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中华普通外科学文献(电子版) ›› 2021, Vol. 15 ›› Issue (06) : 406 -412. doi: 10.3877/cma.j.issn.1674-0793.2021.06.002

论著

大黄素通过miR-429/SOX2轴调控肝癌细胞增殖、迁移和侵袭
李东霞1, 黄丽华1,(), 叶伟1, 钱相君1, 张磊2   
  1. 1. 510655 广州,中山大学附属第六医院胰腺肝胆外科
    2. 510630 广州,中山大学附属第三医院胆胰外科
  • 收稿日期:2021-06-02 出版日期:2021-12-01
  • 通信作者: 黄丽华
  • 基金资助:
    中国博士后科学基金项目(2020TQ0370)

Emodin regulating the proliferation, migration and invasion of hepatocellular carcinoma cells through miR-429/SOX2 axis

Dongxia Li1, Lihua Huang1,(), Wei Ye1, Xiangjun Qian1, Lei Zhang2   

  1. 1. Department of Pancreatic-Hepato-Biliary-Surgery, the Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou 510655, China
    2. Department of Biliary-Pancreatic Surgery, the Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China
  • Received:2021-06-02 Published:2021-12-01
  • Corresponding author: Lihua Huang
引用本文:

李东霞, 黄丽华, 叶伟, 钱相君, 张磊. 大黄素通过miR-429/SOX2轴调控肝癌细胞增殖、迁移和侵袭[J/OL]. 中华普通外科学文献(电子版), 2021, 15(06): 406-412.

Dongxia Li, Lihua Huang, Wei Ye, Xiangjun Qian, Lei Zhang. Emodin regulating the proliferation, migration and invasion of hepatocellular carcinoma cells through miR-429/SOX2 axis[J/OL]. Chinese Archives of General Surgery(Electronic Edition), 2021, 15(06): 406-412.

目的

大黄素具有抗炎、抗氧化、抗肿瘤的作用,本文拟通过医学实验以及生物信息学分析方法探讨大黄素对肝癌细胞的作用以及分子机制。

方法

梯度浓度的大黄素处理肝癌细胞Huh7,通过实时荧光定量聚合酶链式反应(qPCR)与蛋白免疫印迹(Western blotting)方法检测miR-429与SOX2的表达以确定大黄素的处理浓度。合成过表达SOX2的质粒、miR-429 inhibitor、miR-429 mimics,然后转染至Huh7细胞,分别通过qPCR与Western blotting验证miR-429与SOX2的表达变化,并通过CCK-8、迁移和侵袭实验观察细胞功能变化。最后根据TargetScan软件预测的结合位点,利用双荧光素报告基因检测证实miR-429与SOX2的结合。

结果

与对照组相比,大黄素不同浓度(1、10、100 μmol/L)处理组均显著促进了miR-429的表达(P<0.05),抑制了SOX2的表达(P<0.05)。过表达SOX2质粒及miR-429 inhibitor均能促进SOX2的表达(P<0.05),同时促进Huh7细胞的增殖、迁移、侵袭;miR-429 mimics则降低了SOX2的表达(P<0.05)及细胞的增殖、迁移、侵袭能力。此外,miR-429可以直接结合SOX2。

结论

大黄素抑制了肝癌细胞的增殖和侵袭、迁移,具体的机制是通过促进miR-429的表达,进一步抑制SOX2的表达。

Objective

Emodin has anti-inflammatory, antioxidant and antitumor effects. This study discusses the effect and molecular mechanism of emodin on hepatocellular carcinoma cells by medical experimental methods and bioinformatics analysis.

Methods

Huh7 cells were treated with different concentrations, then real-time fluorescence quantitative polymerase chain reaction (qPCR) and Western blotting were used to determine the expression of miR-429 and SOX2. SOX2 overexpression plasmid, miR-429 inhibitor and miR-429 mimics were synthesized and then transfected into Huh7 cells, and the expression changes of miR-429 and SOX2 were verified by qPCR and Western blotting, the functional changes of cells were observed by CCK-8 and transwell assay. Finally, according to the predicted binding site of TargetScan, the binding between miR-429 and SOX2 was confirmed by double luciferase reporter gene detection.

Results

Compared with the control group, emodin at different concentrations (1, 10, 100 μmol/L) significantly promoted the expression of miR-429 and inhibited the expression of SOX2 (P<0.05). Overexpression SOX2 plasmid and miR-429 inhibitor promoted the expression of SOX2 and the ability of proliferation, migration and invasion in Huh7 cells (P<0.05). Furthermore, miR-429 mimics decreased the expression of SOX2 (P<0.05) and the ability of cell migration and invasion. In addition, miR-429 was directly banded with SOX2.

Conclusion

Emodin regulates miR-429/SOX2 axis to inhibit the ability of proliferation, migration and invasion in liver cells.

表1 引物序列信息
图1 大黄素促进肝癌细胞Huh7的miR-429的表达并抑制SOX2的表达 A为qPCR检测不同浓度的大黄素处理后miR-429与SOX2的表达变化;B为Western blotting检测不同浓度的大黄素处理后SOX2的表达变化;* P<0.05
图2 大黄素通过抑制SOX2的表达抑制肝癌细胞Huh7的迁移、侵袭 A为qPCR检测SOX2的表达变化;B为Western blotting检测SOX2的表达变化;C为CCK-8检测细胞的增殖变化;D为Transwell检测Huh7细胞的迁移、侵袭变化;* P<0.05
图3 大黄素通过上调miR-429的表达减少SOX2的表达,进而抑制肝癌细胞Huh7的迁移、侵袭 A为qPCR检测miR-429与SOX2的表达变化;B为Western blotting检测SOX2的蛋白表达变化;C为CCK-8检测Huh7细胞的增殖变化;D为Transwell检测Huh7细胞的迁移、侵袭变化;* P<0.05
图4 miR-429通过直接靶向结合SOX2,以抑制SOX2的表达进而抑制肝癌细胞Huh7的迁移、侵袭 A为qPCR检测miR-429与SOX2的表达变化;B为Western blotting检测SOX2的表达变化;C为CCK-8检测Huh7细胞的增殖变化;D为Transwell检测Huh7细胞的迁移、侵袭变化;* P<0.05
图5 miR-429与SOX2的3’UTR的结合位点 A为TargetScan预测miR-429与SOX2的3’UTR的结合位点;B为双荧光素报告基因检测荧光素酶活性变化;* P<0.05
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