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

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。

Abstract:

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.