过表达LATS1降低食管癌Eca109细胞增殖

Overexpression of LATS1 reduces the proliferation of Eca109 cells

目的运用慢病毒转染技术在食管癌Eca109细胞中过表达LATS1基因,探究LATS1调控Eca109细胞增殖、凋亡及周期的作用及机制。方法构建LATS1基因的慢病毒载体转染Eca109细胞系,RT-PCR检测细胞中LATS1mRNA的表达;Western blot检测LATS1、YAP、BAX/BCL-2的蛋白表达水平;MTS检测细胞增殖;流式细胞术检测细胞凋亡及周期;hochest33258观察细胞凋亡染色。结果 LATS1慢病毒载体转染Eca109细胞后,目的基因组(AdLATS1)LATS1 mRNA及LATS1蛋白表达、BAX蛋白表达明显高于对照组(CON)及阴性对照组(Ad-GFP),而YAP、BCL-2的蛋白表达明显低于对照组及Ad-GFP组(P<0.05);Ad-LATS1组Eca109细胞的增殖率从第5天开始明显低于对照组及Ad-GFP组(P<0.05);Ad-LATS1组细胞较Ad-GFP及对照组G1期比例明显增高而S期比例明显缩短,凋亡率明显增高(P<0.05),细胞荧光染色强度及范围也增高。结论 LATS1基因可上调BAX、下调YAP、BCL-2使Eca109凋亡,并诱导G1期延长、S期缩短来降低其增殖力。

Objective To discuss the impact and mechanism on proliferation,apoptosis and cell cycle of Eca109 cells by overexpress LATS1 in lentivirus transfection technique.Methods Built LATS1-lentivirus vector then transfected the lentivirus vector into Eca109 cells;LATS1 mRNA expression was detected by real-time PCR;LATS1,YAP,BAX/BCL-2 protein expression was detected by western blotting;proliferation ability was detected by MTS assay;the ratio of apoptosis and cell cycle were detected by FCM;the dyeing of apoptosis was observed by hochest33258.Results After transfection of LATS1-lentivirns in Eca109 cells （Ad-LATS1 group）,we found lats1 mRNA expression and protein expression level,BAX protein expression level were all remarkablely higher than control group and Ad-GFP groups while YAP,BCL-2 protein expression level were on the contrary（P ＜0.05）;The ratio of proliferation in Ad-LATS1 group was significantly lower than control group and Ad-GFP groups from the fifth day （P ＜ 0.05）;FCM showed more cells stay in G1 phase in Ad-LATS1 group than in control group and-GFP groups while the ratio of cells in S phase were on the contrary and apoptosis rate was much higher in Ad-LATS1 group than in others（P ＜0.05）;Hoechst 33258 dying also demonstrated that LATS1 could induce apoptosis in Eca109 cells through the degree and range of dyeing.Conclusions LATS1 gene may up-regulate BAX whlle down-regulate YAP and BCL-2 to increase the apoptosis rate of Eca109 cells,more over,it potentially prolong the G1 phase of cell cycle and shorten the S phase to inhibit the proliferation of Eca109 cells.