Mechanism of miR-32-5p targeting TOB1 gene in regulating radiosensitization, migration and invasion of colorectal cancer cells

中华放射肿瘤学杂志 Pub Date : 2020-02-15 DOI:10.3760/CMA.J.ISSN.1004-4221.2020.02.009

Hui Zhang, Hong-Yan Liang

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Abstract

Objection To investigate the effect of miR-32-5p on the radiosensitivity, migration and invasion of colorectal cancer cells and the underlying mechanism. Methods Human colorectal cancer SW480 cells and normal colonic epithelial NCM460 cells were cultured. The colorectal cancer cells were divided into the non-transfected and transfected groups (transfected with anti-miR-NC, anti-miR-32-5p, pcDNA, pcDNA-TOB1, anti-miR-32-5p+ si-NC and anti-miR-32-5p+ si-TOB1, respectively). The expression of miR-32-5p and TOB1 at the mRNA and protein levels was detected by RT-qPCR and Western blot. The radiosensitivity of the transfected cells was determined by colony formation assay. The migration and invasion ability of the transfected cells were detected by Transwell assay. Whether miR-32-5p targeted TOB1 was validated by dual luciferase reporter gene assay and Western blot. Results Compared with human colonic epithelial cells, the expression of miR-32-5p was significantly up-regulated, whereas the expression of TOB1 mRNA and protein was remarkably down-regulated in the colon cancer cells (all P<0.05). Compared with the anti-miR-NC, the quantity of cell migration and invasion was significantly decreased (both P<0.05) and the radiosensitivity ratio was 1.801 in the anti-miR-32-5p group. Compared with the pcDNA group, the quantity of cell migration and invasion was significantly decreased (both P<0.05) and the radiosensitivity ratio was 1.764 in the pcDNA-TOB1 group. Dual luciferase reporter gene assay and Western blot confirmed that miR-32-5p negatively regulated the expression of TOB1 protein. Compared with the anti-miR-32-5p+ si-NC group, the quantity of cell migration and invasion was significantly increased (both P<0.05) and the radiosensitivity ratio was 0.591 in the anti-miR-32-5p+ si-TOB1 group. Conclusions Inhibition of miR-32-5p expression can significantly enhance the radiosensitivity of colorectal cancer cells and suppress cell migration and invasion. The underlying mechanism might be related to the targeted up-regulation of TOB1 expression. Key words: Colorectal cancer cell line; miR-32-5p gene; TOB1 gene; Radiosensitivity; Migration and invasion

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靶向to1基因的miR-32-5p调控结直肠癌细胞放射致敏、迁移和侵袭的机制

目的探讨miR-32-5p对结直肠癌癌症细胞放射敏感性、迁移和侵袭的影响及其机制。方法培养人癌症SW480细胞和正常结肠上皮NCM460细胞。将结直肠癌癌症细胞分为未转染组和转染组（分别用抗-miR-NC、抗-miR-32-5p、pcDNA、pcDNA-TOB1、抗-mmiR-32-5p+si-NC和抗-miR32-5p+si-TOB1转染）。通过RT-qPCR和蛋白质印迹检测miR-32-5p和TOB1在mRNA和蛋白质水平上的表达。通过集落形成试验测定转染细胞的放射敏感性。Transwell法检测转染细胞的迁移和侵袭能力。miR-32-5p是否靶向TOB1通过双荧光素酶报告基因测定和蛋白质印迹进行了验证。结果与人结肠上皮细胞相比，结肠癌癌症细胞中miR-32-5p的表达显著上调，而TOB1 mRNA和蛋白的表达显著下调（均P＜0.05），抗miR-32-5p组的细胞迁移和侵袭量显著减少（均P<0.05），放射敏感性为1.801。与pcDNA组相比，pcDNA-TOB1组的细胞迁移和侵袭量显著减少（均P<0.05），放射敏感性为1.764。双荧光素酶报告基因分析和蛋白质印迹证实miR-32-5p负调控TOB1蛋白的表达。与抗miR-32-5p+si-NC组相比，抗miR-32-5 P+si-TOB1组的细胞迁移和侵袭量显著增加（均P<0.05），放射敏感性为0.591。结论抑制miR-32-5p的表达可显著提高癌症细胞的放射敏感性，抑制细胞的迁移和侵袭。其潜在机制可能与TOB1表达的靶向上调有关。关键词：结直肠癌癌症细胞系；miR-32-5p基因；TOB1基因；放射敏感性；迁移和入侵

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来源期刊

中华放射肿瘤学杂志

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期刊介绍： The Chinese Journal of Radiation Oncology is a national academic journal sponsored by the Chinese Medical Association. It was founded in 1992 and the title was written by Chen Minzhang, the former Minister of Health. Its predecessor was the Chinese Journal of Radiation Oncology, which was founded in 1987. The journal is an authoritative journal in the field of radiation oncology in my country. It focuses on clinical tumor radiotherapy, tumor radiation physics, tumor radiation biology, and thermal therapy. Its main readers are middle and senior clinical doctors and scientific researchers. It is now a monthly journal with a large 16-page format and 80 pages of text. For many years, it has adhered to the principle of combining theory with practice and combining improvement with popularization. It now has columns such as monographs, head and neck tumors (monographs), chest tumors (monographs), abdominal tumors (monographs), physics, technology, biology (monographs), reviews, and investigations and research.