国际肿瘤学杂志

国际肿瘤学杂志 ›› 2019, Vol. 46 ›› Issue (6): 321-326.doi: 10.3760/cma.j.issn.1673-422X.2019.06.001

• 论著 •    下一篇

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   

  1. 昆明医科大学第三附属医院（云南省肿瘤医院）结直肠外科650106
  • 出版日期:2019-06-08 发布日期:2019-07-11
  • 通讯作者: 李强，Email: 1390811139@qq.com E-mail:1390811139@qq.com
  • 基金资助:

    云南省科技计划［2017FE467（072）］；云南省卫生科技计划（2016NS101）

Effects of microRNA-424-5p on proliferation, migration and invasion of colorectal cancer cells by regulating OTX1

Yu Kun, Wang Weiya, Cai Xinyi, Cheng Xianshuo, Zhao Xiaofeng, Bie Yaqin, Li Qiang   

  1. Department of Colorectal Surgery, Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital), Kunming 650106, China
  • Online:2019-06-08 Published:2019-07-11
  • Contact: Li Qiang, Email: 1390811139@qq.com E-mail:1390811139@qq.com
  • Supported by:

    Science and Technology Project of Yunnan Province of China ［2017FE467(072)］; Health Science and Technology Project of Yunnan Province of China (2016NS101)

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摘要： 目的  研究结直肠癌组织中微小RNA-424-5p（miR-424-5p）与orthodenticle人同源盒基因1（OTX1） mRNA表达的关系，并探讨miR-424-5p对结直肠癌HCT116细胞增殖、迁移和侵袭的影响。方法  收集2017年6月至2018年6月在昆明医科大学第三附属医院结直肠外科收治的60例结直肠癌患者，采用实时定量PCR（RT-qPCR）检测miR-4245p和OTX1 mRNA在60对结直肠癌组织和癌旁组织以及结直肠癌HCT116细胞中的表达情况，并分析两者表达的相关性。HCT116细胞分别转染miR-NC（miR-NC组）和miR-424-5p-mimic（miR-424-5p-mimic组），采用CCK-8法、细胞划痕实验、Transwell实验分别检测miR-424-5p对HCT116细胞增殖、迁移和侵袭能力的影响；Western blotting检测过表达miR-424-5p对OTX1蛋白表达的影响。荧光素酶报告实验检测miR-424-5p对OTX1-3′UTR报告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），差异均有统计学意义。结直肠癌组织中miR-424-5p和OTX1 mRNA的表达呈负相关（r=－0.439，P=0.015）。HCT116细胞转染miR-424-5p-mimic后第72、96和120 h时吸光度（A）值分别为0.813±0.064、0.960±0.098、1.287±0.192，转染miR-NC后第72、96和120 h时A值分别为1.163±0.158、1.645±0.117、2.043±0.236，miR-424-5p-mimic组细胞增殖能力显著低于miR-NC组，差异均具有统计学意义（t=3.538，P=0.024；t=7.778，P=0.001；t=4.257，P=0.013）。划痕实验结果显示，划痕24 h后miR-424-5p-mimic组与miR-NC组相比迁移能力显著减弱。miR-NC组和miR-424-5p-mimic组穿膜细胞数分别为（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）。OTX1蛋白在miR-NC组和miR-424-5p-mimic组中的相对表达量分别为0.862±0.121、0.342±0.103，差异有统计学意义（t＝16.286，P<0.001）。荧光素酶实验结果显示，过表达miR-424-5p后，wt-OTX1-3′UTR和mut-OTX1-3′UTR报告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可通过下调OTX1的表达抑制结直肠癌细胞的增殖、迁移和侵袭。

关键词: 结直肠肿瘤, 微RNAs, 细胞增殖, OTX1

Abstract: 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, paracarcinoma 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. ResultsThe 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