LKB1对胃癌细胞增殖、凋亡的影响及相关分子实验

Role of LKB1 in proliferation and apoptosis of gastric cancer cells

目的:探讨LKB1在胃癌发生中的作用及相关分子机制研究,为胃癌化疗药物的研发提供一定的理论基础.方法:利用L K B1过表达质粒以及L K B1s i R N A转染人胃癌细胞株S G C7901,实时荧光定量PCR及Western blot检测质粒以及s i R N A转染效果,流式细胞术检测L K B1过表达以及干扰后的SGC7901细胞(血清饥饿处理2 h)凋亡数量.活性氧检测试剂盒检测L K B1对S G C7901细胞内R O S产生的影响M T T法检测N A C抑制细胞内R O S产生后LKB1过表达以及干扰后SGC7901细胞数量的改变.Western blot检测LKB1过表达以及干扰后SGC7901细胞中凋亡相关蛋白的表达水平.结果:LKB1过表达质粒转染后的人胃癌细胞株SGC7901细胞凋亡增加,胞内ROS产生增加,LKB1过表达后的SGC7901细胞凋亡则明显增多(3.54%vs 1.29%),转染LKB1 si RNA的人胃癌细胞株SGC7901早期凋亡和晚期凋亡的细胞数量占总数较转染scramble si RNA组明显减少(0.54%vs 1.39%)同时Western blot检测发现LKB1过表达后的SGC7901细胞中剪切型Caspase3表达明显升高上升3.12倍,较对照组差异有统计学意义而si RNA干扰后剪切型Caspase3表达则表现为相反的趋势.结论:LKB1通过促进ROS的产生,上调剪切型Caspase3介导的凋亡通路促进胃癌细胞凋亡,因此LKB1在胃癌发生发展过程中具有重要抑制作用,其可能作为胃癌化疗药物研发的重要目标分子.

AIM：To explore the role of LKBl in gastric cancer cells and the related mechanism.METHODS：Real-time PCR and Western blot were used to detect the expression of LKBl in SGC7901 cells carrying LKBl expression vector or siRNA against LKBl.Flow cytometry was used to detect the apoptosis of SGC7901 cells after LKBl overexpression or knockdown.Reactive oxygen detection kits were applied to detect the impact of LKBl on ROS production.MTT method was used to determine intracellular ROS production after NAC inhibition.Western blot was used to detect the expression of apoptosis related proteins in SGC7901 cells after LKBl overexpression or knockdown.RESULTS：LKBl expression was efficiently enhanced or silenced by LKBl expression vector or siRNA against LKBl,respectively.The number of SGC7901 cells decreased as its proliferation rate decreased and apoptosis rate increased（3.54%vs 1.29%）.Intracellular ROS production was increased but blunted by the use of NAC.The apoptosis of SGC7901 cells was significantly reduced following the inhibition of intracellular ROS,but the siRNA transfected group exhibited an opposite trend.Western blot analysis showed that LKBl overexpression up-regulated the expression of cleaved Caspase3 in SGC7901 cells significantly（about 3.12 times）,compared with control cells,but the expression of cleaved Caspase3 in the siRNA transfected group was decreased.CONCLUSION：LKBl raises the production of ROS and up-regulates the expression of cleaved Caspase3 to promote gastric cancer cell apoptosis.Hence,LKB1 plays an important role in the development of gastric cancer and it may be a valuable target for chemotherapy of gastric cancer.