miR-29b2c重组腺病毒的构建及对胃癌细胞增殖和迁移的影响
Effects of recombinant adenovirus Ad-miR-29b2c on HGC-27 cell proliferation and migration
投稿时间:2016-01-04  
DOI:  10.13345/j.cjb.170003
中文关键词:miR-29,胃癌,重组腺病毒,基因治疗
英文关键词:miR-29, gastric cancer, recombinant adenovirus, gene therapy
基金项目:湖北省教育厅科学技术研究计划青年人才项目 (No. Q20141002) 资助。
作者单位E-mail
骆爱玲 湖北大学 中药生物技术省重点实验室 生物资源绿色转化湖北省协同创新中心湖北 武汉 430062  
杜春园 湖北大学 中药生物技术省重点实验室 生物资源绿色转化湖北省协同创新中心湖北 武汉 430062  
赵雪梅 湖北大学 中药生物技术省重点实验室 生物资源绿色转化湖北省协同创新中心湖北 武汉 430062  
梁继超 湖北大学 中药生物技术省重点实验室 生物资源绿色转化湖北省协同创新中心湖北 武汉 430062 liang529114@163.com 
陈勇 湖北大学 中药生物技术省重点实验室 生物资源绿色转化湖北省协同创新中心湖北 武汉 430062 cy101610@qq.com 
摘要点击次数: 168
全文下载次数: 379
中文摘要:
      构建了miR-29b2c重组腺病毒Ad-miR-29b2c,考察了其对HGC-27、MGC-803胃癌细胞增殖及迁移的抑制作用。采用PCR从基因组扩增miR-29b2c片段,并克隆至腺病毒穿梭载体pAdTrack-CMV中,构建穿梭质粒pAdT-29b2c,经酶切及测序鉴定。穿梭质粒经PmeⅠ线性化后与腺病毒骨架载体共转化BJ5183感受态,产生重组腺病毒质粒Ad-miR-29b2c,再经PacⅠ线性化后转染293A细胞进行包装。重组腺病毒扩增后感染HGC-27细胞,通过MTT及细胞迁移实验观察Ad-miR-29b2c对HGC-27、MGC-803细胞增殖及迁移的影响。采用Western blotting检测Ad-miR-29b2c对HGC-27、MGC-803细胞δ-catenin蛋白表达的影响。酶切、测序及荧光定量PCR结果表明重组腺病毒构建成功,miR-29b及miR-29c在HGC-27细胞过表达。MTT实验表明Ad-miR-29b2c能显著抑制HGC-27、MGC-803细胞增殖。细胞迁移实验表明Ad-miR-29b2c能显著抑制HGC-27、MGC-803细胞迁移。此外,Ad-miR-29b2c能显著降低HGC-27、MGC-803细胞δ-catenin蛋白表达水平。综上所述,构建了miR-29b2c的重组腺病毒,并发现其可以抑制胃癌细胞HGC-27和MGC-803的增殖及迁移,该作用可能与miR-29抑制HGC-27、MGC-803细胞δ-catenin蛋白表达有关。
英文摘要:
      We constructed recombinant adenoviruses expressing miR-29b2c (Ad-miR29b2c), and analyzed their effects on the proliferation and migration of HGC-27 and MGC-803 cells. miR-29b2c gene was amplified by PCR from genomic DNA and cloned into the pAdTrack-CMV vector to create the shuttle plasmid pAdT-29b2c. The recombinant plasmid was verified by restriction enzyme digestion and sequencing. The linearized shuttle vector was mixed with an adenoviral backbone plasmid (pAdEasy-1), followed by cotransformation into competent BJ5183 cells to generate the recombinant plasmid pAd-miR-29b2c. Finally, recombinant adenoviral vectors were generated by transfecting the recombinant plasmid into 293A packaging cell line. HGC-27 and MGC-803 cells were infected with the recombinant adenoviruses expressing pAd-miR-29b2c, then MTT and wound-healing assay were used to analyze the effects of pAd-miR-29b2c on the proliferation and migration of HGC-27 and MGC-803 cells. The miR-29b and miR-29c levels were significantly increased in HGC-27 cells after infected with pAd-miR-29b2c. MTT and wound-healing analysis also revealed a significant decrease in proliferation and migration of HGC-27 and MGC-803 cells compared to the control Ad-GFP-infected cells. Furthermore, western blotting results demonstrated that the protein expression level of δ-catenin was reduced in pAd-miR-29b2c transfected HGC-27 and MGC-803 cells. Taken together, the recombinant adenoviral vector was generated, and it can significantly inhibit the proliferation and migration of HGC-27 and MGC-803 cells.
骆爱玲,杜春园,赵雪梅,梁继超,陈勇.miR-29b2c重组腺病毒的构建及对胃癌细胞增殖和迁移的影响[J].生物工程学报,2017,33(7):1136~1144
HTML   查看全文   查看/发表评论  下载PDF阅读器