UROC1在肝细胞癌中的表达及对肿瘤发生的影响

Expression of UROC1 in hepatocellular carcinoma and its effect on tumor development

目的 探究尿刊酸酶(UROC1)在肝细胞癌(HCC)中的表达及其对HCC发生发展的影响。方法 分析蛋白质质谱和TCGA数据库中UROC1在癌和癌旁组织中的表达及预后数据。利用免疫组织化学染色、实时定量PCR(qPCR)和Western blot实验验证HCC中UROC1表达情况。利用HE染色判断肿瘤分化水平,分析UROC1表达水平与其相关性。构建稳定过表达UROC1和其突变体的Hep3B、LM3细胞系,开展体外细胞表型实验。通过细胞计数试剂盒8(CCK-8)、平板克隆实验、EdU实验、划痕实验和Transwell实验探究UROC1对HCC细胞增殖和迁移的影响。结果 TCGA数据库分析结果显示,与癌旁组织相比,UROC1在HCC组织中普遍下调(P<0.000 1),UROC1低表达组较高表达组的患者预后更差(P<0.05)。免疫组织化学染色及评分、qPCR和Western blot实验验证了UROC1在HCC中低表达。不同分化水平癌组织的免疫组织化学染色结果提示分化越差的HCC组织中UROC1表达水平越低(P<0.05)。平板克隆实验结果显示,过表达UROC1的细胞克隆形成数目较对照组明显减少。CCK-8和EdU染色实验结果显示,UROC1过表达的细胞增殖速度显著低于对照组(P<0.001)。划痕实验结果显示,UROC1过表达的HCC细胞迁移距离明显小于对照组细胞(P<0.000 1)。Transwell实验显示,过表达UROC1后细胞穿过小室的数目显著减少(P<0.000 1)。但是UROC1的活性位点突变后上述实验表型的结果和对照组趋于一致。结论 UROC1在HCC组织中低表达,在HCC细胞中过表达UROC1可能对细胞增殖和迁移能力有抑制作用。

Objective To investigate the expression of urocanase domain containing 1(UROC1)in Hepatocellular carcinoma(HCC)and its effect on the development of HCC.Methods UROC1 expression and prognostic data in tumor and para-tumor tissues from protein mass spectrometry and TCGA database were analyzed.Immunohistochemical staining,real time quantitative polymerase chain reaction(qPCR)and Western blot experiments were utilized to verify the expression of UROC1 in HCC.The effect of UROC1 expression level on tumor differentiation was analyzed by HE staining to determine the tumor differentiation level.Hep3B and LM3 cell lines stably overexpressing UROC1 and its mutants were constructed for in vitro phenotyping experiments.Effects of UROC1 on HCC cell proliferation and migration were explored by cell counting kit-8(CCK-8)assay,colony formation,EdU assay,scratch assay and Transwell assay.Results The results of database analysis showed that UROC1 was generally downregulated in HCC tissues,and patients in the UROC1 low-expression group had a worse prognosis.Immunohistochemical staining and scoring,qPCR and Western blot experiments verified the low expression of UROC1 in HCC.Immunohistochemical staining of tumor tissues with different differentiation levels demonstrated that the poorer the differentiation of HCC tissues,the lower the expression level of UROC1.CCK-8,colony formation and EdU assays suggested that overexpression of UROC1 inhibited the proliferation of HCC cells.The results of scratch and Transwell assays showed that overexpression of UROC1 inhibited the migration of HCC cells.However,the results of the above experimental phenotypes after active site mutation converged with those of the control group.Conclusion UROC1 is lowly expressed in HCC tissues,and overexpression of UROC1 in HCC cells may inhibit the ability of cell proliferation and migration.