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

doi:10.3969/j.issn.1674-8115.2022.02.001

摘要/Abstract

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-METTL14后METTL14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

中图分类号:

R735.2

陈鸣, 张靖. 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[J]. 上海交通大学学报(医学版), 2022, 42(2): 135-141.

Ming CHEN, Jing ZHANG. Inhibitory effect of sanguinarine on proliferaton and invasion of gastric cancer cells by upregulating m⁶A methyltransferase 14[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(2): 135-141.

图/表 4

图1 不同浓度SAG对胃癌细胞中 METTL14 表达的影响Note： A/C. Quantitative PCR used to detect the effect of SAG on METTL14 mRNA expression in the gastric cancer cells, MGC-803 (A) and AGS (C). B/D. Western blotting used to detect the effect of SAG on METTL14 protein expression in MGC-803 (B) and AGS (D).

Fig 1 Effect of different concentrations of SAG on METTL14 expression in gastric cancer cells

图2 敲减 METTL14 对SAG抗胃癌细胞增殖作用的影响Note：A. Quantitative PCR used to detect the mRNA levels of METTL14 after transfection of si-METTL14 or si-NC in gastric cancer cells. B. Western blotting used to detect the protein levels of METTL14 after transfection of si-METTL14 or si-NC in gastric cancer cells. C. MTT proliferation test used to detect the cell proliferative potential of the gastric cancer cells in the three different groups.

Fig 2 Effect of knocking down METTL14 on the anti-proliferation activity of SAG in gastric cancer cells

图3 敲减 METTL14 对SAG抗胃癌细胞克隆形成的影响Note：A. The representative pictures of cell clones in the three different groups of gastric cancer cells in the clonal formation assay. B. Comparison of the colony numbers among the three different groups.

Fig 3 Effect of knocking down METTL14 on the anti-colony formation capability of SAG in gastric cancer cells

图4 敲减 METTL14 对SAG抗胃癌细胞侵袭潜能的影响Note：A. The representative pictures of invasive cells in the three different groups of gastric cancer cells in the Transwell invasion assay. B. Comparison of the invasive cell numbers among the three different groups.

Fig 4 Effect of knocking down METTL14 on the anti-invasion potential of SAG in gastric cancer cells

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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

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

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