微小RNA-212-5p调控ATP结合盒E1对结直肠癌细胞生物学的影响

Effect of microRNA-212-5p on the biology of colorectal cancer cells by regulating ATP-binding box E1

目的:探讨miR-212-5p在结直肠癌(CRC)进展中的作用和机制。方法:从新乡医学院第一附属医院收集32例患者的CRC组织及邻近正常组织,体外培养购自上海中科院细胞库的正常结肠黏膜上皮细胞NCM460和CRC细胞系(SW480、SW1116、HT29),实时定量反转录聚合酶链反应(RT-qPCR)检测miR-212-5p和ATP结合盒E1(ABCE1)。将SW480细胞分为miR-NC组、miR-212-5p组、si-NC组、si-ABCE1组、miR-212-5p+pcDNA组、miR-212-5p+pcDNA-ABCE1组。噻唑蓝(MTT)法、Transwell分析用于检测SW480细胞增殖、侵袭迁移。蛋白质印迹法(Western blot)评估ABCE1蛋白表达;双荧光素酶实验分析miR-212-5p和ABCE1靶向关系。两组数据差异用独立样本t检验,多组数据的差异用单因素方差分析和SNK-q检验。结果:CRC组织和细胞中miR-212-5p的表达明显低于对照组(组织:t=100.411,P<0.01;细胞:F=1192.235,P<0.01),ABCE1 mRNA(组织:t=85.952,P<0.01;细胞:F=219.953,P<0.01)和蛋白(组织:t=39.634,P<0.01;细胞:F=125.185,P<0.01)的表达明显升高;miR-212-5p组SW480细胞活力(48 h:0.34±0.01比0.53±0.03,t=18.025,P<0.01;72 h:0.51±0.02比0.97±0.08,t=16.735,P<0.01)、迁移数(40.31±2.12比87.42±6.85,t=19.710,P<0.01)、侵袭数(29.06±1.35比72.19±5.91,t=21.344,P<0.01)、ABCE1蛋白(0.17±0.02比0.56±0.05,t=21.726,P<0.01)表达明显低于miR-NC组。si-ABCE1组SW480细胞活力(48 h:t=10.436,P<0.01;72 h:t=17.332,P<0.01)、迁移数(t=16.741,P<0.01)、侵袭数(t=17.164,P<0.01)明显低于si-NC组。miR-212-5p与ABCE1直接结合。miR-212-5p+pcDNA-ABCE1组SW480细胞活力(48 h:q=21.689,P<0.01;72 h:q=16.709,P<0.01)、迁移数(q=20.273,P<0.01)、侵袭数(q=29.325,P<0.01)、ABCE1蛋白表达(q=24.000,P<0.01)明显高于miR-212-5p+pcDNA组。结论:miR-212-5p靶向ABCE1可抑制CRC细胞生物学行为。

Objective To explore the role and mechanism of microRNA(miR)-212-5p in the progression of colorectal cancer(CRC).Methods CRC tissues and adjacent normal tissues of 32 patients were collected from the First Affiliated Hospital of Xinxiang Medical College,and normal colon mucosal epithelial cells(NCM460)and CRC cell lines(SW480,SW1116,HT29)purchased from the Cell Bank of Shanghai Chinese Academy of Sciences were cultured in vitro.The expression of miR-212-5p and ATP-binding box E1(ABCE1)was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).SW480 cells were divided into miR-NC group,miR-212-5p group,si-NC group,si-ABCE1 group,miR-212-5p+pcDNA group,miR-212-5p+pcDNA-ABCE1 groups.Methyl thiazolyl tetrazolium(MTT)method and Transwell analysis were used to detect the proliferation,migration and invasion ability of SW480 cells in vitro.Western blotting was used to detect the ABCE1 protein expression.The targeting relationship between miR-212-5p and ABCE1 was detected by double luciferase assay.The difference between two groups of data was tested by independent sample t test,and the difference between multiple groups of data was tested by one-way ANOVA and SNK-q test.Results MiR-212-5p expression in CRC tissue and cells was significantly decreased(tissue:t=100.411,P<0.01;cell:F=1192.235,P<0.01),while ABCE1 mRNA(tissue:t=85.952,P<0.01;cell:F=219.953,P<0.01)and protein(tissue:t=39.634,P<0.01;cell:F=125.185,P<0.01)expression was significantly increased.Compared with the miR-NC group,the cell viability(48 h:0.34±0.01 vs.0.53±0.03,t=18.025,P<0.01;72 h:0.51±0.02 vs.0.97±0.08,t=16.735,P<0.01),the number of migrating cells(40.31±2.12 vs.87.42±6.85,t=19.710,P<0.01),the number of invasive cells(29.06±1.35 vs.72.19±5.91,t=21.344,P<0.01)and the expression of ABCE1 protein(0.17±0.02 vs.0.56±0.05,t=21.726,P<0.01)of SW480 cells in the miR-212-5p group were significantly reduced.Compared with the si-NC group,the cell viability(48 h:t=10.436,P<0.01;72 h:t=17.332,P<0.01),the number of migrating cells(t=16.741,P<0.01)and the number of invasive cells(t=17.164,P<0.01)of SW480 cells in the si-ABCE1 group were significantly reduced.Compared with the miR-212-5p+pcDNA group,the cell viability(48 h:q=21.689,P<0.01;72 h:q=16.709,P<0.01),the number of migrating cells(q=20.273,P<0.01),the number of invasive cells(q=29.325,P<0.01)and the expression of ABCE1 protein(q=24.000,P<0.01)of SW480 cells in the miR-212-5p+pcDNA-ABCE1 group were significantly increased.Conclusion MiR-212-5p inhibits the biological behavior of CRC cells by targeting ABCE1.