增殖,并可能通过下调金属基质蛋白酶MMP9的表达,降低巨细胞肺癌细胞迁移侵袭能力。

Objective To study the effect of liver kinase B1(LKB1)on the proliferation,migration and invasion of human lung giant cell cancer PGCL3cell line and its mechanism.Methods Eukaryotic expression vector pCMV-LKB1was constructed and transfected into giant lung cell cancer cell line PGCL3.48h after transfection,the level of LKB1protein was detected by western blotting to determine the efficiency of transfection.The effect of LKB1on the proliferation of the cells was detected every12h for a total of72h.Transwell assay was applied to evaluate cell migration and invasion.Western blotting was performed to detect the expression of matrix metalloproteinase2(MMP2),matrix metalloproteinase9(MMP9),mammalian target of rapamycin(mTOR)and vascular endothelial growth factor(VEGF).Results PCR,double enzyme digestion and DNA sequencing results confirmed that the LKB1fragment was inserted in the right direction into the recombinant eukaryotic vector pCMV-LKB1and that its nucleic acid sequence was the same as published on NCBI.Western blot results showed that the level of LKB1in the transfected cells increased significantly,indicating the success of pCMV-LKB1construction.Growth curve analysis and5-Ethynyl-2’-deoxyuridine(EdU)test showed that the cell proliferative capacity was significantly inhibited by pCMV-LKB1transfection.Transwell assay results showed that LKB1overexpression obviously inhibited the migration and invasion of PGCL3cells.Western blot results showed that of the downstream molecules of LKB1in the transfected PGCL3cells,the total mTOR level remained unchanged,while the expression of phosphorylated m-TOR(p-mTOR)decreased.VEGF,the downstream protein of mTOR,reduced significantly.The level of migration related protein MMP2remained unchanged,while that of MMP9decreased significantly.Conclusion LKB1inhibits the proliferation of PGCL3cells possibly through down-regulating the levels of p-mTOR and VEGF;and also inhibit its migration by down-regulating MMP9expression.