中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (19): 3030-3035.doi: 10.12307/2022.382

• 肿瘤干细胞 cancer stem cells • 上一篇    下一篇

miR-7-5p对人胃癌SGC-7901细胞干样特性的影响

郭隽馥，李乡南，马天驰，苗兰英   

  1. 辽宁中医药大学教学实验中心，辽宁省沈阳市   110847
  • 收稿日期:2021-06-04 修回日期:2021-07-16 接受日期:2021-08-24 出版日期:2022-07-08 发布日期:2021-12-28
  • 通讯作者: 苗兰英，教授，研究员级高级实验师，辽宁中医药大学教学实验中心，辽宁省沈阳市 110847
  • 作者简介:郭隽馥，女，辽宁省沈阳市人，2015年中国医科大学毕业，博士，高级实验师，硕士生导师，主要从事中西医结合肿瘤分子遗传学方面的基础研究工作。
  • 基金资助:
    国家自然科学基金青年项目(81803855)，项目负责人：郭隽馥；辽宁省博士启动项目(2019-BS-166)，项目负责人：郭隽馥；沈阳市中青年科技创新人才支持计划项目(RC190078)，项目负责人：郭隽馥

Effects of miR-7-5p on stem cell-like properties of human gastric cancer SGC-7901 cells

Guo Junfu, Li Xiangnan, Ma Tianchi, Miao Lanying   

  1. Teaching and Experiment Center, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, Liaoning Province, China
  • Received:2021-06-04 Revised:2021-07-16 Accepted:2021-08-24 Online:2022-07-08 Published:2021-12-28
  • Contact: Miao Lanying, Professor, Senior experimentalist, Teaching and Experiment Center, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, Liaoning Province, China
  • About author:Guo Junfu, MD, Senior experimentalist, Master’s supervisor, Teaching and Experiment Center, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, Liaoning Province, China
  • Supported by:
    National Natural Science Foundation of China (Youth Project), No. 81803855 (to GJF); Liaoning Province Doctoral Initiation Project, No. 2019-BS-166 (to GJF); Shenyang Youth Science and Technology Innovation Talent Support Program, No. RC190078 (to GJF)

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摘要：

文题释义：
成球性：具有自我更新能力的肿瘤干细胞能够从癌细胞系和肿瘤活检组织中分离出来，并在三维肿瘤球状体悬浮培养物中生长。为检测肿瘤细胞的成球能力，一般需要将单细胞悬液加入超低吸附多孔培养板中并在培养液中加入多种生长因子以模拟体内肿瘤的生长环境。
软琼脂集落：肿瘤干细胞具有多向分化潜能，在体外培养基中能够由一个祖先细胞增殖形成细胞团也就是集落。利用低熔点琼脂糖制备双层琼脂(上层0.3%流动性较好，下层0.5%相对固定)，再将单细胞悬液加入其中，也是模拟体内肿瘤生长环境的一种手段。

背景：miR-7在胃癌等多种肿瘤组织中表达下调，主要发挥抑癌作用。现已证实，miR-7能够影响胃癌细胞的增殖、凋亡、迁移、侵袭等多种细胞生物学功能。然而，目前有关miR-7与胃癌细胞干样特性关系的研究较少，机制尚不明确。
目的：探讨miR-7-5p对人胃癌SGC-7901细胞干样特性的影响。
方法：以胃癌细胞株SGC-7901为模型，用NC、miR-7-5p mimics、inhibitor NC及miR-7-5p inhibitor转染细胞，转染48 h后采用倒置荧光显微镜观察转染效率，RT-qPCR方法检测miR-7-5p、Nanog和Sox2 mRNA表达水平，采用成球实验和软琼脂集落实验检测细胞成球能力和软琼脂集落形成能力，流式细胞术检测CD24+CD44+细胞亚群比例。
结果与结论：①与inhibitor NC或NC组比较，转染miR-7-5p inhibitor或mimics后，胃癌SGC7901细胞中miR-7-5p的表达量显著降低或升高(P < 0.01)；②与NC组比较，miR-7-5p mimics组成球数量和软琼脂集落形成数量明显减少(P < 0.05，P < 0.01)，而miR-7-5p inhibitor组成球数量和集落形成数量则明显高于miR-7-5p inhibitor NC组(P < 0.05，P < 0.01)；③miR-7-5p mimics组CD24+CD44+细胞比例明显低于NC组(P < 0.01)，而miR-7-5p inhibitor组CD24+CD44+细胞的比例显著高于inhibitor NC组(P < 0.01)；④miR-7-5p mimics组Nanog和Sox2 mRNA表达明显低于NC组，miR-7-5p inhibitor组Nanog和Sox2 mRNA表达显著高于inhibitor NC组(P < 0.05，P < 0.01)；⑤结果表明，miR-7-5p可以通过减弱胃癌细胞的成球能力、软琼脂集落形成能力以及降低胃癌细胞中CD24+CD44+细胞亚群比例等方式抑制胃癌细胞的干样特性，其机制可能与调控干性相关基因Nanog和Sox2的表达有关。

https://orcid.org/0000-0001-5034-2824 (郭隽馥)

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 干样特性, 胃癌, miR-7-5p, CD24, CD44, Nanog, Sox2

Abstract: BACKGROUND: The expression of miR-7 is down-regulated in various tumor tissues, including gastric cancer, and plays a major role in tumor suppression. It has been confirmed that miR-7 can affect a variety of cell biological functions, such as proliferation, apoptosis, migration, and invasion of gastric cancer cells. However, few studies have been conducted on the relationship between miR-7 and stem cell-like properties of gastric cancer cells, and the mechanism remains unclear.  
OBJECTIVE: To investigate the effects of miR-7-5p on stem cell-like properties of human SGC-7901 gastric cancer cells.
METHODS:  The gastric cancer cell line SGC-7901 was used as cell model. NC, miR-7-5p mimics, inhibitor NC and miR-7-5p inhibitor were transfected into cells, separately. The inverted fluorescent microscope was used to observe transfection efficiency at 48 hours post-transfection. The expression levels of miR-7-5p, Nanog and Sox2 mRNA were detected by RT-qPCR. Spheroid and soft-agar colony formation assays were performed to observe the abilities of spheroid and soft-agar colony formation, respectively. The proportion of CD24+CD44+ cell subsets was determined by flow cytometry.  
RESULTS AND CONCLUSION: (1) The expression of miR-7-5p was significantly increased or decreased in SGC7901 cells after transfection of miR-7-5p inhibitor or mimics compared with inhibitor NC or NC group (P < 0.01). (2) The numbers of spheroid and soft-agar colony formation of SGC-7901 cells were decreased in the miR-7-5p mimics group than those in NC group (P < 0.05, P < 0.01), but increased in miR-7-5p inhibitor group than those in inhibitor NC group (P < 0.05, P < 0.01). (3) The proportion of CD24+CD44+ cells in the miR-7-5p mimics group was significantly lower than that in the NC group (P < 0.01). The proportion of CD24+CD44+ cells in miR-7-5p inhibitor group was significantly higher than those in inhibitor NC group (P < 0.01). (4) The mRNA expression levels of Nanog and Sox2 in the miR-7-5p mimics group were significantly lower than those in the NC group, and the mRNA expression levels of Nanog and Sox2 in the miR-7-5p inhibitor group were significantly higher than those in the inhibitor NC group (P < 0.05, P < 0.01). (5) miR-7-5p can inhibit spheroid and soft-agar colony formation abilities of gastric cancer cells, and reduce the proportion of CD24+CD44+ cell subsets in gastric cancer cells. The potential mechanism may be related to the regulation of the expression of stemness genes Nanog and Sox2.

Key words: stem cell-like property, gastric cancer, miR-7-5p, CD24, CD44, Nanog, Sox2

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