驱动蛋白家族成员15（KIF15）对肝细胞癌增殖能力的影响及其作用机制

仇建南; 汪鹏; 曹胤; 王忠夏; 吴俊华; 江春平

doi:10.12449/JCH240217

驱动蛋白家族成员15（KIF15）对肝细胞癌增殖能力的影响及其作用机制

DOI: 10.12449/JCH240217

仇建南¹,
汪鹏¹,
曹胤¹,
王忠夏¹,
吴俊华²,
江春平^1, , ,

1.
南京大学医学院附属鼓楼医院肝胆外科，南京 210008
2.
南京大学医学院基础医学部，南京 210093

基金项目:

国家自然科学基金 (81972888)

伦理学声明：本研究方案于2016年5月7日经由南京大学医学院附属鼓楼医院动物保护与福利委员会批准，批号：2016-057-01，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：仇建南负责实验设计与操作，并撰写论文；汪鹏、曹胤、王忠夏负责数据分析和文章润色；江春平、吴俊华提供课题思路，指导实验内容，审阅最终文稿。

详细信息

通信作者:
江春平， chunpingjiang@126.com （ORCID： 0000-0001-8256-5731）

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出版历程
- 收稿日期: 2023-05-29
- 录用日期: 2023-07-04
- 出版日期: 2024-02-19

Effect of kinesin family member 15 on the proliferation of hepatocellular carcinoma cells and its mechanism of action

1.
Department of Hepatobiliary Surgery，Affiliated Drum Tower Hospital of Nanjing University Medical School，Nanjing 210008，China
2.
Department of Basic Medicine，Nanjing University Medical School，Nanjing 210093，China

Research funding:

National Natural Science Foundation of China (81972888)

More Information

Corresponding author: JIANG Chunping， chunpingjiang@126.com （ORCID： 0000-0001-8256-5731）

摘要

摘要: 目的探究驱动蛋白家族成员15（KIF15）对肝细胞癌（HCC）增殖能力的影响及其作用机制。方法通过分析TCGA和GEPIA数据集确定KIF15在HCC中的表达情况以及对肿瘤分期、生存的影响。采用qRT-PCR和Western Blot检测体外培养的人源HCC细胞系（HepG2、Hep3B、MHCC-97H和LM3）与人正常肝细胞系L02细胞的KIF15表达水平，并选择Hep3B和HepG2进行后续研究。通过对Hep3B慢病毒转染sh-NC/sh-KIF15和对HepG2慢病毒转染LV-vector/LV-KIF15进行CCK-8、平板克隆和EdU染色实验评估细胞的活力和增殖能力。GSEA分析KIF15与HCC相关的作用信号通路并通过Western Blot进行检测。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和GEPIA数据集分析结果显示KIF15在HCC患者癌组织中的表达明显高于正常组织，并且KIF15与HCC分期成正比，KIF15高表达的HCC患者的生存更差。与sh-NC-Hep3B相比，sh3-Hep3B的KIF15 mRNA水平和蛋白水平均下降（P值均<0.05）；与sh-NC-Hep3B相比，sh3-Hep3B的细胞活力、克隆形成数和EdU阳性率均显著降低（P值均<0.05）。与vector-HepG2相比，LV-KIF15-HepG2的KIF15 mRNA水平和蛋白水平均升高（P值均<0.05）；与vector-HepG2相比，LV-KIF15-HepG2的细胞活力、克隆形成数和EdU阳性率均提高（P值均<0.05）。皮下瘤实验结果显示：与sh-NC-Hep3B相比，sh3-Hep3B的瘤体积和瘤重量降低；Ki67的组化评分降低，而TUNEL的组化评分提高（P值均<0.05）。GSEA分析显示在HCC中PI3K/AKT/mTOR通路与KIF15呈正相关（NES=1.59，P<0.001），Western Blot检测发现LY294002能够抑制LV-KIF15-HepG2中上调的PI3K/AKT/mTOR通路，与LV-KIF15-HepG2相比，LY294002+LV-KIF15-HepG2的细胞活力、克隆形成数和EdU阳性率降低（P值均<0.05）。结论 KIF15通过上调PI3K/AKT/mTOR信号通路增强HCC的活力和增殖能力。
- 癌，肝细胞 /
- 驱动蛋白 /
- 信号传导 /
- 细胞增殖
Abstract: Objective To investigate the effect of kinesin family member 15 （KIF15） on the proliferation of hepatocellular carcinoma （HCC） cells and its mechanism of action. Methods TCGA and GEPIA datasets were analyzed to determine the expression of KIF15 in HCC and its effect on tumor stage and survival. Quantitative real-time PCR and Western blot were used to measure the expression level of KIF15 in human-derived HCC cell lines （HepG2， Hep3B， MHCC-97H， and LM3） and human normal liver cell line L02 cultured in vitro， and Hep3B and HepG2 were selected for subsequent studies. CCK-8 assay， plate colony formation assay， and EdU staining were performed for Hep3B cells transfected with shRNA-NC or shRNA-KIF15 and HepG2 cells transfected with LV-vector or LV-KIF15 to evaluate the viability and proliferative capacity of these cells. GSEA was used to analyze the potential signaling pathways associated with KIF15 in HCC， and Western blot was used for detection. 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 The analysis of TCGA and GEPIA datasets showed that in HCC patients， the expression of KIF15 in HCC tissue was significantly higher than that in normal tissue， and the HCC patients with high KIF15 expression tended to have a poorer prognosis. Compared with sh-NC-Hep3B， sh3-Hep3B showed significant reductions in the mRNA and protein levels of KIF15 （P<0.05）， cell viability， clone formation number， and EdU positive rate （all P<0.05）. Compared with vector-HepG2， LV-KIF15-HepG2 showed significant increases in the mRNA and protein levels of KIF15 （P<0.05）， cell viability， clone formation number， and EdU positive rate （all P<0.05）. Subcutaneous tumor assay showed that compared with sh-NC-Hep3B， sh3-Hep3B showed reductions in tumor volume and tumor weight， as well as a significant reduction in the immunohistochemical score of Ki67 and a significant increase in the immunohistochemical score of TUNEL （P<0.05）. GSEA analysis showed that the PI3K/AKT/mTOR pathway was positively correlated with KIF15 in HCC （NES=1.59， P<0.001）. Western blot showed that LY294002 could inhibit the PI3K/AKT/mTOR pathway upregulated in LV-KIF15-HepG2， and compared with LV-KIF15-HepG2， LY294002+LV-KIF15-HepG2 showed significant reductions in cell viability， clone formation number， and EdU positive rate （all P<0.05）. Conclusion KIF15 enhances the viability and proliferative capacity of HCC cells by upregulating the PI3K/AKT/mTOR signaling pathway.
- Carcinoma， Hepatocellular /
- Kinesin /
- Signal Transduction /
- Cell Proliferation

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据估计，长期暴露于甲氨蝶呤(MTX)导致严重肝纤维化的风险约为5%，这促使人们采取强化监测策略。然而，现有证据来源于回顾性研究，这些研究低估了肝病的风险因素。在Atallah等一项纵向队列研究中，使用两种非侵入性标志物评估了长期MTX治疗对肝纤维化的风险。

在2014年—2021年，笔者从6个英国研究中心前瞻性招募了诊断为≥2年类风湿关节炎或银屑病的成年患者。MTX组包括接受MTX治疗≥6个月的患者，而未暴露组包括从未接受MTX的患者。所有的患者都进行了完整的肝脏分析，包括瞬时弹性成像(TE)和增强肝纤维化(ELF)标志物测量。

共纳入999例患者，平均(60.8±12)岁，女性占62.3%。在976例有效TE值中，149例(15.3%)患者肝硬度≥7.9 kPa。在892例有效ELF值中，262例(29.4%)患者ELF≥9.8。年龄和BMI与肝硬度升高和肝纤维化独立相关。MTX累积剂量和持续时间都与肝硬度升高无关。糖尿病是与肝硬度≥7.9 kPa相关性最显著的危险因素(校正比值比=3.19，95%可信区间：1.95~5.20，P＜0.001)。定期使用非甾体类抗炎药与ELF≥9.8的相关性最强(优势比=1.76，95%可信区间：1.20~2.56，P=0.003)，表明类风湿性关节炎的关节炎症程度可能与作为肝纤维化的非侵入性标志物的ELF相混淆。

笔者认为，先前可能高估了MTX本身导致的肝纤维化风险，有必要考虑修改MTX目前的监测准则。

摘译自ATALLAH E, GROVE JI, CROOKS C, et al. Risk of liver fibrosis associated with long-term methotrexate therapy may be overestimated[J]. J Hepatol, 2023. DOI: 10.1016/j.jhep.2022.12.034. [Oline ahead of print]

(吉林大学第一医院肝胆胰内科纪竹慧辛桂杰报道)

注： a、b为TCGA数据集中KIF15在HCC中的表达；c为TCGA数据集中KIF15与HCC分期的关系；d、e为qRT-PCR和Western Blot分析KIF15在HCC细胞株中的表达情况，与L02组比较，*P<0.001；f、g为TCGA和GEPIA数据集中KIF15表达与HCC生存的关系。

图 1 KIF15在HCC中的相对表达情况和对生存率的影响

Figure 1. The relative expression of KIF15 in hepatocellular carcinoma and its effect on survival rate

下载: 全尺寸图片幻灯片

注： a，qRT-PCR验证Hep3B细胞中敲低KIF15的效率，与sh-NC比较，*P<0.01；b，qRT-PCR验证HepG2细胞中过表达KIF15的效率；c，CCK-8实验分析Hep3B和HepG2细胞增殖活力；d，GEPIA数据集中KIF15在HCC中的表达与PCNA的相关性；e、f，克隆形成实验分析Hep3B和HepG2细胞克隆数；g、h，EdU实验分析Hep3B和HepG2细胞增殖能力。

图 2 KIF15对HCC增殖能力的影响

Figure 2. Effect of KIF15 on proliferation of hepatocellular carcinoma

下载: 全尺寸图片幻灯片

注： a，造模后皮下瘤瘤体照片；b，瘤体的体积；c，瘤体的重量；d、e，免疫组化染色分析Hep3B（sh-NC/sh3）瘤体组织中Ki67和TUNEL表达情况。

图 3 KIF15对HCC体内生长能力的影响

Figure 3. Effect of KIF15 on the growth capacity of hepatocellular carcinoma in vivo

下载: 全尺寸图片幻灯片

注： a，GSEA分析KIF15在HCC中潜在的作用通路；b，Western Blot验证Hep3B细胞中PI3K/AKT/mTOR通路表达情况；c、d，Western Blot验证HepG2细胞中PI3K/AKT/mTOR通路表达情况；e，CCK-8实验分析HepG2细胞增殖活力；f，克隆形成实验分析HepG2细胞克隆数；g，EdU实验分析HepG2细胞增殖活力。

图 4 KIF15对HCC增殖能力影响的机制探究

Figure 4. The mechanism of the effect of KIF15 on the proliferation of hepatocellular carcinoma

下载: 全尺寸图片幻灯片

表 1 Western Blot 检测KIF15蛋白在HCC细胞株中的表达水平

Table 1. The protein expression level of KIF15 in HCC cell lines by Western Blot

组别	KIF15
L02	1.00±0.02
HepG2	1.45±0.10^1）
LM3	5.22±0.39^1）
MHCC-97H	3.49±0.25^1）
Hep3B	7.69±0.61^1）
F值	192.40
P值	<0.001
注：与L02组比较，1）P<0.05。

下载: 导出CSV

表 2 Western Blot 检测KIF15蛋白在Hep3B中的表达水平

Table 2. The protein expression level of KIF15 in Hep3B by Western Blot

分组	KIF15
sh-NC	1.00±0.08
sh1	0.54±0.14^1）
sh2	0.66±0.08^1）
sh3	0.15±0.05^1）
F值	41.63
P值	<0.001
注：与sh-NC组比较，1）P<0.05。

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表 3 Western Blot 检测Hep3B细胞中相关蛋白表达结果

Table 3. Western Blot analysis was performed to determine the expression levels of related proteins in Hep3B cells

分组	KIF15	p-PI3K/PI3K	p-AKT/AKT	p-mTOR/mTOR
sh-NC	1.05±0.10	0.99±0.06	0.99±0.06	0.98±0.07
sh3	0.19±0.03	0.14±0.04	0.24±0.05	0.22±0.02
t值	13.88	19.66	16.71	17.48
P值	<0.001	<0.001	<0.001	<0.001

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表 4 Western Blot 检测HepG2细胞中相关蛋白表达结果

Table 4. Western Blot analysis was performed to determine the expression levels of related proteins in HepG2 cells

分组	KIF15	p-PI3K/PI3K	p-AKT/AKT	p-mTOR/mTOR
vector	1.00±0.06	1.04±0.10	1.02±0.06	0.96±0.04
KIF15	6.12±0.20	5.11±0.22	4.56±0.49	4.56±0.55
t值	41.97	29.29	12.46	11.24
P值	<0.001	<0.001	<0.001	<0.001

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表 5 LY294002抑制剂干预对HepG2细胞中相关蛋白表达的影响

Table 5. The effect of inhibitor LY294002 intervention on the expression of related proteins in HepG2 cells

分组	KIF15	p-PI3K/PI3K	p-AKT/AKT	p-mTOR/mTOR
vector	1.06±0.13	1.01±0.09	0.96±0.08	0.97±0.05
KIF15	5.68±0.35	4.64±0.57	4.70±0.51	4.82±0.37
KIF15+LY294002	5.42±0.25^1）	1.17±0.09^1）	1.07±0.12^1）	1.22±0.10^1）
F值	301.05	111.64	145.32	284.41
P值	<0.001	<0.001	<0.001	<0.001
注：与KIF15组比较，1）P<0.05。

下载: 导出CSV

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