亚砷酸钠暴露14周诱导肝癌细胞LM3氧化损伤及恶性迁移的研究

doi:10.3969/j.issn.1674-8115.2022.12.004

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (12): 1677-1684.doi: 10.3969/j.issn.1674-8115.2022.12.004

• 论著 · 基础研究 • 上一篇

亚砷酸钠暴露14周诱导肝癌细胞LM3氧化损伤及恶性迁移的研究

孙金丽¹(), 宋纬巍¹(), 许鸣², 李井泉¹()

^1.上海交通大学公共卫生学院食品安全与毒理学系，上海 200025
^2.内蒙古科技大学包头师范学院生物科学与技术学院，包头 014030

收稿日期:2022-09-29 接受日期:2022-11-05 出版日期:2022-12-28 发布日期:2022-12-28
通讯作者: 李井泉 E-mail:sunjinli1989@shsmu.edu.cn;songweiwei@sjtu.edu.cn;jqli@shsmu.edu.cn
作者简介:孙金丽（1988—），女，实验师，硕士；电子信箱：sunjinli1989@shsmu.edu.cn
宋纬巍（2002—），女，本科生；电子信箱：songweiwei@sjtu.edu.cn。*第一联系人：为共同第一作者。
基金资助:
上海市教育委员会实验技术队伍建设计划(BJ1-3000-22-0090)

Oxidative damage and malignant migration of hepatocellular carcinoma cells LM3 induced by 14 weeks exposure to sodium arsenite

SUN Jinli¹(), SONG Weiwei¹(), XU Ming², LI Jingquan¹()

^1.Department of Food Safety and Toxicology, Shanghai Jiao Tong University School of Public Health, Shanghai 200025, China
^2.College of Biological Science and Technology, Baotou Normal College, Inner Mongolia University of Science and Technology, Baotou 014030, China

Received:2022-09-29 Accepted:2022-11-05 Online:2022-12-28 Published:2022-12-28
Contact: LI Jingquan E-mail:sunjinli1989@shsmu.edu.cn;songweiwei@sjtu.edu.cn;jqli@shsmu.edu.cn
Supported by:
Experimental Technical Team Construction Project of Shanghai Municipal Education Commission(BJ1-3000-22-0090)

摘要/Abstract

摘要：

目的·探究不同剂量的亚砷酸钠暴露对人肝癌细胞株LM3细胞的影响，分析亚砷酸钠促进肝癌细胞恶性迁移的分子机制。方法·建立细胞亚砷酸钠染毒模型，分别用含0、1、10 μmol/L亚砷酸钠的DMEM高糖培养基连续培养LM3细胞14周。暴露结束后，从细胞培养基中去除亚砷酸盐，用不含亚砷酸钠的培养基培养细胞1周使其恢复。以同一批次传代未经过亚砷酸钠处理的细胞作为对照组。分别用CCK-8增殖实验、Transwell迁移实验、DCFH-DA活性氧荧光探针实验验证慢性亚砷酸钠暴露对LM3细胞增殖、迁移和氧化应激的影响；用实时荧光定量PCR和蛋白质印迹法验证慢性亚砷酸钠暴露对LM3细胞相关基因的mRNA和蛋白表达水平的影响。结果·相比对照组，1 μmol/L和10 μmol/L亚砷酸钠染毒能显著增强LM3细胞内的活性氧水平（均P<0.05），并促进LM3细胞的恶性迁移（均P<0.05），且该作用呈剂量依赖性，但亚砷酸钠对LM3细胞的增殖并无显著影响。亚砷酸钠染毒可以上调血管内皮生长因子（vascular endothelial growth factor，VEGF）、还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶2（nicotinamide adenine dinucleotide phosphate oxidases 2，NOX2）、促分裂原活化蛋白激酶8（mitogen-activated protein kinase 8，MAPK8）基因表达水平，上调NOX2、MAPK8蛋白的表达水平。结论·亚砷酸钠暴露能增强LM3细胞的迁移能力和氧化应激水平，也能上调VEGF、NOX2、MAPK8基因的表达，提示亚砷酸钠促进LM3细胞恶性迁移的机制可能与氧化损伤及这些基因的表达上调有关。

关键词: 亚砷酸钠, LM3细胞, 增殖, 氧化应激, 恶性迁移

Abstract:

Objective ·To investigate the effects of different doses of sodium arsenite exposure on human hepatocellular carcinoma cell line LM3, and analyze the underlying potential molecular mechanisms of sodium arsenite-mediated promotion of malignant migration of LM3 cells. Methods ·The model of sodium arsenite exposure was established by continuously culturing LM3 cells in high DMEM glucose medium containing 0, 1 and 10 μmol/L sodium arsenite for 14 weeks. After exposure, the sodium arsenite-containing cell culture medium was discarded, and the cells were allowed to recover in sodium arsenite-free culture medium for one week. Cells from the same batch without sodium arsenite treatment were used as the control group. Subsequently, CCK-8 proliferation assay, Transwell migration assay, and DCFH-DA reactive oxygen species fluorescence probe experiments were performed to verify the effect of chronic sodium arsenite exposure on LM3 cell proliferation, migration, and oxidative stress. Real-time quantitative PCR and Western blotting were performed to elucidate the effect of chronic sodium arsenite exposure on the expression of mRNA and protein related to tumor metastasis in LM3 cells. Results ·Compared with the control group, the sodium arsenite exposure groups (1 μmol/L and 10 μmol/L) had significantly higher reactive oxygen species (ROS) levels (both P>0.05), and sodium arsenite exposure promoted the malignant migration of LM3 cells in a dose-dependent manner (both P>0.05), but sodium arsenite had no significant effect on the proliferation of LM3 cells. Sodium arsenite exposure up-regulated the gene expression levels of vascular endothelial growth factor (VEGF), nicotinamide adenine dinucleotide phosphate oxidases 2 (NOX2), and mitogen-activated protein kinase 8 (MAPK8),and up-regulated the protein expression level of NOX2 and MAPK8. Conclusions ·Sodium arsenite exposure can promote malignant migration and oxidative stress level of LM3 cells, and up-regulate the expression levels of VEGF, NOX2 and MAPK8 genes, suggesting that the mechanism of sodium arsenite promoting malignant migration of LM3 cells may be related to oxidative damage and up-regulation of these genes.

Key words: sodium arsenite, LM3 cells, proliferation, oxidative stress, malignant migration

中图分类号:

孙金丽, 宋纬巍, 许鸣, 李井泉. 亚砷酸钠暴露14周诱导肝癌细胞LM3氧化损伤及恶性迁移的研究[J]. 上海交通大学学报（医学版）, 2022, 42(12): 1677-1684.

SUN Jinli, SONG Weiwei, XU Ming, LI Jingquan. Oxidative damage and malignant migration of hepatocellular carcinoma cells LM3 induced by 14 weeks exposure to sodium arsenite[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(12): 1677-1684.

图/表 6

表1 qRT-PCR引物序列

Tab 1 Primer sequences for qRT-PCR

Gene	Forward sequence （5'→3'）	Reverse sequence （5'→3'）
VEGF	AGGGCAGAATCATCACGAAGT	AGGGTCTCGATTGGATGGCA
NOX2	TGCCAGTCTGTCGAAATCTGC	ACTCGGGCATTCACACACC
MAPK8	TGTGTGGAATCAAGCACCTTC	AGGCGTCATCATAAAACTCGTTC
TGFβ	GGCCAGATCCTGTCCAAGC	GTGGGTTTCCACCATTAGCAC
GAPDH	GGCCTCCAAGGAGTAAGACC	AGGGGAGATTCAGTGTGGTG

图1 慢性亚砷酸钠暴露对LM3细胞增殖的影响

Fig 1 Effects of chronic sodium arsenite exposure on proliferation of LM3 cells

图2 慢性亚砷酸钠暴露对LM3细胞迁移能力的影响Note： A. Crystal violet staining of LM3 cells exposed to 0, 1 and 10 μmol/L sodium arsenite. B. Quantitative analysis of the number of migrating cells in each group with ImageJ software. ①P=0.000.

Fig 2 Effects of chronic sodium arsenite exposure on migration of LM3 cells

图3 DCFH-DA ROS荧光探针检测细胞内ROS水平Note： A. DAPI staining (blue) and DCFH-DA staining (green) of LM3 cells exposed to 0, 1 and 10 μmol/L sodium arsenite. B. Quantitative analysis of DCF fluorescence intensity of LM3 cells in each group. ①P=0.000.

Fig 3 Intracellular ROS levels detected by DCFH-DA ROS fluorescence probe

图4 qRT-PCR检测肿瘤转移相关基因的mRNA表达水平Note： ①P=0.000, ②P=0.007, ③P=0.001, ④P=0.019.

Fig 4 Detection of mRNA expression levels of tumor metastasis-related genes by qRT-PCR

图5 Western blotting实验检测NOX2和MAPK8蛋白表达水平Note：A. Change of protein expression level of NOX2. B. Expression of NOX2 relative to GAPDH. C. Protein expression level change of MAPK8. D. Expression of MAPK8 relative to GAPDH. ①P=0.015, ②P=0.001, ③P=0.000.

Fig 5 Detection of protein expression levels of NOX2 and MAPK8 by Western blotting

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亚砷酸钠暴露14周诱导肝癌细胞LM3氧化损伤及恶性迁移的研究

Oxidative damage and malignant migration of hepatocellular carcinoma cells LM3 induced by 14 weeks exposure to sodium arsenite

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