LASS2/TMSG-1基因过表达对人肺癌95D细胞体外侵袭迁移能力的影响及其机制

Effect of LASS2/TMSG-1 overexpression on the invasion and migration of 95D human pulmonary carcinoma cell and its mechanism

目的分析LASS2/TMSG-1基因过表达对人肺癌95D细胞体外侵袭迁移能力的影响,初步探讨LASS2/TMSG-1基因的作用机制。方法运用脂质体转染法将前期已构建好并冷冻保存的Pc DNA3-TMSG-1质粒通过脂质体转染法瞬时转染入95D细胞(高转移潜能,LASS2/TMSG-1低表达),使外源性LASS2/TMSG-1基因在细胞内过表达即LASS2/TMSG-1过表达组,同时设立转染空白质粒的空白对照组和未转染质粒的阴性对照组。应用体外侵袭及迁移实验检测人肺癌95D细胞侵袭及迁移能力的变化;应用qRT-PCR和Western blot法检测LASS2/TMSG-1 mRNA及蛋白的表达;应用Western blot法检测ATP6L、MMP-2及MMP-9在细胞中的表达水平;应用明胶酶谱法检测肿瘤细胞中MMP-2及MMP-9的活性;使用比色法检测细胞中V-ATPase的活性;使用BCECF H+敏感探针检测细胞外H+浓度的变化。结果 LASS2/TMSG-1过表达组细胞的LASS2/TMSG-1表达水平明显升高,而ATP6L表达水平下降,其V-ATPase活性及细胞外H+浓度降低,与空白对照组和阴性对照组相比差异均具有统计学意义(P<0.05);MMP-2、MMP-9的表达及活性均降低,与其他两组相比差异有统计学意义(P<0.05);并且LASS2/TMSG-1过表达组细胞的体外侵袭及迁移能力明显低于其他两组(P<0.05)。结论 LASS2/TMSG-1可能通过结合ATP6L亚基抑制V-ATPase活性,改变细胞外H+浓度,影响MMP-2、MMP-9的表达及活性,进而改变肿瘤细胞的体外侵袭迁移能力等生物学行为。

Purpose To explore the effect of LASS2/TMSG-1 on invasion and migration of 95D （ a human lung carcinoma cell line with high metastatic potential） and the underlying mechanism. Methods 95D ceils （High metastatic potentiality, LASS2/TMSG-1 low expression） were transfected with LASS2/TMSG-1 mRNA by Lipofectamine to make LASS2/TMSG-1 gene overexpressed. They were divided into three groups, including blank control group, negative control group and LASS2/TMSG-1 gene overexpression group. After transfeetion, the invasion and migration ability of cells were tested by Transwell assay. The expression of LASSS2/TMSG-1 mRNA and protein were detected by qRT-PCR and Western blot. The expression of ATP6L, MMP-2 and MMP-9 was detected by Western blot. The activity of MMP-2 and MMP-9 were determined by gelatin zymography. The activity of V-ATPase was examined by colorimetry. The pH-sensitive fluorescence probes （BCECF） were used to measure the extracellular H ＋ concentrations. Results After trans-fection, the LASS2/TMSG-1 expression level of 95D in lung cancer cells were significantly increased, while ATP6L expression level declined, its V-ATPase activity and extracellular H ＋ concentration decreased; compared with the blank control group and the negative control group, the difference were statistically significant （P 〈0. 05）. The expression and activity of MMP-2/MMP-9 were lower, with significant difference （P 〈 0. 05 ） compared with the other two group. In addition, the invasion and migration ability of LASS2/TMSG- 1 group was significantly lower than the other two groups （P 〈 0. 05）. Conclusion LASS2/TMSG-1 may inhibit the activity of V-ATPase by binding ATP6L subunit, thereby alter the extracellular hydrogen ion concentration, influence the expression and activity of MMP-2, MMP-9, and therefore change the invasion and migration ability of the tumor cells in vitro.