姜黄素通过下调microRNA-211表达促进结肠癌细胞的凋亡及其机制

Curcumin promotes apoptosis in colon cancer by downregulating miR-211 expression

摘要:

目的  检测姜黄素对结肠癌细胞中微小RNA 211（microRNA 211，miR-211）表达的影响并探讨miR-211调控结肠癌细胞增殖和凋亡的可能机制。方法  使用MTT法和流式细胞术检测不同浓度姜黄素在不同时间点对结肠癌细胞增殖和凋亡的影响；使用实时荧光定量PCR（qRT-PCR）检测不同浓度姜黄素对结肠癌细胞中miR-211表达的影响；使用生物信息学预测miR-211下游靶基因TP53INP1并使用荧光素酶报告基因法验证其结合；用miR-211模拟物（miR-211 mimics）检测其对姜黄素处理的结肠癌细胞增殖的影响，同时使用Western blot法检测其对TP53INP1蛋白表达的影响；用TP53INP1小干扰RNA检测其对姜黄素处理的结肠癌细胞增殖和凋亡的影响。结果  相较于未处理组，姜黄素在10～50 μmol/L浓度可抑制肿瘤细胞增殖并促进其凋亡（P <0.05）。姜黄素可抑制肿瘤细胞中miR-211的表达，并具有时间和剂量依赖性（P <0.05）；此外，miR-211下游靶基因TP53INP1蛋白表达量上调（P <0.05）。用miR-211转染结肠癌细胞可逆转姜黄素对其增殖的抑制作用，并且TP53INP1表达下调（P <0.05）；用TP53INP1小干扰RNA可逆转姜黄素对结肠癌细胞增殖和凋亡的影响（P <0.05）。结论  姜黄素可通过下调miR-211抑制结肠癌细胞增殖并促进其凋亡，并且TP53INP1是miR-211下游调控蛋白之一。

Abstract:

Objective To study the effect of curcumin on miR-211 in colon cancer cells and clarify a miR-211-mediated mechanism which plays a role in the anti-tumor effects of curcumin. Methods The dose-effect and time-effect relationship of curcumin on HCT116 was tested by MTT assay and flow cytometry. Expression level of miR-211 in curcumin-treated HCT116 was detected by qRT-PCR. TP53INP1 was predicted as a target of miR-211 using GeneTarget database and confirmed by dual luciferase reporter assays. Additionally, the effect of upregulating miR-211 by miR-211 mimics or silencing TP53INP1 by siRNA on curcumin-treated HCT116 was examed by MTT and flow cytometry. Results Compared with untreated HCT116 cells, curcumin at 10-50 μmol/L inhibited cell proliferation and induced apoptosis in a dose-dependent and time-dependent manner. Curcumin also produced a dose and time dependent suppression of miR-211 (P < 0.05). Moreover, the protein level of TP53INP1 was significantly elevated in crucumin-treated HCT116 cells (P < 0.05). Transfection of HCT116 with miR-211 mimics or TP53INP1 small interfering RNA significantly reversed the effect of curcumin on HCT116, compared to corresponding controls (P < 0.05). Conclusions Our data suggest a novel molecular mechanism in which inhibition of miR-211 and upregulation of TP53INP1 mediate the anticancer activities of curcumin in colon cancer cells.