微小RNA-424-5p调控OTX1对结直肠癌细胞增殖、迁移和侵袭的影响

余昆, 王伟雅, 蔡昕怡, 程先硕, 赵笑枫, 别雅琴, 李强
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CCK-8 assay, scratch assay and Transwell assay were used to detect the effects of miR-424-5p on the proliferation, migration and invasion of HCT116 cells. The effect of miR-424-5p on the expression of OTX1 protein was detected by Western blotting. The luciferase report assay was used to detect the influence of miR-424-5p on the luciferase activity of OTX1-3′UTR vector. \n \n \nResults \nThe relative expressions of OTX1 mRNA in CRC tissues and para-carcinoma tissues were 1.049±0.446 and 0.639±0.178 (t=-6.583, P<0.001); and the relative expression of miR-424-5p in CRC tissues and para-carcinoma tissues were 0.865±0.261 and 1.329±0.387 (t=7.705, P<0.001), with statistically significant differences. Negative correlation was found between the expression of miR-424-5p and OTX1 mRNA in CRC tissues (r=-0.439, P=0.015). The absorbance values of HCT116 cells transfected with miR-424-5p-mimic were 0.813±0.064, 0.960±0.098, 1.287±0.192 on 72, 96 and 120 hours respectively, and those of HCT116 cells transfected with miR-NC were 1.163±0.158, 1.645±0.117 and 2.043±0.236. The proliferation ability of miR-424-5p-mimic group was lower than that of miR-NC group and the differences between the two groups were statistically significant (t=3.538, P=0.024; t=7.778, P=0.001; t=4.257, P=0.013). The scratch assay showed that the migration of HCT116 cells in miR-424-5p-mimic group was inhibited as compared with miR-NC group. The numbers of cells permeating septum of miR-NC and miR-424-5p-mimic group were 177.104±17.834 and 35.667±13.634, and the difference was statistically significant (t=15.246, P<0.001). The relative expressions of OTX1 protein in miR-NC and miR-424-5p-mimic group were 0.862±0.121 and 0.342±0.103 respectively, and the difference was statistically significant (t=16.286, P<0.001). Luciferase report assay showed that the luciferase activities of wt-OTX1-3′UTR and mut-OTX1-3′UTR vector were 0.305±0.095 and 0.898±0.080 after over expression of miR-424-5p, and the difference was statistically significant (P<0.001). \n \n \nConclusion \nThe expressions of miR-424-5p and OTX1 mRNA are negatively correlated in CRC tissue. miR-424-5p can inhibit the proliferation, migration and invasion of CRC cells by down-regulating the expression of OTX1. \n \n \nKey words: \nColorectal neoplasms; MicroRNAs; Cell proliferation; OTX1","PeriodicalId":16120,"journal":{"name":"国际肿瘤学杂志","volume":"280 1","pages":"321-326"},"PeriodicalIF":0.0000,"publicationDate":"2019-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"微小RNA-424-5p调控OTX1对结直肠癌细胞增殖、迁移和侵袭的影响\",\"authors\":\"余昆, 王伟雅, 蔡昕怡, 程先硕, 赵笑枫, 别雅琴, 李强\",\"doi\":\"10.3760/CMA.J.ISSN.1673-422X.2019.06.001\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Objective \\nTo investigate the correlation of the expression of microRNA-424-5p (miR-424-5p) and orthodenticle homeobox 1 (OTX1) gene in colorectal cancer (CRC) tissue, and the effects of miR-424-5p on the proliferation, migration and invasion of CRC cells HCT116. \\n \\n \\nMethods \\nA total of 60 patients with CRC were collected from June 2017 to June 2018 in Department of Colorectal Surgery of Third Affiliated Hospital of Kunming Medical University. 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引用次数: 0

摘要

目的探讨microRNA-424-5p (miR-424-5p)与OTX1基因在结直肠癌(CRC)组织中表达的相关性,以及miR-424-5p对结直肠癌细胞HCT116增殖、迁移和侵袭的影响。方法收集昆明医科大学第三附属医院结直肠外科2017年6月至2018年6月收治的结直肠癌患者60例。采用实时定量PCR (RT-qPCR)检测60例结直肠癌组织、癌旁组织和结直肠癌细胞系中miR-424-5p和OTX1 mRNA的表达。进一步分析miR-424-5p与OTX1基因表达的相关性。将miR-NC (miR-NC组)和miR-424-5p-mimic (miR-424-5p-mimic组)转染HCT116细胞。采用CCK-8法、scratch法和Transwell法检测miR-424-5p对HCT116细胞增殖、迁移和侵袭的影响。Western blotting检测miR-424-5p对OTX1蛋白表达的影响。荧光素酶报告法检测miR-424-5p对OTX1-3'UTR载体荧光素酶活性的影响。结果OTX1 mRNA在结直肠癌组织和癌旁组织中的相对表达量分别为1.049±0.446和0.639±0.178 (t=-6.583, P<0.001);miR-424-5p在结直肠癌组织和癌旁组织中的相对表达量分别为0.865±0.261和1.329±0.387 (t=7.705, P<0.001),差异有统计学意义。miR-424-5p与OTX1 mRNA在结直肠癌组织中的表达呈负相关(r=-0.439, P=0.015)。转染miR-424-5p-mimic的HCT116细胞在72、96和120 h的吸光度值分别为0.813±0.064、0.960±0.098、1.287±0.192,转染miR-NC的HCT116细胞在72、96和120 h的吸光度值分别为1.163±0.158、1.645±0.117和2.043±0.236。miR-424-5p-mimic组的增殖能力低于miR-NC组,两组间差异有统计学意义(t=3.538, P=0.024;t = 7.778, P = 0.001;t = 4.257, P = 0.013)。划痕实验显示,与miR-NC组相比,miR-424-5p-mimic组抑制了HCT116细胞的迁移。miR-NC组和miR-424-5p-mimic组通过隔膜的细胞数分别为177.104±17.834个和35.667±13.634个,差异有统计学意义(t=15.246, P<0.001)。miR-NC组和miR-424-5p-mimic组OTX1蛋白相对表达量分别为0.862±0.121和0.342±0.103,差异有统计学意义(t=16.286, P<0.001)。荧光素酶报告实验显示,过表达miR-424-5p后wt- otx1 -3’utr载体和mutt - otx1 -3’utr载体的荧光素酶活性分别为0.305±0.095和0.898±0.080,差异有统计学意义(P<0.001)。结论miR-424-5p与OTX1 mRNA在结直肠癌组织中的表达呈负相关。miR-424-5p通过下调OTX1的表达抑制CRC细胞的增殖、迁移和侵袭。关键词:结直肠肿瘤;小分子核糖核酸;细胞增殖;OTX1
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微小RNA-424-5p调控OTX1对结直肠癌细胞增殖、迁移和侵袭的影响
Objective To investigate the correlation of the expression of microRNA-424-5p (miR-424-5p) and orthodenticle homeobox 1 (OTX1) gene in colorectal cancer (CRC) tissue, and the effects of miR-424-5p on the proliferation, migration and invasion of CRC cells HCT116. Methods A total of 60 patients with CRC were collected from June 2017 to June 2018 in Department of Colorectal Surgery of Third Affiliated Hospital of Kunming Medical University. Real time quantitative PCR (RT-qPCR) was used to detect the expression of miR-424-5p and OTX1 mRNA in 60 cases of CRC tissues, para-carcinoma tissues and CRC cell lines. The correlation between the expression of miR-424-5p and OTX1 gene was further analyzed. miR-NC (miR-NC group) and miR-424-5p-mimic (miR-424-5p-mimic group) were transfected into HCT116 cells. CCK-8 assay, scratch assay and Transwell assay were used to detect the effects of miR-424-5p on the proliferation, migration and invasion of HCT116 cells. The effect of miR-424-5p on the expression of OTX1 protein was detected by Western blotting. The luciferase report assay was used to detect the influence of miR-424-5p on the luciferase activity of OTX1-3′UTR vector. Results The relative expressions of OTX1 mRNA in CRC tissues and para-carcinoma tissues were 1.049±0.446 and 0.639±0.178 (t=-6.583, P<0.001); and the relative expression of miR-424-5p in CRC tissues and para-carcinoma tissues were 0.865±0.261 and 1.329±0.387 (t=7.705, P<0.001), with statistically significant differences. Negative correlation was found between the expression of miR-424-5p and OTX1 mRNA in CRC tissues (r=-0.439, P=0.015). The absorbance values of HCT116 cells transfected with miR-424-5p-mimic were 0.813±0.064, 0.960±0.098, 1.287±0.192 on 72, 96 and 120 hours respectively, and those of HCT116 cells transfected with miR-NC were 1.163±0.158, 1.645±0.117 and 2.043±0.236. The proliferation ability of miR-424-5p-mimic group was lower than that of miR-NC group and the differences between the two groups were statistically significant (t=3.538, P=0.024; t=7.778, P=0.001; t=4.257, P=0.013). The scratch assay showed that the migration of HCT116 cells in miR-424-5p-mimic group was inhibited as compared with miR-NC group. The numbers of cells permeating septum of miR-NC and miR-424-5p-mimic group were 177.104±17.834 and 35.667±13.634, and the difference was statistically significant (t=15.246, P<0.001). The relative expressions of OTX1 protein in miR-NC and miR-424-5p-mimic group were 0.862±0.121 and 0.342±0.103 respectively, and the difference was statistically significant (t=16.286, P<0.001). Luciferase report assay showed that the luciferase activities of wt-OTX1-3′UTR and mut-OTX1-3′UTR vector were 0.305±0.095 and 0.898±0.080 after over expression of miR-424-5p, and the difference was statistically significant (P<0.001). Conclusion The expressions of miR-424-5p and OTX1 mRNA are negatively correlated in CRC tissue. miR-424-5p can inhibit the proliferation, migration and invasion of CRC cells by down-regulating the expression of OTX1. Key words: Colorectal neoplasms; MicroRNAs; Cell proliferation; OTX1
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