DNA-PKcs短发夹RNA载体的构建及其表达

目的构建DNA-PKcsshRNA表达载体,观察该基因的抑制对A2780细胞增殖活性的影响。方法将针对人DNA-PKcs基因的不同部位设计的shRNA插入到真核表达质粒并转染人卵巢A2780细胞,RT-PCR和WesterBlot检测其对该基因mRNA和蛋白水平的抑制效率,MTT法测定DNA-PKcs基因的抑制对肿瘤细胞增殖活性的影响。结果 DNA测序分析证实DNA-PKcs shRNA表达载体pSIRENDNA-PKcs shRNA构建成功并在细胞中表达,转染重组载体的A2780细胞DNA-PKcs的表达在mRNA和蛋白水平均明显下降;重组载体转染细胞增殖活性明显降低。结论成功构建DNA-PKcs shRNA表达载体为进一步研究DNA损伤修复基因DNA-PKcs奠定了基础;DNA-PKcs表达的抑制可能会导致肿瘤细胞增殖活性降低。

The Construction of DNA-Pkcs Short-hairpin siRNA Recombinant Vector and Its Expression in Vitro

Objective To construct eukaryotic vector expressing shRNA(short hairpin RNA) of DNA-PKcs and study the significance of superexpression of DNA-PKcs in A2780 cells. Methods Designing two different shRNA targeting the coding sequence of DNA-PKcs, the pSIREN-DNA-PKcs shRNA was constructed by inserting the designed shRNA to the plasmid vector pSIREN.The human ovarian cancer cell strain A2780 was transfected by pSIREN-DNA-PKcs shRNA. The Expression of DNA-Pkcs in transfercted cells was detected by RT-PCR and Westernblot. The prolixferation activeities of transfercted cells were assayed by MTT. Results It was verified by partial nucleotide sequenceing and restriction endonuclease digestion that the constructed vector expressing shRNA of DNA-PKcs was correct. The DNA-PKcs expression was significantly suppressed in A2780 cells transfected by pSIRENDNA-PKcs shRNA compared with untransfected A2780 cells. The A2780 cells transfected with pSIRENDNA-PKcs shRNA had Lower Cellula proliferation activities compared with untransfected A2780 cells. Conclusion The studing results laid the foundation for further studing on the biological functions of DNA-PKcs.The superexpression of DNA-PKcs should decrease the proliferation activities of carcinoma cells.