TAT-E4orf4融合蛋白对胃癌细胞增殖、迁移和凋亡的影响  

Effects of TAT-E4orf4 fusion protein on migration and apoptosis of gastric cancer cells

作　　者：殷洁烽[1] 徐统球 张喆[1] 孙元水[1] YIN Jie-feng;XU Tong-qiu;ZHANG Zhe;SUN Yuan-shui(Department of General Surgery,Tongde Hospital,Hangzhou,Zhejiang 310012,China;Department of Anus and Intestine Surgery,Xianju County Hospital of Chinese Medicine,Taizhou 317300,China)

机构地区：[1]浙江省立同德医院普外科,杭州310012 [2]浙江省仙居县中医院肛肠外科,台州317300

出　　处：《浙江中西医结合杂志》2021年第2期111-115,124,共6页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省医药卫生科技计划项目(No.2018KY329)。

摘　　要：目的分析TAT-E4orf4融合蛋白对胃癌AGS细胞凋亡的影响及其作用机制。方法PCR法构建pET28-his-TAT-E4orf4表达载体,经PCR酶切鉴定后,诱导表达。并分别用pET28、pET28-his-E4orf4和pET28-his-TAT-E4orf4处理胃癌AGS细胞,分别采用MTT法和Transwell小室检测TAT-E4orf4对胃癌细胞增殖和迁移的影响,以流式细胞仪检测刺激后胃癌细胞的凋亡,以Western blot检测半胱氨酸天冬氨酸蛋白酶(Caspases)和酪氨酸激酶(Src)信号轴上关键蛋白表达的变化。结果PCR和Western blot实验结果证实成功构建pET28-TAT-E4orf4表达载体,MTT法显示pET28-his-E4orf4和pET28-his-TAT-E4orf4刺激后,胃癌AGS细胞增殖活性较空白对照组(1.01±0.12)分别下降至(0.69±0.44)和(0.41±0.06),且后者下降更为明显(P<0.05)。与MTT实验结果趋势相似,Transwell实验的结果,pET28-his-TAT-E4orf4刺激后胃癌细胞迁移数目显著减少到(163.21±36.77)个,与Blank组(796.48±114.35)个、pET28组(803.71±127.39)个、pET28-his-E4orf4组(287.06±78.51)个比较,差异具有统计学意义(P均<0.05)。流式细胞术检测结果显示,TAT-E4orf4融合蛋白干预后,AGS细胞凋亡率显著增加(33.40%),与Blank组(6.28%)、pET28组(8.40%)、pET28-his-E4orf4组(20.50%)比较,差异具有统计学意义(P<0.05)。Western blot实验结果表明,Caspase-3和Caspase-9的表达水平无明显变化(P>0.05),而Src-ERK-AKT信号轴相关蛋白的表达在TAT-E4orf4融合蛋白干预后出现明显下调,差异具有统计学意义(P<0.05)。结论TAT-E4orf4融合蛋白能抑制胃癌细胞增殖和迁移,促进胃癌细胞凋亡,与Caspase途径无关,主要依赖对Src-ERK-AKT信号通路的调控。Objective To assess the effects of TAT-E4orf4 fusion protein on the migration and apoptosis of gastric cancer cells.Methods A recombinant expression vector pET28-his-TAT-E4orf4 containing cDNA of cell penetrating peptide-adenovirus early region 4 open reading frame 4 protein was constructed using PCR and cloning techniques,and the expression of fusion protein was induced.Gastric cancer AGS cells were treated with expressed proteins from pET28,pET28-his-E4orf4,or pET28-his-TAT-E4orf4 vector,respectively.Cell viability and migration capacity were measured using MTT and Transwell assays,respectively,while cell apoptosis was detected using flow cytometry.Western blot was used to detect the expression of key proteins on the Caspases and tyrosine kinase Src signaling pathways.Results PCR and Western blot confirmed the successful construction of pET28-TAT-E4orf4 expression vector and the expression of the fusion protein,respectively.MTT assay showed AGS cell viability was reduced obviously after treatment with proteins from pET28-his-E4orf4 and pET28-his-TAT-E4orf4(0.69±0.44 and 0.41±0.06 vs.1.01±0.12,respectively).Moreover,Transwell assay showed significant inhibition effects of pET28-his-TAT-E4orf4 on tumor cell migration compared to the Blank,pET28,and pET28-his-E4orf4 groups(163.21±36.77 vs.796.48±114.35,803.71±127.39 and 287.06±78.51,respectively;P<0.05).Flow cytometry data showed that TAT-E4orf4 fusion protein treatment significantly increased the apoptosis rate of AGS cells(33.4%),compared to the Blank(6.28%),pET28(8.40%),and pET28-his-E4orf4 groups(20.5%)(P<0.05).Western blot data showed no significant change in the expression of Caspase-3 and-9(P>0.05),while the expression of Scr-ERK-AKT pathway proteins was significantly decreased with TAT-E4orf4 fusion protein treatment(P<0.05).Conclusion The TAT-E4orf4 fusion protein was able to inhibit gastric cancer cell viability and migration and promote tumor cell apoptosis,which was not associated with Caspase pathway while might be through the Scr-ERK-AKT signa

关 键 词：胃癌 凋亡 细胞穿膜肽 腺病毒早期区4第4编码蛋白 迁移 

分 类 号：R735.2[医药卫生—肿瘤]