锚蛋白重复序列22（ANKRD22）对人肝癌细胞的影响及其机制

蔡浚哲; 刘松柏; 费晓斌; 刘鹏; 朱昌毫; 王兴; 潘耀振

doi:10.12449/JCH240519

锚蛋白重复序列22（ANKRD22）对人肝癌细胞的影响及其机制

DOI: 10.12449/JCH240519

蔡浚哲¹,
刘松柏²,
费晓斌¹,
刘鹏³,
朱昌毫⁴,
王兴^{1, 4},
潘耀振^{1, 4, , ,}

1.
贵州医科大学临床医学院，贵阳 550000
2.
贵州医科大学附属白云医院肝胆外科，贵阳 550000
3.
贵州医科大学附属医院肝胆外科，贵阳 550000
4.
贵州医科大学附属肿瘤医院肝胆外科，贵阳 550000

基金项目:

国家自然科学基金 (81960431);

国家自然科学基金 (81960535);

贵州省科技计划项目 (QKHZC［2021］YB444)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：蔡浚哲、刘松柏、费晓斌负责实验操作；刘鹏、朱昌毫负责资料收集与数据分析；王兴负责课题设计；潘耀振指导撰写论文并最后定稿。

详细信息

通信作者:
潘耀振， panyaozhen@gmc.edu.cn （ORCID： 0000-0002-9300-382X）

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- 被引次数: 0
出版历程
- 收稿日期: 2023-10-13
- 录用日期: 2023-11-17
- 出版日期: 2024-05-25

Effect of ankyrin-repeat domain-containing protein 22 on human hepatoma cells and its mechanism

Junzhe CAI¹,
Songbai LIU²,
Xiaobin FEI¹,
Peng LIU³,
Changhao ZHU⁴,
Xing WANG^{1, 4},
Yaozhen PAN^{1, 4
, ,
,}

1.
College of Clinical Medicine，Guizhou Medical University，Guiyang 550000，China
2.
Department of Hepatobiliary Surgery，The Affiliated Baiyun Hospital of Guizhou Medical University，Guiyang 550000，China
3.
Department of Hepatobiliary Surgery，The Affiliated Hospital of Guizhou Medical University，Guiyang 550000，China
4.
Department of Hepatobiliary Surgery，The Affiliated Cancer Hospital of Guizhou Medical University，Guiyang 550000，China

Research funding:

National Natural Science Foundation of China (81960431);

National Natural Science Foundation of China (81960535);

Science and Technology Fund of Guizhou Province (QKHZC［2021］YB444)

More Information

Corresponding author: PAN Yaozhen， panyaozhen@gmc.edu.cn （ORCID： 0000-0002-9300-382X）

摘要

摘要: 目的探讨锚蛋白重复序列22（ANKRD22）对人肝癌细胞增殖、侵袭和迁移的影响及其分子机制。方法通过TCGA数据库分析正常肝组织及肝细胞癌组织中ANKRD22的表达水平及其与预后的关系。通过qRT-PCR和Western Blot检测人正常肝细胞（L-02）和人肝癌细胞系（Huh7、Hep G2、MHCC-97H、SK-HEP-1、SMMC-7721）中ANKRD22的表达情况。通过CCK-8、EdU、划痕实验及Transwell检测ANKRD22对肝癌细胞增殖、侵袭和迁移能力的影响。通过Western Blot检测ANKRD22与细胞周期蛋白、EMT相关蛋白之间的关系。通过KEGG、ssGSEA分析进一步探究ANKRD22在肝癌细胞中的作用机制，并进行实验验证。计量资料两组间比较采用成组t检验，多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果 TCGA数据库中ANKRD22在肝细胞癌组织中较正常肝组织高表达（t=5.083，P<0.05），且ANKRD22高表达患者的总生存期及疾病相关生存期均显著低于ANKRD22低表达的患者（P值均<0.05）。肝癌细胞系中ANKRD22的表达量均高于正常肝细胞（P值均<0.05）。增殖实验结果提示，ANKRD22过表达组的EdU阳性率、增殖速度均高于空载对照（Vector）组（t值分别为19.60、6.72，P值均<0.001）；si-ANKRD22#2组及si-ANKRD22#3组的EdU阳性率、增殖速度较si-NC组均降低（P值均<0.001）。Cyclin E1、Cyclin D1、CDK7、CDK4在过表达组中表达高于Vector组（t值分别为3.54、4.95、6.34、5.19，P值均<0.01）；在si-ANKRD22#2组及si-ANKRD22#3组中表达均低于si-NC组（P值均<0.001）。P27在过表达组中表达低于Vector组（t=6.12，P<0.001），在si-ANKRD22#2组及si-ANKRD22#3组中表达均高于si-NC组（P值均<0.001）。侵袭、迁移实验结果提示，ANKRD22过表达组的迁移速度、穿膜数量（迁移组和侵袭组）均高于Vector组（t值分别为5.01、25.60、3.67，P值均<0.05）；si-ANKRD22#2组及si-ANKRD22#3组的迁移速度、穿膜数量（迁移组和侵袭组）较si-NC组均降低（P值均<0.01）。N-cadherin、Vimentin、Snail在过表达组中表达高于Vector组（t值分别为12.13、8.85、13.97，P值均<0.001），在si-ANKRD22#2组及si-ANKRD22#3组中表达均低于si-NC组（P值均<0.001）；E-cadherin在过表达组中表达低于Vector组（t=4.98，P<0.01），在si-ANKRD22#2组及si-ANKRD22#3组中表达均高于si-NC组（P值均<0.001）。KEGG富集分析及ssGSEA分析提示，ANKRD22在肝细胞癌中与PI3K/AKT/mTOR信号通路相关，在过表达组中，p-AKT/AKT、p-PI3K/PI3K、p-mTOR/mTOR均较Vector组升高（t值分别为12.21、3.43、9.75，P值均<0.01）；在si-ANKRD22#2组及si-ANKRD22#3组中表达均低于si-NC组（P值均<0.001）。结论 ANKRD22在肝癌细胞中高表达，能促进肝癌细胞的增殖、侵袭和迁移能力，并且能促进PI3K/AKT/mTOR信号通路的激活。
- 癌，肝细胞 /
- 锚蛋白重复 /
- 细胞增殖 /
- 细胞运动
Abstract: Objective To investigate the effect of ankyrin-repeat domain-containing protein 22 （ANKRD22） on the proliferation， invasion， and migration of human hepatoma cells and its molecular mechanism. Methods The TCGA database was used to analyze the expression level of ANKRD22 in normal liver tissue and hepatocellular carcinoma tissue and its association with prognosis. Western Blot and qRT-PCR were used to measure the expression of ANKRD22 in human normal liver cells （L-02） and human hepatoma cells （Huh7， HepG2， MHCC-97H， SK-HEP-1， and SMMC-7721）； CCK-8 assay， EdU， wound healing assay， and Transwell assay were used to observe the effect of ANKRD22 on the proliferation， invasion， and migration of hepatoma cells； Western Blot was used to investigate the association of ANKRD22 with cyclins and EMT-related proteins； KEGG and ssGSEA analyses were performed to investigate the mechanism of action of ANKRD22 in hepatoma cells， and related experiments were conducted for validation. The independent-samples t-test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results In the TCGA database， the expression level of ANKRD22 in hepatoma tissue was significantly higher than that in normal liver tissue （t=5.083， P<0.05）， and the patients with a high expression level of ANKRD22 had longer overall survival and disease-related survival than those with a low expression level of ANKRD22 （P<0.05）. The expression level of ANKRD22 in various human hepatoma cell lines was higher than that in human normal liver cells （all P<0.05）. Cell proliferation assay showed that the ANKRD22 overexpression group had significantly higher EdU positive rate and proliferation rate than the Vector group （t=19.60 and 6.72， both P<0.001）， and compared with the si-NC group， the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower EdU positive rate and proliferation rate （all P<0.001）. Compared with the Vector group， the overexpression group had significantly higher expression levels of Cyclin E1， Cyclin D1， CDK7， and CDK4 （t=3.54， 4.95， 6.34， and 5.19， all P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）. The overexpression group had a significantly lower expression level of P27 than the Vector group （t=6.12， P<0.001）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group （both P<0.001）. Invasion and migration experiments showed that compared with the Vector group， the ANKRD22 overexpression group had significantly higher migration rate and number of crossings through the membrane （migration group and invasion group）（t=5.01， 25.60， and 3.67， all P<0.05）， and compared with the si-NC group， thesi-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower migration rate and number of crossings through the membrane （migration group and invasion group）（all P<0.01）. The overexpression group had significantly higher expression levels of N-cadherin， Vimentin， and Snail than the Vector group （t=12.13， 8.85， and 13.97， all P<0.001）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）； the overexpression group had a significantly lower expression level of E-cadherin than the Vector group （t=4.98， P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group （both P<0.001）. The KEGG enrichment analysis and the ssGSEA analysis showed that ANKRD22 was associated with the PI3K/AKT/mTOR signaling pathway in hepatocellular carcinoma， and the overexpression group had significantly higher expression levels of p-AKT/AKT， p-PI3K/PI3K， and p-mTOR/mTOR than the Vector group （t=12.21， 3.43， and 9.75， all P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）. Conclusion ANKRD22 is highly expressed in hepatoma cells and can promote the proliferation， invasion， and migration of hepatoma cells and the activation of the PI3K/AKT/mTOR signaling pathway.
- Carcinoma， Hepatocellular /
- Ankyrin Repeat /
- Cell Proliferation /
- Cell Movement

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图 1 ANKRD22在HCC中的表达情况

注： a，ANKRD22在正常肝组织与HCC组织中的表达水平；b，ANKRD22不同表达水平患者的总生存期；c，ANKRD22不同表达水平患者的疾病相关生存期；d，ANKRD22在正常肝细胞与肝癌细胞系中的mRNA水平；e，ANKRD22在正常肝细胞与肝癌细胞系中的蛋白表达水平。

Figure 1. Expression of ANKRD22 in hepatocellular carcinoma

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图 2 ANKRD22转染后的表达情况

注： a，ANKRD22在Vector组及过表达组MHCC-97H中的mRNA水平；b，ANKRD22在Vector组及过表达组MHCC-97H中的蛋白表达水平；c，ANKRD22在si-NC组及各敲低组Huh7中的mRNA水平；d，ANKRD22在si-NC组及各敲低组Huh7中的蛋白表达水平。

Figure 2. Expression of ANKRD22 after transfection

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图 3 ANKRD22对肝癌细胞增殖能力的影响

注： a，EdU增殖试验结果；b，EdU阳性率统计结果；c，CCK-8试验统计结果；d，各分组中周期蛋白表达情况。

Figure 3. Effect of ANKRD22 on proliferation of hepatocellular carcinoma cells

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图 4 ANKRD22对肝癌细胞侵袭、迁移能力的影响

注： a，划痕实验结果；b，划痕处迁移面积； c，Transwell实验结果；d，Transwell迁移细胞数；e，Transwell侵袭细胞数；f，各分组中EMT相关蛋白表达情况。

Figure 4. Effect of ANKRD22 on invasion and migration of hepatocellular carcinoma cells

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图 5 ANKRD22在肝癌细胞中的作用机制

注： a，ANKRD22在HCC中的KEGG富集分析情况；b，ANKRD22在HCC中与PI3K/AKT/mTOR信号通路的ssGSEA富集分析情况；c，各分组中PI3K/AKT/mTOR信号通路相关蛋白表达情况。

Figure 5. Mechanism of ANKRD22 in hepatocellular carcinoma cells

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