miR-210通过Wnt/β-catenin促进结直肠癌对顺铂的药物敏感性研究

miR-210 promoted cisplatin drug sensitivity in colorectal cancer through Wnt/β-catenin pathway

目的 探讨miR-210对结直肠癌细胞增殖及顺铂(DDP)药物敏感性及其机制影响。方法 RT-qPCR检测人正常结肠上皮细胞(HCoEpiC)、结直肠癌细胞(HCT-116、HT-29、SW620)及顺铂耐药细胞株(HCT-116/DDP)中miR-210的表达;使用CCK-8和Annexin V-FITC/PI法检测miR-210过表达或干扰后,HCT-116细胞增殖及凋亡情况。将HCT-116/DDP细胞随机分成Control组、miR-210 mimics组、miR-210 inhibitor组,在DDP(80μmol/L)或对照作用24 h后,CCK-8检测HCT-116/DDP细胞活力;Annexin V-FITC/PI双染检测细胞凋亡,Western blot检测细胞凋亡相关指标(Bad、Bcl-2、Cleaved Caspase-3)、耐药相关指标(ABCG2、MRP2、P-gp)及Wnt/β-Catenin通路中重要蛋白的表达。结果 与HCoEpiC细胞相比,结直肠癌细胞(HCT-116、HT-29、SW620)中miR-210的表达显著降低,而HCT-116/DDP细胞中miR-210的表达比HCT-116细胞中进一步降低。miR-210 mimics能够显著抑制结直肠癌细胞及其顺铂耐药株的细胞活力,促进细胞凋亡,而inhibitor作用相反。在HCT-116/DDP细胞中,与DDP组比较,DDP+mimics联合组能够显著抑制细胞活力,促进细胞凋亡,促进Bad、Cleaved Caspase-3的表达,抑制Bcl-2的表达,抑制耐药相关蛋白(p-gp、MRP2、ABCG2)的表达,抑制Wnt3a、β-Catenin的表达;miR-210 inhibitor则得到相反的结果。结论 miR-210可能通过调控Wnt/β-catenin通路,影响耐药相关蛋白(p-gp、ABCG2、MRP2)的表达,最终促进HCT-116/DDP耐药细胞对顺铂的药物敏感性。

Objective To investigate the effect and mechanism of miR-210 on the proliferation and cisplatin(DDP)sensitivity of colorectal cancer cells.Methods The expression of miR-210 in human normal colonic epithelial cells(HCoEpiC),colorectal cancer cells(HCT-116,HT-29,SW620)and cisplatin resistant cell lines(HCT-116/DDP)were detected by RT-qPCR.Proliferation and apoptosis of HCT-116 cells after miR-210 overexpression or interference were detected by CCK-8 and Annexin V-FITC/PI assay,respectively.HCT-116/DDP cells were randomly divided into Control group,miR-210 mimics group and miR-210 inhibitor group.After DDP(80μmol/L)or Control treatment for 24 h,the cell activity of HCT-116/DDP cells was detected by CCK-8.Apoptosis was detected by Annexin V-FITC/PI double staining,and expression of apoptosis-related proteins(Bad,Bcl-2,Cleaved caspase-3),drug-resistance related proteins(ABCG2,MRP2,P-gp)and Wnt/β-catenin pathway proteins were detected by Western blot assay.Results Compared with HCoEpiC cells,the expression of miR-210 in colorectal cancer cells(HCT-116,HT-29,SW620)was significantly decreased,and the expression of miR-210 in HCT-116/DDP cells was further decreased than that in HCT-116 cells.miR-210 mimics significantly inhibited the cell viability and promote apoptosis of colorectal cancer cells and cisplatin resistant strains.miR-210 inhibitor had the opposite effect.In HCT-116/DDP cells,compared with the DDP group,the DDP+mimics group could significantly inhibit cell activity,promote cell apoptosis,promote the expression of Bad and Cleaved caspase-3,inhibit expression of Bcl-2,inhibit expression of drug-resistant related proteins(P-gp,MRP2,ABCG2),and inhibit the expression of Wnt3a andβ-catenin.With miR-210 inhibitor,the opposite effect was found.Conclusion miR-210 may affect the expression of drug-resistant related proteins(P-gp,ABCG2,MRP2)by regulating Wnt/β-catenin pathway,and finally promote the drug sensitivity of HCT-116/DDP resistant cells to cisplatin.