上海交通大学学报(医学版) ›› 2022, Vol. 42 ›› Issue (2): 135-141.doi: 10.3969/j.issn.1674-8115.2022.02.001

• 论著 · 基础研究 •    

血根碱通过上调m6A甲基转移酶14对胃癌细胞增殖和侵袭的抑制作用

陈鸣(), 张靖()   

  1. 上海交通大学医学院附属第六人民医院消化科,上海 200233
  • 收稿日期:2021-07-27 出版日期:2022-02-28 发布日期:2022-03-17
  • 通讯作者: 张靖 E-mail:1349262506@qq.com;jing5522724@163.com
  • 作者简介:陈 鸣(1997—),女,硕士生;电子信箱:1349262506@qq.com
  • 基金资助:
    国家自然科学基金面上项目(82074161);上海市自然科学基金面上项目(21ZR1448700);上海市教育委员会高峰高原学科建设计划(20191831)

Inhibitory effect of sanguinarine on proliferaton and invasion of gastric cancer cells by upregulating m6A methyltransferase 14

Ming CHEN(), Jing ZHANG()   

  1. Department of Gastroenterology, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China
  • Received:2021-07-27 Online:2022-02-28 Published:2022-03-17
  • Contact: Jing ZHANG E-mail:1349262506@qq.com;jing5522724@163.com
  • Supported by:
    National Natural Science Foundation of China(82074161);Shanghai Natural Science Foundation(21ZR1448700);Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support(20191831)

摘要:

目的·探讨血根碱(sanguinarine,SAG)对胃癌细胞MGC-803和AGS增殖和侵袭的影响,及其作用机制与N6-甲基腺嘌呤(m6A)甲基转移酶(methyltransferase 14,METTL14)的关系。方法·利用不同浓度SAG(0、10、20 μmol/L)处理胃癌细胞系MGC-803和AGS,48 h后通过定量PCR和Western blotting检测SAG对METTL14表达的影响。然后用METTL14的小干扰RNA(si-METTL14)慢病毒载体或其空载对照si-NC慢病毒载体转染上述2个胃癌细胞系,再以10 μmol/L SAG或PBS处理48 h,将细胞分为si-METTL14+SAG组、si-NC+SAG组和si-NC+PBS组。定量PCR和Western blotting验证si-METTL14转染后METTL14在胃癌细胞的表达水平。噻唑蓝(MTT)细胞增殖实验、细胞克隆形成实验及Transwell侵袭实验分别观察3组细胞的增殖水平、克隆形成数量及侵袭潜能。2组数据间比较采用独立样本t检验,2组以上数据间比较采用单因素方差分析。结果·与对照组(0 μmol/L)比较,10 μmol/L、20 μmol/L SAG在2种胃癌细胞中均可上调METTL14的mRNA和蛋白表达水平(均P<0.05),且呈一定的浓度依赖性。定量PCR和Western blotting结果证实,转染si-METTL14METTL14在胃癌细胞的表达水平显著降低。MTT细胞增殖实验结果显示,si-NC+SAG组较si-NC+PBS组细胞增殖率显著降低(均P=0.000);克隆形成实验结果显示,si-NC+SAG组较si-NC+PBS组细胞克隆数显著减少(均P<0.01);Transwell侵袭实验结果显示,si-NC+SAG组较si-NC+PBS组穿过基底胶(Matrigel)的细胞数量显著减少(均P<0.01)。而si-METTL14可以部分逆转SAG的上述对胃癌细胞的抑制作用。结论·SAG可以抑制胃癌细胞的增殖活性、克隆形成及侵袭潜能,这些作用可能是通过上调METTL14表达水平实现的。

关键词: 胃癌, 血根碱, 甲基转移酶14, N6-甲基腺嘌呤, 细胞增殖, 细胞侵袭

Abstract:

Objective·To investigate the effect of sanguinarine (SAG) on the proliferation and invasion of gastric cancer cells MGC-803 and AGS, and the relationship between the mechanism and N6-methyladenosine (m6A) methyltransferase 14 (METTL14).

Methods·After the gastric cancer cell lines (MGC-803 and AGS) were exposed to different concentrations of SAG (0, 10, 20 μmol/L) for 48 h, quantitative PCR and Western blotting analysis were used to detect the effect of SAG on the expression of METTL14. Then, MGC-803 and AGS cells transfected with lentiviruses-mediated small interfering RNA of METTL14 (si-METTL14) or control (si-NC) were treated with 10 μmol/L SAG or PBS for 48 h, and thus the two cell lines were divided into si-METTL14+SAG group, si-NC+SAG group and si-NC+PBS group, respectively. Quantitative PCR and Western blotting were used to verify the expression levels of METTL14 after it was transfected with si-METTL14 in gastric cancer cells. The proliferation level, number of clones formed and invasion potential of the 3 groups in both MGC-803 cells and AGS cells were observed by MTT proliferation assay, cell clone formation test and Transwell invasion assay, respectively. Independent samples t test was used for comparison between two groups of data, and one-way ANOVA was used for comparison between more than two groups of data.

Results·Compared with the control group (0 μmol/L), 10 μmol/L SAG and 20 μmol/L SAG up-regulated METTL14 mRNA and protein expression levels (P<0.05), showing a certain concentration dependence. Quantitative PCR and Western blotting results confirmed that the expression levels of METTL14 in gastric cancer cells were significantly reduced after transfection of si-METTL14 in both MGC-803 cells and AGS cells. MTT cell proliferation assay showed that the cell proliferation rate of the si-NC+SAG group was significantly lower than that of the si-NC+PBS group in each cell line (P=0.000). The cell clone formation test showed that the number of cell clones of the si-NC+SAG group was significantly smaller than that of the si-NC+PBS group in each cell line (P<0.01). The Transwell invasion assay showed that the cells crossing Matrigel gel in the si-NC+SAG group was significantly less than that in the si-NC+PBS group in each cell line (P<0.01). si-METTL14 could partially reverse the inhibitory effects of SAG on gastric cancer cells.

Conclusion·SAG can inhibit the proliferation activity, clonal formation and invasion potential of gastric cancer cells, which may be realized by upregulating METTL14 expression level.

Key words: gastric carcinoma, sanguinarine (SAG), methyltransferase 14 (METTL14), N6-methyladenosine (m6A), cell proliferation, cell invasion

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