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.
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。.
Keywords: gastric cancer; gene therapy; miR-29; recombinant adenovirus.