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

doi:10.11958/20240221

Abstract

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

CLC Number:

R735.7

Figures/Tables 12

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

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

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＊＊。

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

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^＊＊

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^＊＊

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

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

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

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

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

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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