Objective: To investigate the effects of LASS2/TMSG1 gene overexpression on proliferation and apoptosis of human lung cancer A549 cells and explore the possible mechanism.
Methods: We examined LASS2/TMSG1 expression level in a previously constructed A549 cell line overexpressing LASS2/TMSG1 using Western blotting. The proliferation and apoptosis of the cells were detected using colony-forming assay, CCK-8 assay, Hoechst/PI double staining and flow cytometry. Fourteen nude mice were randomized into 2 groups (n=7) to receive subcutaneous injection of A549 cells with or without LASS2/TMSG1 overexpression on the back of the neck, and the cell proliferation in vivo was observed. The expression levels of p38 MAPK protein and p-p38 MAPK protein in the xenografts were detected with Western blotting. ELISA was used to detect the levels of ceramide and p38 MAPK protein in cultured A549 cell supernatants and the xenografts in nude mice.
Results: Compared with the negative control cells, A549 cells with LASS2/TMSG1 overexpression had significantly lowered proliferation ability in vitro with increased early apoptosis rate (P < 0.05), and showed obvious growth inhibition after inoculation in nude mice(P < 0.05). Western blotting showed that in both cultured A549 cells and the xenografts in nude mice, LASS2/TMSG1 gene overexpression significantly increased the expression levels of p38 MAPK protein and p-p38 MAPK protein (P < 0.05); the results of ELISA also revealed significantly increased levels of ceramide and p38 MAPK protein in the cell supernatant andxenografts as well (P < 0.05).
Conclusion: Overexpression of LASS2/TMSG1 gene can significantly inhibit the proliferation and promote early apoptosis of human lung cancer A549 cells both in vitro and in vivo possibly by upregulating the expressions of ceramide and p38 MAPK protein to activate a signal transduction cascade.
目的: 探究人源性长寿保障基因2/肿瘤转移抑制相关基因1（LASS2/TMSG1）过表达对人肺癌A549细胞增殖、凋亡的影响并探讨相关机制。
方法: 对课题组先前构建好的人肺癌A549细胞阴性对照组和LASS2/TMSG1基因过表达组进行如下实验：Western blot检测LASS2/TMSG1蛋白表达水平，平板克隆形成实验、CCK-8法、Hoechst/PI双染、流式细胞术检测A549细胞体外增殖能力及凋亡情况。将14只裸鼠随机分为阴性对照组和LASS2/TMSG1基因过表达组，7只/组，分别向对应组别的裸鼠颈背部皮下注射A549细胞阴性对照组和LASS2/TMSG1过表达组细胞悬液，构建裸鼠移植瘤模型，检测LASS2/TMSG1基因过表达对A549细胞体内增殖能力的影响。Western blot检测A549细胞及裸鼠移植瘤中p38 MAPK、p-p38 MAPK蛋白表达水平。ELISA法分析A549细胞上清液及裸鼠移植瘤组织匀浆中神经酰胺和p38 MAPK蛋白含量。
结果: 与阴性对照组相比，LASS2/TMSG1基因过表达组A549细胞LASS2/TMSG1蛋白表达量显著升高（P < 0.05），体外增殖能力下降（P < 0.05），早期凋亡率上升（P < 0.05），移植瘤细胞体内增殖受到抑制（P < 0.05），A549细胞及裸鼠移植瘤中p38 MAPK、p-p38 MAPK蛋白表达量均显著升高（P < 0.05），A549细胞上清液及裸鼠移植瘤组织匀浆中神经酰胺和p38 MAPK含量显著增加（P < 0.05）。
结论: LASS2/TMSG1基因过表达能显著抑制人肺癌A549细胞的体内、外增殖能力，促进肿瘤细胞早期凋亡，这可能与LASS2/TMSG1基因过表达后上调了神经酰胺及其下游的效应分子p38 MAPK蛋白进而启动级联信号传导通路有关。
Keywords: LASS2/TMSG1; apoptosis; ceramide; lung cancer; p38 MAPK; proliferation.