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

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.