环腺苷酸反应元件结合蛋白3样蛋白1在骨肉瘤组织的表达及其临床意义

Clinical significance of cAMP responsive element binding protein 3 like 1 expression in osteosarcoma

目的分析环腺苷酸反应元件结合蛋白3样蛋白1(CREB3L1)在骨肉瘤中的表达,探究CREB3L1在骨肉瘤中的作用及临床意义。方法收集2019年9月至2021年12月于郑州大学第一附属医院骨科住院并接受手术治疗的骨肉瘤患者组织5例,采用转录组测序分析差异表达基因;实时荧光定量聚合酶链反应(RT-qPCR)检测CREB3L1在另外12对匹配的骨肉瘤和癌旁组织中的表达;IHC验证CREB3L1表达;采用Kaplan-Meier方法分析公共数据中骨肉瘤队列中CREB3L1和患者预后的关系。组间比较采用t检验。结果对临床采集的5对匹配骨肉瘤和正常骨组织进行转录组测序分析。比较分析共发现1809个差异表达基因(|FC|>1.5,P<0.01),其中有1111个基因在骨肉瘤组织中表达升高,697个基因在骨肉瘤组织中表达下调;基因富集分析结果显示,差异表达的基因主要涉及细胞外基质组织、胶原蛋白分解代谢、细胞黏附等信号通路。参与胶原蛋白生物合成、修饰和降解,细胞外基质胶原纤维形成等多个通路的基因在骨肉瘤中显著富集,其中CREB3L1等在骨肉瘤中表达升高明显。RT-qPCR分析匹配的骨肉瘤及对应正常骨组织中CREB3L1的mRNA表达表明(n=12),大部分标本中(9/12)CREB3L1在骨肉瘤中的表达明显高于对应的正常骨组织(4.408±3.230比1.000±0.000,t=2.256,P<0.05);IHC表明,CREB3L1在骨肉瘤细胞核与细胞质的表达高于正常骨组织(细胞质:2.750±1.290比1.750±0.683,t=2.739,P<0.05;细胞核:2.125±0.806比1.250±0.775,t=3.130,P<0.01);Kaplan-Meier生存分析表明,CREB3L1的表达与骨肉瘤患者的总体生存率、无转移生存率负相关(P<0.01,P<0.001);通过比较有和无肺转移患者病例中CREB3L1细胞核IHC评分,CREB3L1在有肺部转移肿瘤细胞核内表达量高于非转移者(2.500±0.527比1.700±0.949,t=2.331,P<0.05)。结论CREB3L1在骨肉瘤中表达升高且与患者较差的生存预后密切相关。

Objective To analyze the expression of cAMP responsive element binding protein 3 like 1(CREB3L1)in osteosarcoma and to investigate the mechanism and clinical significance of CREB3L1 in osteosarcoma.Methods Paired tumor and precancerous bone tissues were collected from 5 patients with osteosarcoma who were hospitalized and surgically treated at the Department of Orthopedics,First Affiliated Hospital of Zhengzhou University from September 2019 to December 2021.RNA sequencing(RNA-seq)identified CREB3L1 as one of the most upregulated genes in osteosarcoma tissues compared with healthy bone tissues.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to validate CREB3L1 expression in additional 12 pairs of matched osteosarcoma and precancerous bone tissues.The protein expression of CREB3L1 was verified by immunohistochemistry(IHC).The prognostic value of CREB3L1 in osteosarcoma was assessed by Kaplan-Meier analysis using publicly available data set.T test was used for comparison between groups.Results RNA-seq analysis identified a total of 1809 differentially expressed genes between the precancerous and cancerous bone tissues,among which 1111 genes were up-expressed and 697 genes were down-expressed in osteosarcoma(|FC|>1.5,P<0.01).Gene enrichment analysis showed that the differentially expressed genes were mainly involved in signaling pathways of extracellular matrix remodeling,collagen catabolism and cell adhesion.Genes involved in collagen biosynthesis,modification and degradation,extracellular matrix collagen fibril formation pathways were significantly enriched in osteosarcoma.CREB3L1 expression was significantly increased in osteosarcoma.RT-qPCR analysis of additional 12 pairs of osteosarcoma samples showed that in most osteosarcoma specimens,CREB3L1 mRNA expression was significantly higher(4.408±3.230 vs.1.000±0.000,t=2.256,P<0.05)than that in precancerous bone tissues(9/12).IHC showed that the protein level of CREB3L1 was significantly increased in both the nucleus and cytoplasm of osteosarcoma tissues as compared with that in precancerous bone tissues(cytoplasm:2.750±1.290 vs.1.750±0.683,t=2.739,P<0.05;nucleus:2.125±0.806 vs.1.250±0.775,t=3.130,P<0.01).Kaplan-Meier survival analysis showed that CREB3L1 expression was inversely correlated with overall survival and metastasis-free survival of osteosarcoma patients(P<0.01,P<0.001).By comparing the nuclear immunohistochemistry scores of CREB3L1 between patients with and without lung metastasis,it was shown that the nuclear expression of CREB3L1 in tumor cells was significantly higher in patients with lung metastasis than those without lung metastasis(2.500±0.527 vs.1.700±0.949,t=2.331,P<0.05).Conclusion CREB3L1 is highly expressed in osteosarcoma and is closely associated with poor survival.