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目的构建小RNA193前体(pre-miR-193b)重组腺病毒,观察miR-193b对慢性髓系白血病K562细胞增殖的影响。方法化学合成miR-193b cDNA序列,克隆进入pAdTrack-CMV穿梭质粒;重组穿梭质粒经PmeⅠ酶切线性化后,转入含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183感受态细胞中进行同源重组,获得pAd-miR-193b重组腺病毒质粒,经PacI线性化后转入HEK293细胞包装、扩增。倍比稀释法测定病毒滴度。实时荧光定量PCR检测K562细胞中miR-193b表达水平,MTT法检测K562细胞增殖能力。结果pAd-miR-193b重组腺病毒质粒,经限制性内切酶鉴定和测序分析显示构建成功,并能够高效转染K562细胞;与对照组相比,荧光定量PCR检测显示重组腺病毒载体感染K562细胞后,miR-193b基因表达明显增加;同时高表达miR-193b基因的K562细胞增殖水平低于对照组。结论K562细胞高效表达的miR-193b可抑制K562细胞的增殖。
Objective To construct the recombinant adenovirus small pre-miR-193b pre-miR-193b and observe the effect of miR-193b on the proliferation of chronic myeloid leukemia K562 cells. Methods The cDNA sequence of miR-193b was chemically synthesized and cloned into pAdTrack-CMV shuttle plasmid. The recombinant shuttle plasmid was digested with PmeI and transformed into E. coli BJ5183 competent cells containing adenovirus backbone plasmid pAdEasy-1 for homologous recombination , Obtained pAd-miR-193b recombinant adenovirus plasmid, after PacI linearized into HEK293 cell packaging, amplification. Multiplicity dilutions were used to determine virus titer. The expression of miR-193b in K562 cells was detected by real-time fluorescence quantitative PCR, and the proliferation of K562 cells was detected by MTT assay. Results The recombinant plasmid pAd-miR-193b was identified by restriction enzyme analysis and sequencing analysis showed that the recombinant plasmid pAd-miR-193b was successfully constructed and transfected into K562 cells efficiently. Compared with the control group, fluorescence quantitative PCR showed that the recombinant adenovirus vector infected K562 The expression of miR-193b gene was significantly increased in the cells, while the proliferation of K562 cells with high expression of miR-193b gene was lower than that in the control group. Conclusion The miR-193b highly expressed in K562 cells can inhibit the proliferation of K562 cells.