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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。
The miR-29b2c recombinant adenovirus Ad-miR-29b2c was constructed and its effects on the proliferation and migration of HGC-27 and MGC-803 gastric cancer cells were investigated. The fragment of miR-29b2c was amplified by PCR from the genome and cloned into the adenoviral shuttle vector pAdTrack-CMV to construct the shuttle plasmid pAdT-29b2c, which was identified by restriction analysis and sequencing. The shuttle plasmid was linearized with Pme I and then co-transformed with adenovirus backbone vector into BJ5183 competent cells to generate the recombinant adenovirus plasmid Ad-miR-29b2c. The recombinant plasmid was linearized with PacⅠ and then transfected into 293A cells for packaging. HGC-27 cells were infected with recombinant adenovirus after amplification. The effects of Ad-miR-29b2c on the proliferation and migration of HGC-27 and MGC-803 cells were observed by MTT and cell migration assay. The effect of Ad-miR-29b2c on the expression of δ-catenin in HGC-27 and MGC-803 cells was detected by Western blotting. The results of restriction enzyme digestion, sequencing and real-time PCR showed that recombinant adenovirus was successfully constructed and miR-29b and miR-29c were overexpressed in HGC-27 cells. MTT assay showed that Ad-miR-29b2c significantly inhibited the proliferation of HGC-27 and MGC-803 cells. Cell migration experiments showed that Ad-miR-29b2c significantly inhibited the migration of HGC-27 and MGC-803 cells. In addition, Ad-miR-29b2c significantly decreased the expression of δ-catenin protein in HGC-27 and MGC-803 cells. In summary, the recombinant adenovirus miR-29b2c was constructed and found to inhibit the proliferation and migration of gastric cancer cells HGC-27 and MGC-803, which may be related with miR-29 inhibition of HGC-27, MGC-803 cells δ-catenin protein expression.