P300低表达食管癌细胞株TE-1的Survivivn蛋白乙酰化、增殖迁移侵袭能力变化及其机制

Changes of Survivivn protein acetylation,proliferation,migration and invasion of esophageal cancer TE-1 cell line with low expression of P300 and the mechanism

目的观察乙酰基转移酶P300低表达食管癌细胞株TE-1细胞中的Survivivn蛋白乙酰化、增殖迁移侵袭能力变化,并探讨其机制。方法取对数生长期TE-1细胞分为si-P300组、si-P300-NC组、NC组。si-P300组采用脂质体介导法转染乙酰基转移酶P300下调质粒,si-P300-NC组采用脂质体介导法转染阴性对照质粒,NC组不转染质粒,转染后继续孵育48 h进行后续实验。取各组细胞,采用免疫共沉淀(Co-IP)实验检测Survivin蛋白乙酰化水平。取各组细胞,继续培养12、24、36、48 h时,采用CCK-8法检测细胞增殖能力(以OD值表示)。取各组细胞,采用细胞划痕实验检测迁移能力(以细胞迁移距离表示),采用Transwell小室实验检测侵袭能力(以穿膜细胞数表示),采用Western blotting法检测VEGF信号通路相关蛋白AKT1、MDM2、Bcl-2、Caspase3。结果si-P300组细胞Survivin蛋白乙酰化水平均低于si-P300-NC组、NC组(P均<0.05)。si-P300组细胞培养12、24、36、48 h时的OD值均低于si-P300-NC组、NC组(P均<0.05)。si-P300组细胞迁移距离和穿膜细胞数均低于si-P300-NC组、NC组(P均<0.05)。si-P300组细胞AKT1、MDM2、Bcl-2蛋白相对表达量均低于si-P300-NC组、NC组(P均<0.05),Caspase3蛋白相对表达量均高于si-P300-NC组、NC组(P均<0.05)。结论转染P300下调质粒抑制P300表达后,TE-1细胞乙酰化水平下降,细胞增殖、迁移、侵袭能力降低,其机制可能与调控VEGF信号通路有关。

Objective To observe the changes of Survivivn protein acetylation,proliferation,migration and invasion of esophageal cancer cell line TE-1 with low expression of acetyltransferase P300,and to explore the mechanism.Methods TE-1 cells in the logarithmic growth phase were divided into the si-P300 group,SI-P300-NC group and NC group,respectively.TE-1 cells in the Si-P300 group were transfected with acetyl transferase P300 down-regulated plasmid by liposome mediated method,TE-1 cells in the SI-P300-NC group were transfected with negative control plasmid by liposome mediated method,and TE-1 cells in the NC group were transfected without plasmid and were incubated for 48 h after transfection for follow-up experiment.Cells from each group were collected and Survivin protein acetylation level was detected by Co-IP assay.Cells were taken from each group and cultured for 12,24,36 and 48 h.The cell proliferation ability(expressed by OD value)was detected by CCK-8.Cells in each group were selected,and the migration ability(represented by cell migration distance)was detected by cell Scratch test,and the invasion ability(represented by the number of transmembrane cells)was detected by Transwell chamber test.VEGF signaling pathway-related proteins AKT1,MDM2,Bcl-2,and Caspase3 were detected by Western blotting.Results The acetylation level of Survivin protein was lower in the si-P300 group than in the si-P300-NC group and NC group(both P<0.05).The OD values of the si-P300 group were lower than those of the si-P300-NC group and NC group at 12,24,36 and 48 h after cell culture(all P<0.05).The cell migration distance was shorter and the number of transmembrane cells was smaller in the si-P300 group than in the si-P300-NC group and NC group(all P<0.05).The relative expression levels of AKT1,MDM2 and Bcl-2 protein in the si-P300 group were lower than those in the si-P300-NC group and NC group(all P<0.05),and the relative expression level of Caspase3 protein was higher than those in the si-P300-NC group and NC group(all P<0.05).Conclusion After transfection with P300 down-regulated plasmid inhibits the expression of P300,the acetylation level of TE-1 cells decreases,and the cell proliferation,migration and invasion decrease,which may be related to the regulation of VEGF signaling pathway.