ATOX1通过JAK2/STAT3通路促进肝癌细胞生物学行为的机制探讨

doi:10.11958/20240221

天津医药 ›› 2024, Vol. 52 ›› Issue (9): 907-912.doi: 10.11958/20240221

ATOX1通过JAK2/STAT3通路促进肝癌细胞生物学行为的机制探讨

马佳佳¹^,²^,³(), 张亚苹¹^,²^,³, 杨斌²^,³, 赵美琪²^,⁴, 蒋璐⁵^,⁶, 黄小玉²^,⁴, 范路畅¹^,²^,³, 王凤梅⁵^,⁶^,^△()

1 天津医科大学三中心临床学院（邮编300170）
2 天津市重症疾病体外生命支持重点实验室
3 天津市肝胆疾病研究所
4 南开大学医学院
5 天津市第一中心医院，天津医科大学一中心临床学院
6 天津市肝癌分子诊断与治疗重点实验室

收稿日期:2024-02-21 修回日期:2024-04-17 出版日期:2024-09-15 发布日期:2024-09-06
通讯作者: ^△E-mail：wangfengmeitj@126.com
作者简介:马佳佳（1996），女，硕士在读，主要从事肝脏肿瘤方面研究。E-mail：Majiajia@tmu.edu.cn
基金资助:
天津市卫生健康委科技重点学科专项(TJWJ2022XK027);天津市医学重点学科（专科）建设项目(TJYXZDXK-034A)

Mechanism study of ATOX1 promoting biological behavior of hepatocellular carcinoma cells through JAK2/STAT3 pathway

MA Jiajia¹^,²^,³(), ZHANG Yaping¹^,²^,³, YANG Bin²^,³, ZHAO Meiqi²^,⁴, JIANG Lu⁵^,⁶, HUANG Xiaoyu²^,⁴, FAN Luchang¹^,²^,³, WANG Fengmei⁵^,⁶^,^△()

1 The Third Central Clinical College of Tianjin Medical University, Tianjin 300170, China
2 Tianjin Key Laboratory of Extracorporeal Life Support for Critical Diseases
3 Tianjin Institute of Hepatobiliary Disease
4 School of Medicine, Nankai University
5 Tianjin First Central Hospital, the First Central Clinical College of Tianjin Medical University
6 Tianjin Key Laboratory of Molecular Diagnosis and Treatment of Liver Cancer

Received:2024-02-21 Revised:2024-04-17 Published:2024-09-15 Online:2024-09-06
Contact: ^△E-mail：wangfengmeitj@126.com

马佳佳, 张亚苹, 杨斌, 赵美琪, 蒋璐, 黄小玉, 范路畅, 王凤梅. ATOX1通过JAK2/STAT3通路促进肝癌细胞生物学行为的机制探讨[J]. 天津医药, 2024, 52(9): 907-912.

MA Jiajia, ZHANG Yaping, YANG Bin, ZHAO Meiqi, JIANG Lu, HUANG Xiaoyu, FAN Luchang, WANG Fengmei. Mechanism study of ATOX1 promoting biological behavior of hepatocellular carcinoma cells through JAK2/STAT3 pathway[J]. Tianjin Medical Journal, 2024, 52(9): 907-912.

摘要/Abstract

摘要：

目的探究肝细胞癌（HCC）中抗氧化剂1铜伴侣蛋白（ATOX1）表达的临床意义及其与肿瘤细胞增殖、迁移和侵袭的关系。方法分析人类基因组图谱数据库HCC癌组织和正常肝组织ATOX1 mRNA的表达。采用免疫组织化学染色检测15例HCC癌和癌旁组织中ATOX1的表达。人肝癌细胞株Hep3B和HepG2分为对照组（NC组）、ATOX1敲低1组（si-ATOX1#1组）和ATOX1敲低2组（si-ATOX1#2组）。通过CCK-8细胞增殖实验、划痕实验、Transwell侵袭实验观察敲低ATOX1对癌细胞恶性生物学行为的影响。构建裸鼠异种皮下移植瘤模型，分析敲低ATOX1表达对移植瘤质量和体积的影响。Western blot检测移植瘤中Janus激酶2/信号转导和转录激活因子3（JAK2/STAT3）通路蛋白表达水平。结果生物信息学分析显示HCC癌组织中ATOX1 mRNA表达高于癌旁组织（P<0.05）。免疫组化染色发现HCC癌组织中ATOX1蛋白阳性率高于癌旁组织（93.33% vs. 13.33%，P<0.01）。体外实验结果显示，siRNA敲低Hep3B、HepG2细胞中ATOX1蛋白表达后，癌细胞的增殖、迁移及侵袭能力均显著降低（均P<0.05）。小鼠体内实验中，sh-ATOX1敲低组裸鼠皮下移植瘤的体积和质量均显著小于sh-con组，JAK2/STAT3通路相关蛋白p-JAK2、p-STAT3、细胞周期蛋白D1（CyclinD1）及基质金属蛋白酶2表达均下降。结论 ATOX1能够通过JAK2/STAT3通路促进HCC的增殖、迁移和侵袭，可能成为潜在的肿瘤标志物和治疗靶点。

关键词: 癌, 肝细胞, 细胞运动, 生物标记, 肿瘤, 抗氧化剂1铜伴侣蛋白, JAK2/STAT3通路

Abstract:

Objective To investigate the clinical significance of the expression of antioxidant 1 copper chaperone protein (ATOX1) in hepatocellular carcinoma (HCC) and its relationship with tumor proliferation, migration and invasion. Methods The expression of ATOX1 mRNA in HCC cancer tissue and normal liver tissue was analyzed using the Human Genome Atlas database. Immunohistochemical experiment was used to detect the expression of ATOX1 in 15 cases of HCC cancer tissue and adjacent tissue. Human HCC cell lines Hep3B and HepG2 were divided into the control group (NC), the ATOX1 knockdown group 1 (si-ATOX1#1) and the ATOX1 knockdown group 2 (si-ATOX1#2). The effects of ATOX1 knockdown on the malignant biological behavior of HCC cells were observed through CCK-8 cell proliferation experiment, scratch experiment and Transwell invasion experiments. A nude mouse xenograft tumor model was constructed to analyze the effect of ATOX1 knockdown on the quality and volume of transplanted tumors. Western blot assay was used to detect the relationship between ATOX1 and JAK2/STAT3 pathway protein expression. Results Bioinformatics analysis showed that expression of ATOX1 mRNA in HCC cancer tissue was higher than that in adjacent normal tissue (P<0.05). The immunohistochemical staining results showed that the positive rate of ATOX1 protein was higher in HCC cancer tissue than that in adjacent tissue (93.33% vs. 13.33%, P<0.01). In vitro experimental results showed that siRNA knockdown of ATOX1 protein expression in Hep3B and HepG2 cells significantly reduced the proliferation, migration and invasion abilities of cancer cells (P<0.05). In vivo experiments in mice showed that the volume and weight of subcutaneous xenograft tumors were significantly smaller in the sh-ATOX1 group than those in the sh-con group (P<0.05). The expression levels of JAK2/STAT3 pathway-related proteins p-JAK2, p-STAT3, CyclinD1 and MMP2 were significantly lower in the subcutaneous transplanted tumor tissue of the sh-ATOX1 group than that of the sh-con group (P<0.05). Conclusion ATOX1 can promote the proliferation, migration and invasion of HCC through JAK2/STAT3 pathway, which can potentially become a potential tumor marker and therapeutic target.

Key words: carcinoma, hepatocellular, cell movement, biomarkers, tumor, antioxidant 1 copper chaperone protein, JAK2/STAT3 pathway

中图分类号:

R735.7

图/表 12

图1 免疫组化染色检测HCC癌及癌旁组织中ATOX1蛋白表达（×400）

Fig.1 Detection of ATOX1 protein expression in HCC cancerous tissue and adjacent tissue by immunohistochemical staining (× 400)

图2 Western blot实验验证siRNA转染后ATOX1表达效率

Fig.2 Western blot assay verified the expression efficiency of ATOX1 after siRNA transfection

图3 CCK-8实验检测敲低ATOX1后HepG2和Hep3B细胞增殖能力的变化

Fig.3 Changes of proliferation capacity of HepG2 and Hep3B cells after ATOX1 knockdown detected by CCK-8 assay ＊P<0.05，＊＊P<0.01。HepG2：F12 h=1.590，F24 h=1.991，F36 h=15.830＊＊，F48 h=33.380＊＊；Hep3B：F12 h=137.290＊＊，F24 h=111.591＊＊，F36 h=9.960＊，F48 h=24.541＊＊。

图4 敲低ATOX1后HepG2 和Hep3B细胞的迁移能力变化

Fig.4 Changes in migration ability of HepG2 and Hep3B cells after ATOX1 knockdown

表1 各组HepG2和Hep3B细胞ATOX1蛋白表达水平比较（n=6，$\bar{x}±s$）

Tab.1 Comparison of ATOX1 expression in HepG2 and Hep3B cells between the three groups

组别	ATOX1蛋白（HepG2）	ATOX1蛋白（Hep3B）
NC组	0.74±0.02	0.49±0.04
si-ATOX1#1组	0.52±0.01^a	0.16±0.02^a
si-ATOX1#2组	0.38±0.01^a	0.18±0.01^a
F	998.001^＊＊	293.431^＊＊

表2 各组HepG2和Hep3B细胞迁移和侵袭能力比较（n=6，$\bar{x}±s$）

Tab.2 Comparison of migration and invasion ability of HepG2 and Hep3B cells between the three groups

组别	Hep3B		HepG2
组别	划痕愈合率/%	侵袭细胞数/ （个/视野）	划痕愈合率/%	侵袭细胞数/ （个/视野）
NC组	61.34±4.45	257.68±34.36	53.54±4.50	188.35±22.21
si-ATOX1#1组	34.47±3.20^a	113.42±12.45^a	32.30±3.04^a	156.78±14.53^a
si-ATOX1#2组	37.67±2.25^a	106.23±13.42^a	28.78±2.11^a	146.98±12.16^a
F	110.450^＊＊	86.691^＊＊	187.672^＊＊	9.870^＊＊

图5 Transwell实验检测敲低ATOX1对HepG2和Hep3B癌细胞侵袭能力的影响

Fig.5 Effects of ATOX1 knockdown on the invasion of HepG2 and Hep3B cancer cells detected by Transwell assay

图6 Western blot实验检验sh-ATOX1的敲低效率

Fig.6 The knockdown efficiency of sh-ATOX1 by Western blot assay

图7 sh-ATOX1组和对照组成瘤6周后皮下移植瘤的大体表现 A：sh-con组；B：sh-ATOX1敲低组。

Fig.7 Comparison of the gross appearance of subcutaneous transplanted tumors 6 weeks after tumor formation between the sh-ATOX1 group and the NC group

图8 sh-ATOX1组和sh-con组成瘤6周后皮下移植瘤的肿瘤质量和肿瘤体积比较 A：肿瘤质量比较；B：肿瘤体积比较：t9 d=0.121，t12 d=0.381，t15 d=5.605＊＊，t18 d=7.077＊＊，t21 d=9.189＊＊，t 24 d=7.071＊＊，＊＊P<0.01。

Fig.8 Comparison of subcutaneous transplanted tumor mass and tumor volume after 6 weeks between the sh-ATOX1 group and the sh-con group

图9 Western blot实验检验敲低ATOX1对JAK2/STAT3通路蛋白表达的影响

Fig.9 The effect of ATOX1 knockdown on the JAK2/STAT3 pathway detected by Western blot assay

表3 各组小鼠肿瘤组织中ATOX1及JAK2/STAT3通路相关蛋白表达水平比较（n=6，$\bar{x}±s$）

Tab.3 Comparison of expression levels of ATOX1 and JAK2/STAT3 pathway related proteins between the two groups of tumor tissue

组别		ATOX1		Cyclin D1		MMP2
sh-con组		1.10±0.21		1.22±0.22		1.33±0.33
sh-ATOX1组		0.43±0.11		0.63±0.17		0.79±0.20
t		6.803^＊＊		5.097^＊＊		3.390^＊＊
组别	p-JAK2		JAK2		p-STAT3		STAT3
sh-con组	1.23±0.21		1.13±0.23		1.22±0.30		1.03±0.22
sh-ATOX1组	0.66±0.17		1.20±0.22		0.45±0.16		1.00±0.13
t	5.081^＊＊		0.601		5.848^＊＊		0.286

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ATOX1通过JAK2/STAT3通路促进肝癌细胞生物学行为的机制探讨

Mechanism study of ATOX1 promoting biological behavior of hepatocellular carcinoma cells through JAK2/STAT3 pathway

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