IL-24-TRAIL基因载体的构建及其对乳腺癌干细胞迁移和凋亡的影响

Construction of IL-24-TRAIL Vector and Its Influences on Migration and Apoptosis of Breast Cancer Stem Cells

目的构建真核表达载体pIRES-IL-24-TRAIL,探讨其对乳腺癌干细胞迁移和凋亡的影响。方法以人胎盘组织总RNA为模板,采用RT-PCR二步法,扩增TRAIL的cDNA序列,将其克隆入pIRES载体,扩增IL-24的cDNA序列,将其克隆入pIRES-TRAIL载体,构建重组真核表达载体pIRESIL-24-TRAIL,以Lipofectamine2000转染技术,将质粒pIRES-IL-24-TRAIL导入乳腺癌干细胞MCF-7和MDA-MB-231,流式细胞仪检测各组细胞凋亡情况,划痕实验比较不同组间细胞迁移情况。结果(1)克隆到TRAIL、IL-24基因的cDNA序列,成功构建其真核表达载体。(2)流式细胞仪发现,实验组比对照组细胞凋亡率高(P<0.05)。(3)细胞划痕实验显示实验组细胞迁移能力明显低于对照组(P<0.05)。结论 pIRES-IL-24-TRAIL构建成功,并能诱导乳腺癌干细胞凋亡、降低其迁移能力。

Objective To construct the eukaryotic expression vector pIRES-IL-24-TRAIL and investigate its influences on the migration and apoptosis of breast cancer stem cells. Methods The total mRNA isolated from human placenta tissue was used as template and reverse transcriptase-polymerase chain reaction （RT PCR） was applied, eDNA fragment encoding human TRAIL gene was amplified and cloned into pIRES vector, cDNA fragment encoding human IL-24 gene was amplified and cloned into the recombinant eukaryotic expression vector pIRES-TRAIL to construct pIRES-IL-24-TRAIL plasmid after sequencing. Subsequently, plasmid DNA of pIRES-IL-24-TRAIL was transfected into breast cancer stem cells MCF-7 and MDA MB-231 mediated by Lipofectamine2000 transfection reagent. Cell apoptosis was measured with flow cytometry. Wound healing test was performed for cell motility assay. Results （1） eDNA sequence encoding human TRAIL and IL-24 gene was successfully cloned, and recombinant eukaryotic expression vector pIRES-IL-24-TRAIL was constructed. （2） The apoptosis rate in the transfected group tested by flow cytomtry was higher than that in the control group（P〈0.05）. （3） Wound-healing assay showed that cell migration in the transfected group could be more effectively suppressed than that in the control group（P〈0.05）. Conclusion pIRES-IL-24-TRAIL is constructed successfully and could promote the apoptosis of breast cancer stem cells and impede tumor cell migration.