psiRNA—hHDEK的构建及其对CasKi细胞生物学特性影响的研究

目的观察DEK基因沉默对宫颈癌CaSki细胞增殖、细胞周期及凋亡、衰老的影响，为探索防治人类宫颈癌的新方法提供依据。方法根据siRNA设计原则和Genebank中DEK核苷酸序列，利用软件设计出针对DEK位点的特异性小干扰RNA，克隆至siRNA真核表达载体psiRNA-hHlneo中，应用脂质体介导重组质粒转染宫颈癌CaSki细胞株，用RT-PCR和Western blot检测转染后DEKmRNA和蛋白表达，转染后48hMTr法检测细胞增殖、流式细胞仪检测细胞凋亡和细胞周期改变、SA-β-半乳糖苷酶细胞化学染色鉴定细胞衰老。结果转染质粒psiRNA—hHDEK组DEKm RNA及蛋白的表达明显减少（0．28±0．02），DEK基因沉默后CaSki细胞增殖能力下降，细胞周期阻抑，细胞凋亡率增加，衰老细胞增多。结论成功构建表达DEK siRNA的真核表达质粒psiRNA-hHDEK，并能够有效沉默CaSki细胞中DEK基因表达。DEK基因沉默后能够诱导CaSki细胞凋亡和细胞衰老。DEK基因在宫颈癌发生和演变中发挥重要作用，其既是衰老抑制基因，又是凋亡抑制基因。

The construction of psiRNA-hHDEK and its impact on CasKi cells biological characteristics

Objective The cell proliferation, cell cycle, apoptosis and the impact of senescence after RNAi were performed. This research provided a theoretical and experimental basis of the prevention and treatment of cervical cancer. Methods According to DEK nucleotide sequence in G enebank, 56 nt o1igonucleotide fragment containing specific target DEK sequence was designed with computer software, and the fragnent was synthesized and cloned into the eukaryotic expressed plasmid vector psiRNA-hH ineo.Then it was transfected into CaSki cells by lipofectamine. DEK mRNA and protein expression were detected to verify the gene silence effect by RT-PCR and Western blot analysis. CaSki cell proliferative inhibition rates were accessed by MTT assay at the 48th hour after DEK siRNA transfection, at the same time cell cycle and cell apoptosis were analyzed by flow cytometry, and SA - β-galactosidase enzyme cytochemiatry was used to identify cell senescence. Results DEK mRNA and protein expression was significantly reduced in psiRNA- hHDEK transfected CaSki cells(0. 28 ±0. 02). Cell proliferation was decreased, cell cycle was inhibited, and cell apoptosis and senescent cells were increased afar DEK gene was silenced. Concluslon DEK siRNA eukaryotic expression vector was successfully constructed and DEK gene expression of CaSki cells was effectively silenced. DEK gene silencing could induce CaSki cells into apoptosis and senescence.DEK gene played an important role in the occurrence and evolution of cervical cancer, which was not only a senescence suppressor gene but also an apeptosis suppressor gene.