摘要
目的 观察胃癌细胞中转移相关基因1(MTA1)是否可被泛素化修饰及其对MTA1表达的影响.方法 将未转染的胃癌MKN-45细胞[Myc-MTA1(-)、HA-Ub(-)]设为对照组,3个实验组MKN-45细胞分别转染入带Myc标记的MTA1质粒[Myc-MTA1(+)、HA-Ub(-)]和带HA标记的泛素质粒[Myc-MTA1(-)、HA-U(+)]以及共同转染入该两种质粒的胃癌MKN-45细胞[Myc-MTA1(+)、HA-Ub(+)].向每个培养皿内转染2μg质粒,培养24h后即可收获转染细胞,以蛋白酶体抑制剂MG-132处理实验组胃癌细胞MKN-45,以免疫沉淀法、Western blot法等检测MTA1表达水平,并将带Myc标记的MTA1质粒和带HA标记的泛素(HA-Ub)质粒分别转入胃癌细胞MKN-45,设为实验组,未转染的胃癌细胞MKN-45为对照组,检测各组细胞中Myc-MTA1的表达.同时采用流式细胞仪法检测MG-132诱导各组胃癌MKN-45细胞的凋亡.结果 (1)以蛋白酶体抑制剂MG-132处理胃癌细胞MKN-45后,MTA1表达水平显著增加;2、6h分别为(0.78 ±0.15) μg/ml和(1.37±0.33)μg/ml,与对照组比较[(0.28 ±0.09) μg/ml],差异有统计学意义(t=2.134,5.562,P <0.05).(2)免疫共沉淀实验结果显示,6h后,实验组泛素化程度为(77.28±9.38)%,与对照组的(15.41±2.14)%比较,实验组泛素化程度明显增加(P<0.05).(3)在胃癌MKN-45细胞[Myc-MTA1(+)、HA-Ub(+)]中,可检测到Myc-MTA1可被强烈泛素化,泛素化程度为(64.59±7.61)%,与另外两组比较,差异有统计学意义(P<0.05).(3)将MKN-45细胞以siMTA1处理封闭MTA1的表达后,将Myc-MTA1质粒以及HA-Ub质粒转入该细胞,可见当后者存在时Myc-MTA1可被强烈泛素化.转染两种质粒的胃癌MKN-45细胞[Myc-MTA1(+)、HA-Ub(+)]凋亡数量显著多于对照组MKN-45 细胞(t =2.436,3.537,P<0.05).(4)c-聚腺苷二磷酸核糖聚合酶(PARP)的水平显示MKN-45细胞[Myc-MTA1(+)、HA-Ub(+)]凋亡程度显著高于对照组MKN-45细胞(t=1.845,2.374,P<0.05).结论 在胃癌细胞中,MTA1是可被泛素化途径修饰的蛋白,其泛素化途径在胃癌的发展与转移中发挥重要作用.
Objective To observe the ubiquitination of metastasis associated 1 (MTA1) in gastric cancer cells and its effect on the expression of MTA1.Methods The non-transfected MKN-45 cells [Myc-MTA1 (-),HA-Ub (-)] served as the control group,and three experimental groups of MKN-45 cells were transfected with the myc tag of MTA1 plasmid [Myc-MTA1 (+),HA-Ub (-)],HA tagged ubiquitin plasmid [Myc-MTA1 (-),HA-U (+)],or co-transfected with the two plasmids [Myc-MTA1 (+),HA-Ub (+)].2 μg plasmid was transfected into each culture dish,and then the transfected cells were harvested after 24 h culture.MKN-45 cells in the experimental group were treated with proteasome inhibitor MG-132.The expression level of MTA1 was detected by immunoprecipitation (IP) and Western blotting.MTA1 expressing plasmid containing the Myc tag and HAtagged ubiquitin plasmid (HA-Ub) was co-transfected into cells,and the expression of Myc-MTA1 was detected.Flow cytometry was used to detect the apoptosis of MKN-45 cells induced by MG-132.Results (1) With the proteasome inhibitor MG-132 treatment of gastric cancer cell line MKN-45,MTA1 expression levels increased significantly in experimental group,there were (0.78 ± 0.15) μg/ml and (1.37 ± 0.33) μg/ml after 2 h and 6 h,compared with the control group [(0.28 ±0.09) μg/ml],the difference was statistically significant (t =2.134,5.562,P 〈0.05).(2) After 6 h,the degree of ubiquitination in experimental group was (77.28 ± 9.38) %,and the control group was (15.41 ± 2.14) %,the difference was statistically significant (P 〈 0.05).(3) In gastric cancer MKN-45 cells [Myc-MTA1 (+),HA-Ub (+) group,Myc-MTA1 can be strongly ubiquitin,the degree of ubiquitin was (64.59 ± 7.61)%,compared with the other two groups,the difference was statistically significant (P 〈0.05).(4) MG-132 treatment of the experimental group and the control group of MKN-45 gastric cancer cells after 24 h of gastric cancer MKN-45 cells transfected with [Myc-MTA1 (+) 、HA-Ub(+)],the apoptosis ratio was (22.90 ± 4.17)%,significantly higher than control group MKN-45 cell apoptosis ratio (8.25 ± 1.04) %.C-poly adenosine diphosphate-ribose polymerase (c-PARP) levels showed that [Myc-MTA1 (±),HA-Ub (+)] the degree of apoptosis was (29.77 ±5.04) %,significantly higher than the control group MKN-45 cells (13.10 ±3.28)% (t =1.845,2.374,P 〈0.05).Conclusion MTA1 in human gastric cancer cells can be regulated through ubiquitination pathway.Its ubiquitination pathway plays an important role in the development and metastasis of gastric cancer.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第5期1234-1237,共4页
Chinese Journal of Experimental Surgery
关键词
胃癌
转移相关基因1
泛素化
Gastric cancer
Metastasis associated 1
Ubiquitination