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

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。

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.