沉默circ0000745通过靶向miR-296-5p对结直肠癌细胞增殖及转移的影响

Effect of circ;000745 on the Proliferation and Metastasis of Colorectal Cancer Cells by Targeting miR-296-5p

目的探讨circ;000745对结直肠癌细胞增殖及转移的影响及其可能的作用机制。方法收集2013年3月~2016年7月在四川省人民医院确诊的结直肠癌及癌旁组织65例,采用实时定量PCR(qRT-PCR)法检测结直肠癌组织、癌旁组织中circ;000745与miR-296-5p表达,分析circ;000745与miR-296-5p表达与结直肠癌患者预后的关系。将人结直肠癌细胞HCT-116分为si-NC组、si-circ;000745组、miR-NC组、miR-296-5p过表达组、si-circ;000745+anti-miR-NC组、si-circ;000745+anti-miR-296-5p组;采用CCK-8法、平板克隆形成实验、Transwell实验分别检测各组细胞增殖、克隆形成、迁移及侵袭;采用双荧光素酶报告实验检测circ;000745与miR-296-5p的靶向关系;采用Western blot法检测基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)蛋白表达量。结果与癌旁组织比较,结直肠癌组织中circ;000745的表达升高(P<0.05),miR-296-5p的表达降低(P<0.05);随访5年,65例患者中死亡9例,存活56例,与存活患者相比,死亡患者的circ;000745明显增高(P<0.05),miR-296-5p明显降低(P<0.05)。与HT-29细胞相比,HCT-116细胞的circ;000745 mRNA表达升高(P<0.05),miR-296-5p的表达降低(P<0.05),选择HCT-116细胞进行下一步研究。与si-NC组比较,si-circ;000745组细胞存活率、MMP-2、MMP-9蛋白、细胞克隆形成数、迁移及侵袭细胞数减少(均P<0.05);circ;000745可靶向调控miR-296-5p的表达;与miR-NC组比较,miR-296-5p过表达组细胞存活率、MMP-2、MMP-9蛋白、细胞克隆形成数、迁移及侵袭细胞数减少(均P<0.05);与si-circ;000745+anti-miR-NC组比较,si-circ;000745+anti-miR-296-5p组细胞存活率、MMP-2、MMP-9蛋白水平、细胞克隆形成数、迁移及侵袭细胞数增多(均P<0.05)。结论结直肠癌组织中circ;000745呈高表达,miR-296-5p呈低表达,高表达circ;000745及低表达miR-296-5p可用于评估结直肠癌患者预后,circ;000745通过靶向miR-296-5p促进结直肠癌细胞增殖及转移,沉默circ;000745可为结直肠癌治疗提供新思路。

Objective To explore the effect of circ;000745 on the proliferation and metastasis of colorectal cancer cells and its possible mechanism.Methods Colorectal cancer tissue and adjacent tissue samples were collected from 65 cases diagnosed in Sichuan Provincial People’s Hospital from March 2013 to July 2016.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of circ;000745 and miR-296-5 p in colorectal cancer tissues and adjacent tissues.The correlation between the expression of circ;000745 and miR-296-5 p and the prognosis of colorectal cancer patients was analyzed.Human colorectal cancer cells HCT-116 were divided into si-NC group, si-circ;000745 group, miR-NC group, miR-296-5 p group, si-circ;000745+anti-miR-NC group, and si-circ;000745+anti-miR-296-5 p group.The CCK-8,plate clone formation, and Transwell were used to detect cell proliferation, clone formation, migration and invasion in each group.The dual luciferase reporter experiment was used to detect the targeting relationship between circ;000745 and miR-296-5 p.Western blotting was used to detect the protein expression of matrix metalloproteinase 2(MMP-2)and matrix metalloproteinase 9(MMP-9).Results Compared with adjacent tissues, the expression of circ;000745 in colorectal cancer tissues was increased(P<0.05),while the expression of miR-296-5 p was decreased(P<0.05).After 5 years of follow-up, 9 of the 65 patients died and 56 survived.Compared with surviving patients, circ;000745 levels in dead patients were significantly increased(P<0.05),while miR-296-5 p levels were significantly decreased(P<0.05).Compared with HT-29 cells, the expression of circ;000745 in HCT-116 cells was increased(P<0.05),while the expression of miR-296-5 p was decreased(P<0.05),and HCT-116 cells were selected for the subsequent study.Compared with the si-NC group, cell survival rates, the protein levels of MMP-2,MMP-9,and the number of cell clone formation, the number of migration and invasion cells in the si-circ;000745 group were decreased(all P<0.05).And circ;000745 could target and regulate the expression of miR-296-5 p.Compared with the miR-NC group, cell survival rate, the protein levels of MMP-2,MMP-9,and the number of cell clone formation, the number of migration and invasion cells in the miR-296-5 p group were decreased(all P<0.05).Compared with the si-circ;000745+anti-miR-NC group, cell survival rate, the protein levels of MMP-2,MMP-9,and the number of cell clone formation, the number of migration and invasion cells in the si-circ;000745+anti-miR-296-5 p group were increased(all P<0.05).Conclusion The expression of circ;000745 in colorectal cancer tissues was increased, and the expression of miR-296-5 p was decreased.The high expression of circ;000745 and low expression of miR-296-5 p could be used to evaluate the prognosis of colorectal cancer patients.Moreover, circ;000745 promoted the proliferation and metastasis of colorectal cancer cells through targeting regulation of miR-296-5 p.Silencing circ;000745 could provide new ideas for the research and treatment of colorectal cancer.