miR-125b通过下调HAX-1的表达提高CD133~+ SW480细胞对顺铂的敏感性

目的:探讨miR-125b在CD133~+结直肠癌细胞中发挥的作用并研究其是否和顺铂的体外治疗有关。方法:用RT-qPCR方法检测miR-125b在常规SW480肿瘤细胞及CD133~+ SW480肿瘤细胞中的表达水平。流式细胞术检测miR-125b和顺铂对SW480细胞系中CD133~+ 细胞比例的影响。MTT法检测miR-125b对顺铂杀伤CD133~+ SW480细胞能力的影响。利用生物信息学及Western blot方法验证miR-125b是否调节CD133~+ SW480细胞中HAX-1的表达。运用JC-1染色、Annexin V染色及Western blot方法研究miR-125b影响顺铂疗效的信号通路的效应。结果:CD133~+ SW480细胞中的miR-125b表达水平显著低于正常结直肠上皮细胞系FHC和常规SW480肿瘤细胞。顺铂体外单独治疗能提高SW480细胞系中CD133~+细胞的比例,然而联用miR-125b模拟物后CD133~+ SW480细胞的比例显著下降。MTT实验结果表明miR-125b可显著增强顺铂对CD133~+ SW480细胞的杀伤活性。Western blot实验表明miR-125b的靶基因可能为HAX-1。miR-125b联合顺铂可引起CD133~+ SW480细胞线粒体膜电位的丧失并诱导线粒体内细胞色素C的释放,进而引起细胞凋亡。转染HAX-1表达载体后miR-125b联合顺铂对CD133~+ SW480细胞的凋亡诱导效应显著降低。结论:miR-125b通过下调HAX-1的表达提高CD133~+结直肠癌细胞对顺铂的敏感性。

miR-125b increases sensitivity of CD133+ colorectal cancer cells to cisplatin by down-regulating HAX-1 expression

AIM: To investigate the role of miR-125b in regulating the sensitivity of CD133⁺ colorectal cancer cells to cisplatin.METHODS: The expression of miR-125b was detected by RT-qPCR in the routine SW480 cells and CD133⁺ SW480 cells. Flow cytometry analysis was performed to measure the percentage of CD133⁺ cell population in the SW480 cell line treated with miR-125b and cisplatin. MTT assay was performed to evaluate the effect of miR-125b on the cisplatin-induced cell death in the CD133⁺ SW480 cells. Bioinformatics and Western blot were performed to determine whether the expression of HAX-1 was regulated by miR-125b. JC-1 staining, Annexin V staining and Western blot analysis were used to study the pathway of apoptosis in the CD133⁺ SW480 cells co-treated with miR-125b and cisplatin.RESULTS: The expression of miR-125b was significantly lower in the CD133⁺ SW480 cells than that in the routine SW480 cells and normal colonic epithelial FHC cells. Treatment with cisplatin alone increased the percentage of CD133⁺ SW480 cell population. However, miR-125b significantly inhibited the enrichment of CD133⁺ cell population induced by cisplatin. In addition, the results of MTT assay showed that the anti-tumor effect of cisplatin was significantly enhanced when the miR-125b was transfected into the CD133⁺ SW480 cells. The results of Western blot indicated that the HAX-1 gene was the target of miR-125b. Furthermore, the apoptosis induced by the combination of miR-125b and cisplatin was dependent on the dysfunction of mitochondrial membrane, leading to the release of cytochrome C into the cytoplasm and the subsequently activation of apoptosis in the CD133⁺ SW480 cells.CONCLUSION: miR-125b increased the sensitivity of CD133⁺ colo-rectal cancer cells to cisplatin by down-regulating the expression of HAX-1.