从miRNA-17-92途径探讨小檗碱和吴茱萸碱对大肠癌HT29细胞周期调控机制

Regulation mechanism of berberine and evodiamine on cell cycle by miR-17-92 cluster in colorectal cancer HT29 cells

目的:探讨盐酸小檗碱和吴茱萸碱通过调控miR-17-92基因簇对大肠癌HT29细胞周期的作用机制,为中医药防治大肠癌提供实验依据。方法:体外培养人结肠癌HT29细胞,盐酸小檗碱(2.5,5.0,10.0μM)和吴茱萸碱(1.8,3.75,7.5μM)作用于稳定转染miR-17-5p和miR-20a的HT29细胞后,采用MTT法、流式细胞术、实时荧光定量PCR及Western印记法检测药物对HT29细胞增殖、细胞周期分布情况、mRNA表达水平及E2F1蛋白表达的影响。结果:盐酸小檗碱和吴茱萸碱可以抑制转染的HT29细胞增殖,具有时间依赖性;并且盐酸小檗碱和吴茱萸碱分别以浓度5.0、3.75μM作用转染HT29细胞72h后抑制率超过50%,因此盐酸小檗碱取2.5、10.0μM,吴茱萸碱取1.8、7.5μM用于后续实验,二者能够将细胞阻滞在S期,抑制miR-17-5p和miR-20a的表达水平及E2F1的蛋白表达。结论:盐酸小檗碱和吴茱萸碱通过靶向miR-17-92抑制E2F1蛋白的表达,将细胞阻滞在S期,进而抑制大肠癌HT29细胞的增殖,为中医药防治大肠癌提供新的靶点及实验依据。

Objective： To research the regulation mechanism of berberine and evodiamine on cell cycle by miR-17-92 cluster in colorectal cancer HT29 cells. Methods： HT29 cells transfected with miR-17-5p and miR-20a were treated with berberine and evodiamine at different concentrations. The proliferation of HT29 cells was measured by MTT assay. Cell cycle distribution was examined by flow cytometry. Expression level of miR-17-92 was detected by real-time PCR and expression of E2F1 protein was examined by western blot. Results： Berberine and evodiamine displayed a significant growth inhibitory effect on HT29 cells transfected in time-dependent manner. When berberine and evodiamine treated treasfected HT29 about 72h with the conceentration of 5. 0 and 3. 75μM respectively,the inhibition rate was more than 50%. So berberine at the density of 2. 5,10. 0μM and evodiamine at 1. 8,7. 5μM was in subsequent experiments. The flow cytometry analysis showed HT29 cells could arrested in the phase S by berberine and evodiamine. Furthermore the expression of miR-17-92 mRNA and E2F1 protein was decreased by two alkaloids. Conclusion： These data suggested that berberine and evodiamine could inhibit cell proliferation by down-regulating the miR-17-92 and E2F1 protein expression in colorectal cancer HT29 cells.