BRD7逆转录病毒稳定系的构建及其功能评价

Construction of BRD7 retroviral stable cell line and the evaluation of its biological functions

目的探索BRD7(bromodomain-containing protein7)是否具有抑癌基因的功能。方法根据NCBI提供的BRD7基因序列设计特异性引物扩增其编码区序列,经酶切、连接转化后进一步挑取单克隆菌落进行菌液PCR筛选及测序鉴定得到阳性重组质粒。共转293T细胞后收集浓缩病毒上清并感染U2OS细胞,24h后用嘌呤霉素(puromycin)筛选稳定表达BRD7的细胞株。结果菌落PCR扩增及DNA测序分析证明重组pMSCV-BRD7-GFP质粒克隆成功。GFP绿色荧光结果显示,绝大部分细胞成功整合了pMSCV-GFP或pMSCV-BRD7-GFP外源质粒;Western blotting检测发现感染重组质粒pMSCV-BRD7-GFP的细胞株中BRD7蛋白的表达水平显著高于对照组。功能结果显示,过表达BRD7显著抑制U2OS细胞的增殖,促进下游靶基因p21与MDM2的表达。结论本研究成功构建了针对BRD7基因的逆转录病毒稳定系,且初步证明了BRD7发挥抑癌基因的功能,为下一步深入研究其被调控的机制与功能奠定了基础。

Objective To study the tumor suppressor role of BRD7 （bromodomain-containing protein 7）. Methods The BRD7 gene based on the coding sequence from GenBank was amplified by RT-PCR. Then the PCR harvested product was suffered to endonuclease digestion and ligation. After transformation to competent cell DH5 et, the recombinant positive plasmid clones were identified by colony-PCR and sequencing analysis. The expressing plasmid were cotransfected into 293T cells, the harvested supematant was subjected to infect U2OS cells, which will further be selected with puromycin. Results Colony-PCR and DNA sequencing result indicated that pMSCV-BRDT-GFP vector has been constructed successfully. The GFP fluorescence showed that there are nearly no untransfected cells remained. Westem blotting showed that the protein level of BRD7 in pMSCV-BRD7-GFP transfected cancer cell line is significantly up-regulated compared with control cancer cell lines. Biological function study showed that overexpression of BRD7 significantly inhibited the proliferation of U2OS cells and enhanced the expression of target genes p21 and MDM2. Conclusion The overexpression cancer cell line of pMSCV-BRD7-GFP is successfully constructed. BRD7 may function as a tumor suppressors. Therefore, it provided a new foundation for further mechanism and functional study.