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构建miRNA-29a/c的重组腺病毒并观察其对膀胱癌T24细胞增殖能力的调控。以人全基因组DNA为模板,PCR扩增miR-29a、miR-29c,克隆至腺病毒穿梭载体pAdtrace-TO4-CMV。重组穿梭载体经pme I线性化后与腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌BJ5183,通过同源重组获得重组腺病毒质粒pAdEasy-1-miR-29a、pAdEasy-1-miR-29c,pac I线性化后转染HEK-293细胞,进行包装和扩增。实时荧光定量PCR检测感染腺病毒的膀胱癌T24细胞中miR-29a、miR-29c的表达水平,并利用CCK-8实验检测细胞增殖能力。经DNA测序和限制性内切酶分析显示,重组腺病毒质粒pAdEasy-1-miR-29a、pAdEasy-1-miR-29c构建成功;感染腺病毒Ad-miR-29a和Ad-miR-29c后,经实时荧光定量PCR检测,膀胱癌细胞中miR-29a、miR-29c表达显著增高(P<0.01);过表达miR-29a/c后的CCK-8实验显示,细胞增殖能力明显低于对照组(P<0.05)。以上说明已成功构建miR-29a、miR-29c腺病毒,过表达miR-29a/c可抑制膀胱癌细胞的增殖。
The recombinant adenovirus of miRNA-29a / c was constructed and the proliferation of bladder cancer T24 cells was observed. MiR-29a and miR-29c were amplified by PCR using human whole genome DNA as a template, and cloned into the adenovirus shuttle vector pAdtrace-TO4-CMV. The recombined shuttle vector was transformed into competent E. coli BJ5183 by adenovirus backbone plasmid pAdEasy-1 after pme I linearization. The recombinant adenovirus plasmids pAdEasy-1-miR-29a, pAdEasy-1-miR- Pac I was linearized and transfected into HEK-293 cells for packaging and amplification. The expression of miR-29a and miR-29c in bladder cancer T24 cells infected with adenovirus was detected by real-time fluorescence quantitative PCR. The cell proliferation was detected by CCK-8 assay. DNA sequencing and restriction endonuclease analysis showed that recombinant adenovirus plasmids pAdEasy-1-miR-29a and pAdEasy-1-miR-29c were successfully constructed. After adenovirus Ad-miR-29a and Ad-miR- The expression of miR-29a and miR-29c in bladder cancer cells was significantly increased by real-time fluorescent quantitative PCR (P <0.01). The CCK-8 assay after overexpression of miR-29a / c showed that the cell proliferation ability was significantly lower than that of the control group (P <0.05). The above description has been successfully constructed miR-29a, miR-29c adenovirus, overexpression of miR-29a / c can inhibit bladder cancer cell proliferation.