基于转录组学和蛋白质组学技术分析缺氧对肝细胞癌肿瘤微环境的影响

An analysis of the effect of hypoxia on the tumor microenvironment of hepatocellular carcinoma based on transcriptomics and proteomics techniques

目的探讨缺氧对肝细胞癌(hepatocellular carcinoma,HCC)肿瘤微环境的影响,为HCC患者的诊治提供指导。方法收集2018年5月至2019年7月在广西医科大学附属肿瘤医院行根治性肝切除术的116例HCC患者组织样本及相应的临床病理资料,基于HALLMARK-HYPOXIA基因集和单样本基因集富集分析方法计算HCC患者的缺氧评分,并使用转录组测序数据和深度学习模型检测的免疫组化染色结果对评分结果进行验证。通过Log-rank检验、单因素和多因素Cox比例风险模型及卡方检验分析缺氧评分与HCC患者预后和临床病理参数的关系。利用转录组学和蛋白质组学技术探讨高、低缺氧评分HCC患者的分子特征和肿瘤微环境异质性。结果根据缺氧评分将116例HCC患者分为高缺氧评分组(n=58)和低缺氧评分组(n=58)。与低缺氧评分组相比,高缺氧评分组的缺氧诱导因子1α(hypoxia-inducible factor1α,HIF-1α)表达水平明显升高(P<0.001);HCC患者的缺氧评分与HIF-1α的表达水平呈正相关(r=0.672,P<0.001)。高缺氧评分组患者的总生存期(P=0.001)和无复发生存期(P=0.006)均明显低于低缺氧评分组患者,且高缺氧评分与HCC患者更高的巴塞罗那分期(P=0.040)和肿瘤复发率(P=0.020)有关。高缺氧评分是HCC患者预后不良的独立因素(HR=2.074,95%CI:1.036~4.153,P=0.040)。高缺氧评分组患者主要富集调控HCC干细胞的信号通路(包括Wnt、Hedgehog、TGF-β等信号通路),且该组患者的肝癌干细胞相关标志物(包括CD24、CD44、CXCL12、EpCAM、ICAM1、KRT19、PROM1和THY1)表达上调。高缺氧评分组和低缺氧评分组的肿瘤微环境细胞组成存在异质性,且高缺氧评分组患者的CD45-CD326+类肿瘤干细胞亚群和CD45+CD326+恶性双阳性细胞亚群的HCC干细胞相关标志物(包括CD54、CK19、CD34、OV6、CD13、CD133)表达水平较低缺氧评分组更高。结论高缺氧评分患者的生存预后较低缺氧评分患者差且具有更强的肿瘤干性特征的肿瘤微环境。

Objective To investigate the effect of hypoxia on the tumor microenvironment in hepatocellular carcinoma(HCC),providing the guidance for the diagnosis and treatment of HCC patients.Methods Tissue samples and corresponding clinicopathological data of 116 HCC patients who underwent radical hepatectomy in Guangxi Medical University Cancer Hospital from May 2018 to July 2019 were collected.The hypoxia scores of HCC patients were calculated based on the HALLMARK‐HYPOXIA gene set and single‐sample gene set enrichment analysis,and the scores were validated by using transcriptome sequencing data and immunohistochemical staining results detected by a deep learning model.The relationship between the hypoxia score and the prognostic and clinicopathological parameters was analyzed by Log‐rank,univariable and multivariable Cox proportional hazards model,and chi‐square test.The molecular features and tumor microenvironment heterogeneity of HCC patients with high hypoxia scores and low hypoxia scores were analyzed by using transcriptomics and proteomics techniques.Results 116 HCC patients were divided into a high hypoxia score group(n=58)and a low hypoxia score(n=58)group according to the hypoxia score.Compared with the low hypoxia score group,the high hypoxia score group showed a significantly higher expression level of hypoxia‐inducible factor 1α(HIF‐1α)(P<0.001),and the hypoxia score of HCC patients was positively correlated with the expression level of HIF‐1α(r=0.672,P<0.001).The overall survival(P=0.001)and recurrence‐free survival(P=0.006)of the patients in the high hypoxia score group were significantly lower than those in the low hypoxia score group,and the high hypoxia score was associated with higher Barcelona stage(P=0.040)and tumor recurrence rate(P=0.020)in HCC patients.High hypoxia score was an independent factor of poor prognosis in HCC patients(HR=2.074,95%CI:1.036-4.153,P=0.040).The high hypoxia score mainly enriched in the signaling pathways regulating HCC stem cells(including Wnt,Hedgehog,TGF‐βand other signaling pathways)and the expression levels of liver cancer cells related markers(including CD24,CD44,CXCL12,EpCAM,ICAM1,KRT19,PROM1 and THY1)were up‐regulated in this group.The cell compositions of tumor microenvironment in high and low hypoxia groups were heterogeneous,and the expression levels of HCC stem cell‐associated markers(including CD54,CK19,CD34,OV6,CD13,and CD133)in the CD45-CD326+tumor stem cell subsets and CD45+CD326+malignant double positive cell subsets in the high hypoxia group were higher than those in the low hypoxia group.Conclusions The patients with high hypoxia scores have a poorer survival prognosis than those with low hypoxia scores,and have a tumor microenvironment with stronger tumor stem characteristics.