同源盒基因A6（HOXA6）调控肝癌HepG2细胞增殖、侵袭、转移和凋亡的作用机制

刘玉婷; 麦静愔; 侯天禄; 成扬

doi:10.12449/JCH250414

同源盒基因A6（HOXA6）调控肝癌HepG2细胞增殖、侵袭、转移和凋亡的作用机制

DOI: 10.12449/JCH250414

刘玉婷¹,
麦静愔²,
侯天禄¹,
成扬^1, , ,

1.
上海中医药大学附属曙光医院肝病研究所，上海 201203
2.
上海中医药大学附属上海市中医医院急诊科，上海 200071

基金项目:

国家自然科学基金面上项目 (82374189);

国家自然科学基金面上项目 (81774098)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：刘玉婷、侯天禄负责实验操作；麦静愔负责数据统计分析；成扬负责设计研究方案，指导撰写文章并最后定稿。

详细信息

通信作者:
成扬， drchengyang@126.com （ORCID： 0009-0000-2266-5757）

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出版历程
- 收稿日期: 2024-08-08
- 录用日期: 2024-09-23
- 出版日期: 2025-04-25

Mechanism of action of Homebox A6 in regulating the proliferation， invasion， metastasis， and apoptosis of HepG2 hepatoma cells

1.
Institute of Hepatology，Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine，Shanghai 201203，China
2.
Department of Emergency，Shanghai Traditional Chinese Medicine Hospital，Shanghai University of Traditional Chinese Medicine，Shanghai 200071，China

Research funding:

General Projects of the National Natural Science Foundation of China (82374189);

General Projects of the National Natural Science Foundation of China (81774098)

More Information

Corresponding author: CHENG Yang， drchengyang@126.com （ORCID： 0009-0000-2266-5757）

摘要

摘要: 目的研究同源盒基因A6（HOXA6）对肝癌HepG2细胞增殖、侵袭、转移和凋亡的影响，以及与PI3K/AKT信号通路的关系。方法培养肝癌HepG2细胞，构建HOXA6过表达质粒和干扰siRNA并转染细胞，随机分成4组：空白质粒组、HOXA6过表达组、siRNA阴性对照组和siRNA HOXA6干扰组。采用CCK-8法检测细胞增殖，Transwell法检测细胞侵袭，划痕愈合实验检测细胞迁移（相关蛋白TIMP3、MMP9和MMP3），流式细胞仪检测HepG2肝癌细胞凋亡（相关蛋白BAX和BCL2），BCA法测定蛋白质浓度，Western Blot检测蛋白表达水平。计量资料多组间比较采用单因素方差分析，进一步两两比较采用SNK-q检验。结果与空白质粒组比，HOXA6过表达显著促进了肝癌HepG2细胞的增殖、侵袭和迁移（P值均<0.001）；TIMP3蛋白表达水平显著下降（P<0.001），而MMP9和MMP3表达水平均显著升高（P值均<0.001）。与siRNA阴性对照组比，干扰HOXA6表达显著抑制了肝癌HepG2细胞的增殖、侵袭和迁移（P值均<0.001）；TIMP3蛋白表达水平显著升高（P<0.001），MMP9和MMP3表达均显著下降（P值均<0.001）。流式细胞仪检测结果显示，与空白质粒组比，HOXA6过表达抑制了肝癌HepG2细胞的凋亡（P<0.001）；凋亡相关蛋白BAX表达下降而BCL2表达升高（P值均<0.001）。与siRNA阴性对照组相比，干扰HOXA6表达则促进了肝癌HepG2细胞的凋亡（P<0.001）；BAX表达升高而BCL2表达下降（P值均<0.001）。HOXA6过表达组p-AKT/AKT和p-PI3K/PI3K均显著高于空白质粒组（P值均<0.001），siRNA HOXA6干扰组p-AKT/AKT和p-PI3K/PI3K均显著低于siRNA阴性对照组（P值均<0.001）。结论 HOXA6通过磷酸化激活PI3K/AKT信号通路促进HepG2肝癌细胞增殖、侵袭和转移，抑制肝癌细胞凋亡。
- Hep G2细胞 /
- 基因，同源盒 /
- 细胞增殖 /
- 细胞凋亡
Abstract: Objective To investigate the effect of Homebox A6 （HOXA6） on the proliferation， invasion， metastasis， and apoptosis of HepG2 hepatoma cells and its association with the PI3K/AKT signaling pathway. Methods HepG2 hepatoma cells were cultured， and HOXA6 overexpression plasmid and siRNA were constructed and transfected into cells. The cells were randomly divided into empty plasmid group， HOXA 6 overexpression group， siRNA negative control group， and siRNA HOXA6 interference group. CCK8 assay was used to measure cell proliferation， Transwell assay was used to observe cell invasion， and wound healing assay was used to observe cell migration （related proteins TIMP3， MMP9， and MMP3）. Flow cytometry was used to measure cell apoptosis （related proteins BAX and BCL2）， the BCA method was used to measure protein concentration， and Western Blot was used to measure the expression of related proteins. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the SNK-q test was used for further comparison between two groups. Results Compared with the empty plasmid group， HOXA6 overexpression significantly promoted the proliferation， invasion， and migration of HepG2 hepatoma cells （all P<0.001）， and there was a significant reduction in the protein expression of TIMP3 （P<0.001）， while there were significant increases in the expression levels of MMP9 and MMP3 （both P<0.001）. Compared with the siRNA negative control group， HOXA6 interference significantly inhibited the proliferation， invasion， and migration of HepG2 hepatoma cells （all P<0.001）， and there was a significant increase in the protein expression of TIMP3 （P<0.001）， while there were significant reductions in the expression levels of MMP9 and MMP3 （both P<0.001）. Flow cytometry showed that compared with the empty plasmid group， HOXA6 overexpression inhibited the apoptosis of HepG2 hepatoma cells （P<0.001）， with a significant reduction in the expression of the apoptosis-related protein BAX and a significant increase in the expression of BCL2 （both P<0.001）. Compared with siRNA negative control group， HOXA6 interference promoted the apoptosis of HepG2 hepatoma cells （P<0.001）， with a significant increase in the expression of BAX and a significant reduction in the expression of BCL2 （both P<0.001）. Compared with the empty plasmid group， the HOXA6 overexpression group had significantly higher ratios of p-AKT/AKT and p-PI3K/PI3K （both P<0.001）， and compared with the siRNA negative control group， the siRNA HOXA6 interference group had significantly lower ratios of p-AKT/AKT and p-PI3K/PI3K （both P<0.001）. Conclusion HOXA6 can promote the proliferation， invasion， and metastasis of HepG2 hepatoma cells and inhibit their apoptosis by activating the PI3K/AKT signaling pathway through phosphorylation.
- Hep G2 Cells /
- Genes， Homeobox /
- Cell Proliferation /
- Apoptosis

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图 1 各组细胞不同培养时间点的OD值

Figure 1. OD values of cells in each group at different cultivation time points

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注： a，空白质粒组；b，HOXA6过表达组；c，siRNA阴性对照组；d，siRNA HOXA6干扰组。

图 2 各组细胞Transwell实验结果（结晶紫染色，×200）

Figure 2. Transwell detection results in each group

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图 3 各组细胞侵袭数量

Figure 3. Relative invaded cells in each group

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图 4 各组细胞划痕实验

Figure 4. Cell scratch-healing results in each group

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图 5 各组细胞迁移数量

Figure 5. Relative migrated cells in each group

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图 6 各组细胞TIMP3、MMP9和MMP3的蛋白表达情况

Figure 6. Protein expression of TIMP3， MMP9 and MMP3 in each group

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注： a，空白质粒组；b，HOXA6过表达组；c，siRNA阴性对照组；d，siRNA HOXA6干扰组。

图 7 各组HepG2肝癌细胞凋亡的检测结果

Figure 7. Detection results of cell apoptosis in each group

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图 8 各组HepG2肝癌细胞的凋亡率

Figure 8. Apoptotic rates of cells in each group

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图 9 各组细胞BAX和BCL2的蛋白表达情况

Figure 9. Protein expression of BAX and BCL2 in each group

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图 10 各组细胞AKT、p-AKT、PI3K和p-PI3K的蛋白表达情况

Figure 10. Protein expression of AKT， p-AKT， PI3K and p-PI3K in each group

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