TGF-β1对胃癌细胞增殖、凋亡及miR-302a和AKT通路的影响

Effects of TGF-β1 on the Proliferation and Apoptosis of Gastric Canc-er Cells and the miR-302a and AKT Pathways

目的探究转化生长因子-β1(TGF-β1)对胃癌细胞增殖、凋亡及miR-302a和AKT通路的影响.方法在含有TGF-β1(0、1、5、10 ng/ml)的培养基培养胃癌细胞系BGC-823,MTT法检测细胞增殖情况;平板克隆形成实验检测细胞克隆形成数量;Hoechst33258染色观察细胞形态;流式细胞术检测细胞凋亡以及周期阻滞情况;实时荧光定量PCR(qRT-PCR)检测细胞miR-302a表达;蛋白印迹(WB)法检测p-PI3K、AKT、p-AKT蛋白表达情况.结果与Control组相比,TGF-β1处理组细胞增殖抑制率、细胞凋亡率升高,细胞克隆数量降低,且随着TGF-β1浓度的增加,细胞增殖抑制率、细胞凋亡率逐渐升高,细胞克隆数量逐渐降低(P<0.05).与Control组相比,TGF-β1处理组G1期细胞比例显著增加,G2期细胞比例显著降低,且随着TGF-β1浓度的增加,G1期细胞比例逐渐增加,G2期细胞比例逐渐降低(P<0.05).与Control组相比,TGF-β1处理组miR-302a表达升高,p-PI3K、p-AKT蛋白表达水平均显著降低,且随着TGF-β1浓度的增加,miR-302a表达逐渐升高,p-PI3K、p-AKT蛋白表达逐渐降低(P<0.05).结论TGF-β1可能通过上调miR-302a表达、抑制AKT通路,使细胞G1期阻滞,从而抑制胃癌细胞增殖,促进其凋亡.

Objectivek To investigate the effects of transforming growth factor-β1(TGF-β1)on the proliferation and apoptosis of gastric cancer cells and the miR-302a and AKT path-ways.Methodsk Gastric cancers(BGC-823)were cultured in the medium containing TGF-β1(0,1,5,10 ng/ml).MTT assay was used to detect cell proliferation;plate colony formation as-say was used to detect the number of cell clones;cell morphology was observed by Hoechst33258 staining;cell apoptosis and cell cycle arrest were detected by flow cytometry;real-time fluores-cence quantitative PCR(qRT-PCR)was used to detect miR-302a expression;Western blotting(WB)was used to detect the expressions of p-PI3K,AKT and p-AKT.Resultsk With the increase of TGF-β1 concentration,the cell proliferation inhibition rate and apoptosis rate increased,and the number of cell clones decreased in the TGF-β1 treatment groups.The difference in the cell prolifera-tion inhibition rate and apoptosis rate was significant between the TGF-β1 treatment groups and the control group(P<0.05).The proportion of G1 phase cells increased significantly,and that of G2 phase cells decreased significantly in TGF-β1 treatment groups as compared with those in the control group(P<0.05).The expression of miR-302a was much higher,and the expression of p-PI3K and p-AKT much lower in TGF-β1 treatment groups than in control group(P<0.05).Conclu-sionk TGF-β1 may inhibit the proliferation and promote apoptosis of gastric cancer cells by up-regu-lating the expression of miR-302a,suppressing the AKT pathway,and arresting cells at G1 phase.