MiR-19反义核酸与CD133+HT29细胞亚群对多柔比星敏感性关系的研究

Study the Relationship Between MiR-19 Antisense Oligonucleotides and the Sensitivity of CD133+HT29 Cell Subsets to Doxorubicin

关键词:  miR-19  PTEN  多柔比星  结直肠癌干细胞  Bad

Key words:  miR-19  PTEN  doxorubicin  colorectal cancer stem cells  Bad

摘要:

目的 探讨miR-19反义核酸与CD133⁺HT29细胞亚群对多柔比星敏感性的关系并研究其机制。方法 用RT-qPCR方法检测miR-19在结直肠癌细胞中的表达水平。流式细胞术检测miR-19反义核酸和多柔比星对HT29细胞系的CD133⁺细胞亚群种群比例的影响。MTT法检测miR-19反义核酸对多柔比星杀伤CD133⁺HT29细胞亚群能力的影响。利用生物信息学及Western blot法验证miR-19是否调节CD133⁺HT29细胞亚群中PTEN的表达。运用Western blot、免疫共沉淀、流式细胞术研究miR-19反义核酸影响多柔比星疗效的信号通路。结果 结直肠癌细胞系的miR-19表达水平显著高于正常结直肠上皮细胞系，并且CD133⁺细胞中的miR-19表达水平显著高于常规结直肠癌细胞。多柔比星体外单独治疗能提高HT29细胞系中CD133⁺细胞亚群的比例，然而联用miR-19反义核酸后CD133⁺HT29细胞亚群的种群比例显著下降。MTT结果表明miR-19反义核酸可显著增强多柔比星对CD133⁺HT29细胞亚群的杀伤活性。Western blot实验表明miR-19的靶基因可能为PTEN。MiR-19反义核酸可显著抑制CD133⁺HT29细胞亚群PI3K、AKT和Bad的磷酸化，增强Bad与Bcl-2和Bcl-xl的结合，从而提高CD133⁺HT29细胞亚群对多柔比星依赖的凋亡信号的敏感性，促进caspase-9和caspase-3的活化。结论 MiR-19反义核酸通过PTEN/PI3K/AKT/Bad途径提高CD133⁺HT29细胞亚群对多柔比星的敏感性。

Abstract:

OBJECTIVE To investigate the relationship between the miR-19 and the sensitivity of colorectal cancer stem cells to doxorubicin.METHODS The expression of miR-19 was detected by RT-qPCR assay in the colorectal cancer stem cells. Flow cytometry analysis was performed to measure the percentage of CD133⁺ population in the HT29 cell line treated with miR-19 and doxorubicin. MTT assay was performed to evaluate the effect of miR-19 antisense oligonucleotides on the doxorubicin-induced cell death in the CD133⁺ HT29 cell subsets. Bioinformatics and Western blot assays were performed to determine whether the expression of PTEN is regulated by miR-19. Western blot, co-immunoprecipitation and flow cytometry assays were performed to study the pathway of apoptosis in the CD133⁺ HT29 cell subsets co-treated with miR-19 antisense oligonucleotides and doxorubicin.RESULTS The expression of miR-19 was significantly higher in the colorectal cancer cell lines than that in the normal colorectal epithelial cell line. In addition, the expression of miR-19 was up-regulated in the cancer stem cells compared with the routine colorectal cancer cells. Single treatment of doxorubicin increased the percentage of CD133⁺ HT29 cell population. However, the combination with miR-19 antisense oligonucleotides significantly inhibited the enrichment of CD133⁺ cell population induced by the doxorubicin. In addition, the results of MTT assay showed that the anti-tumor effect of doxorubicin could be significantly enhanced when the miR-19 antisense oligonucleotides were transfected into the CD133⁺ HT29 cell subsets. The results of western blot indicated that the PTEN gene was the target of miR-19. Furthermore, the miR-19 antisense oligonucleotides significantly inhibited the phosphorylation of PI3K, ATK and Bad and increased the interaction with the Bad and Bcl-2 as well as Bcl-xl. Subsequently, the sensitivity of CD133⁺ HT29 cell subsets to the doxorubicin-induced apoptosis was significantly enhanced, and the activation of caspase-9 and caspase-3 was promoted.CONCLUSION MiR-19 antisense oligonucleotides increased the sensitivity of CD133⁺ HT29 cell subsets to doxorubicin through PTEN/PI3K/AKT/Bad pathway.