姜黄素通过调控microRNA-21表达影响结肠癌细胞自噬和凋亡的研究

Effect of curcumin on autophagy and apoptosis of colon cancer cells by regulating microRNA-21 expression

目的 探讨姜黄素通过调控micro RNA-21表达影响结肠癌细胞自噬和凋亡的机制。方法 使用姜黄素处理人结直肠癌细胞HCT116后,通过实时荧光定量PCR检测mi R-21-3p和mi R-21-5p的相对表达量,免疫印迹检测细胞自噬标志蛋白p62与LC3B的表达量,Annexin V染色检测细胞的凋亡水平。mi RDB在线分析mi R-21-3p和mi R-21-5p的潜在底物并通过荧光素酶报告系统检测mi R-21-3p和mi R-21-5p是否与底物结合。通过过表达或敲低mi R-21-3p或mi R-21-5p,检测人结直肠癌细胞的自噬水平和凋亡水平。结果 随着姜黄素的浓度上升细胞的凋亡水平提高,自噬关键基因LC3B的表达量下降、p62的表达量上升,自噬水平上升,mi R-21-3p与mi R-21-5p的表达下降(P <0. 05)。mi R-21-3p靶向ATG10,而mi R-21-5p靶向APAF1。过表达mi R-21-3p后,结直肠癌细胞HCT116的ATG10表达量下降、p62的表达量上升、LC3B的表达量下降,自噬水平下降。敲低mi R-21-3p后,结直肠癌细胞HCT116的ATG10表达量上升、p62的表达量下降、LC3B的表达量上升,自噬水平上升。过表达mi R-21-5p后,结直肠癌细胞HCT116的APAF1表达量下降、细胞凋亡水平下降(P <0. 05)。敲低mi R-21-5p后,结直肠癌细胞HCT116的APAF1表达量上升、细胞凋亡水平上升(P <0. 05)。结论 姜黄素通过抑制mi R-21-3p和mi R-21-5p的表达量以减少ATG10和APAF1的表达量,最终促进人结肠癌细胞HCT116自噬和凋亡。

Objective To investigate the mechanism of curcumin affecting autophagy and apoptosis of colon cancer cells by regulating microRNA-21 expression.Methods After treatment of human colorectal cancer cells HCT116 with curcumin,the relative expressions of miR-21-3p and miR-21-5p were detected by real-time fluorescence.quantitative PCR,the expressions of autophagy marker p62 and LC3B were detected by Western blot,and the apoptosis levels were detected by Annexin V staining.Potential substrates of miR-21-3p and miR-21-5p were analyzed online by miRDB and whether miR-21-3p and miR-21-5p bound to the substrates were detected by luciferase reporting system.Autophagy and apoptosis levels of human colorectal cancer cells were detected by overexpression or knockdown of miR-21-3p or miR-21-5p.Results With the increase of curcumin concentration,the expression of LC3B,p62,autophagy,miR-21-3p and miR-21-5p decreased(P<0.05).In addition,miR-21-3p targets ATG10,while miR-21-5p targets APAF1.After overexpression of miR-21-3p,ATG10 expression of HCT116 decreased,p62 expression increased,LC3B expression decreased and autophagy level decreased in colorectal cancer cells.After knockdown of miR-21-3p,ATG10 expression level of HCT116,p62 expression level,LC3B expression level and autophagy level of colorectal cancer cells increased.After overexpression of miR-21-5p,APAF1 expression and apoptosis level of HCT116 in colorectal cancer cells decreased(P<0.05).After miR-21-5p was knocked down,APAF1 expression and apoptosis level of HCT116 were increased in colorectal cancer cells(P<0.05).Conclusion Curcumin reduces the expression of ATG10 and APAF1 by inhibiting the expression of MiR-21-3p and MiR-21-5p,and ultimately promotes autophagy and apoptosis of human colon cancer cell HCT116.