期刊文献+

胃癌细胞MKN-45中肿瘤转移相关基因1的泛素化修饰及对其表达的影响 被引量:1

Ubiquitination and expression of metastasis associated 1 in gastric cancer MKN-45 cells
原文传递
导出
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
  • 相关文献

参考文献15

  • 1Yaguchi M, Wada Y,Toh Y,et al. Identification and characterization of the variants of metastasis-associated protein 1 generated following alter- native splicing[J]. Biochim Biophys Acta,2005,1732(1-3) :8-14. DOI : 10. 1016/j. bbacxp. 2005.12. 001.
  • 2李新涛,马鑫,逄海港,高宇,范阳,张旭.TD—IκBα病毒包装及其对肾癌细胞株ACHN增殖和侵袭的影响[J].中华实验外科杂志,2014,31(6):1184-1186. 被引量:3
  • 3Manavathi B, Kumar R. Metastasis tumor antigens, an emerging family of muhifaceted master coregulators [ J ]. J Biol Chem,2007,282 ( 3 ) :1529-1533. DOI: 10. 1002/pros. 20563.
  • 4Toh Y, Ohga T, Endo K, et al. Expression of the metastasis-associated MTA1 protein and its relationship to deaeetylation of the histone H4 in esophageal squamous cell carcinomas [ J]. Int J Cancer,2004,110 (3) :362-367. DOI:10. 1002/ije. 20154.
  • 5Hofer MD, Kuefer R, Varambally S, et al. The role of ciated protein 1 in prostate cancer progression [ J ]. Cancer Res,2004, 64 ( 3 ) : 825 -829. DOI: 10.1002/cr. 2004.0813. a.
  • 6Iai AZ,Durrant M,Zuo D,et al. Met kinase-dependent loss of the E3 lig- ase Cbl in gastric cancer[J]. J Biol Chem,2012,287 (11 ) :8048-8059. DOI : 10. 1074/jbe. M112. 339820.
  • 7Wang ZJ,Yang JL, Wang YP, et al. Decreased histone H2B monou- biquitination in malignant gastric carcinoma [ J ]. World J Gastroen- terol,2013,19 (44) :8099-8107. DOI : 10. 3748/wjg. v19. i44. 8099.
  • 8Yi C,Li X,Xu W,et al. Relationship between the expression of MTA-1 gene and the metastasis and invasion in human osteosarcoma[J]. J Hua- zhong Univ Sci Technolog Med Sci ,2005,25(4) :445-447. DOI:10. 1074/jbc. M206743200.
  • 9Cong L,Pakala SB,Ohshiro K,et al. SUMOylation and SUMO-interacting motif (SIM) of metastasis tuwr antigen 1 (MTA1) synergistically regulate its transcriptional repressor function [ J ]. J Biol Chem, 2011,286 ( 51 ) : 43793-438. DOI, 10.1074/ibc. M111. 267237.
  • 10卢秀波,刘洋,殷德涛,邱新光,王庆兆.乳头状甲状腺癌中S100A4、E-钙黏素的表达及其意义[J].中华实验外科杂志,2007,24(1):31-32. 被引量:8

二级参考文献18

  • 1卢秀波,安兆峰,王庆兆,殷德涛,邱新光.Survivin和c-myc在分化型甲状腺癌中的表达及临床意义[J].中华实验外科杂志,2004,21(6):705-706. 被引量:18
  • 2李琦,于颖彦,朱正纲,刘炳亚,纪玉宝,张奕,程琳玲,林言箴.核转录因子-κB组成性激活对胃癌细胞增殖的影响及其机制[J].中华实验外科杂志,2004,21(8):957-960. 被引量:12
  • 3张东伟,杨维良.甲状腺癌基因治疗的现状及展望[J].中华实验外科杂志,2005,22(2):255-256. 被引量:16
  • 4Navab F.Stress ulcer.Is routme prophylaxis necessary? Gastroenterol,1995.90:708.
  • 5Sou EY,Clark OH.Surgical considerations and approach to thyroid cancer.Endocrinol Metal Clin North Am,1996,25:115-118.
  • 6Mazzucchelli L.Protein S100A4:too long overlooked by pathologists? Pathol,2002,160:7-13.
  • 7Rudland PS,Platt-Higgins A,Renshaw C,et al.Prognostic significance of the metastasis-inducing protein S100A4 (p9ka) in human breast cancer.Cancer Res,2000,60:1595-1603.
  • 8Kimura Y,Endo Y,Yonemura Y,et al.Clinical significance of S100A4 and E-cadherin-related adhesion molecules in nonsmall cell lung cancer.Oncol,2000,16:1125-1131.
  • 9Pietase A,Schluns K,Marenholz I,et al.Molecular cloning and characterization of the human S100A4 gene encoding a novel member of the 5100 family.Genomics,2002,79:513-522.
  • 10Yonemura Y,Endou Y,Kimura K,et al.Inverse expression of S100A4 and E-cadherin is associated with metastatic potential in gastric cancer.Clin Cancer Res,2000,6:4234-4242.

共引文献9

同被引文献4

引证文献1

二级引证文献2

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部