遗传 ›› 2012, Vol. 34 ›› Issue (2): 190-197.doi: 10.3724/SP.J.1005.2012.00190

• 研究报告 • 上一篇    下一篇

食管癌细胞抗失巢凋亡基因UBCH7的发现与鉴定

杨扬1, 2, 王博石2, 汪晓敏2, 张钰2, 王明荣2, 贾雪梅1   

  1. 1. 安徽医科大学组织胚胎学教研室, 合肥 230032 2. 中国医学科学院北京协和医学院 肿瘤医院肿瘤研究所, 分子肿瘤学国家重点实验室, 北京 100021
  • 收稿日期:2011-10-18 修回日期:2011-12-10 出版日期:2012-02-20 发布日期:2012-02-25
  • 通讯作者: 王明荣 E-mail:wangmr2015@126.com
  • 基金资助:

    国家高技术研究发展规划(863计划)项目(编号:2009AA022706)和中国医学科学院分子肿瘤学国家重点实验室开放基金(编号:SKL-KF-2011-01)资助

Screening and identification of anoikis-resistant gene UBCH7 in esophageal cancer cells

YANG Yang1, 2, WANG Bo-Shi2, WANG Xiao-Min2, ZHANG Yu2, WANG Ming-Rong2, JIA Xue-Mei1   

  1. 1. Department of Histology and Embryology, Anhui Medical University, Hefei 230032, China 2. State Key Laboratory of Molecular Oncology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China
  • Received:2011-10-18 Revised:2011-12-10 Online:2012-02-20 Published:2012-02-25
  • Contact: Mingrong Wang E-mail:wangmr2015@126.com

摘要: 失巢凋亡(Anoikis)是细胞失去与细胞外基质(Extra-cellular matrix, ECM)粘附时发生的特殊形式的凋亡, 是机体维持组织稳态的关键机制之一。抗失巢凋亡能力的获得是肿瘤细胞发生远处转移的前提条件之一。为了鉴定与食管癌细胞抗失巢凋亡相关的基因, 文章首先构建食管癌细胞系的逆转录病毒文库, 感染对失巢凋亡敏感的NIH3T3细胞, 利用感染病毒cDNA文库的混合细胞系进行软琼脂集落形成实验, 挑取在悬浮条件下仍可生长成为较大集落的细胞单克隆(潜在具有抗失巢凋亡能力的细胞), 通过逆转录病毒载体特异的引物PCR扩增失巢凋亡抗性克隆基因组中的插入cDNA片段, 以此获得食管癌细胞系cDNA文库中潜在的具有失巢凋亡抗性的基因。经测序发现其中一个失巢凋亡克隆中整合的cDNA片段包括人UBCH7/UBE2L3基因全长的编码序列(开放阅读框)。利用携带pMSCV-UBCH7的逆转录病毒感染NIH3T3细胞进行验证, 结果显示细胞失巢凋亡抗性增强, 并且在具有高转移潜能的食管癌细胞系MLuC1中降调UBCH7表达可减弱其失巢凋亡抗性。这些结果表明, UBCH7/UBE2L3是一个与食管癌失巢凋亡抗性相关的基因。

关键词: 失巢凋亡, 食管癌(ESCC), 逆转录病毒cDNA文库筛选, UBCH7

Abstract: Anoikis is a kind of programmed cell death induced by loss of extracellular matrix (ECM) adhesion, which is one of key factors for homestasis. Resistance to anoikis is required for tumor cell metastasis. We have previously shown several anoikis-resistance genes in esophageal squamous cell carcinoma (ESCC). In order to find novel anoikis-resistant genes in ESCC, we constructed retroviral cDNA library using total RNA from ESCC cell lines. NIH 3T3 cells, which are sensitive to anoikis, were infected with the library constructed. The cells were cultured in soft agar, and the clones which can survive in detached states were selected. The cDNAs inserted into the anoikis-resistant NIH3T3 clones were amplified using retroviral specific primers. Sequencing analysis showed that a cDNA fragment inserted into the anoikis-resistant clone contains full coding sequence (ORF) of human UBCH7/UBE2L3 gene. By infection with retrovirus encoding UBCH7 ORF (pMSCV-UBCH7), forced expression of UBCH7 increased the anoikis-resistance of NIH3T3 cells. More importantly, knockdown of UBCH7 expression by siRNA transfection reduced the anoikis-resistant ability of esophageal cancer MLuC1 cells. The data suggest that UBCH7/UBE2L3 gene would be involved in anoikis-resistance in ESCC.

Key words: anoikis, esophageal squamous cell carcinoma (ESCC), retroviral cDNA library screen, UBCH7