背景:食管鳞状细胞癌(ESCC)是全球最常见的恶性肿瘤之一,其发展包括从上皮内瘤变(IN)到癌(CA)的多步骤过程。然而,关键的调节因子和潜在的分子机制在很大程度上仍然未知.
目的:探索与ESCC多阶段进展相关的微环境中的基因和浸润免疫细胞,以利于诊断和早期干预。
方法:通过向C57BL/6小鼠提供含有4-硝基喹啉1-氧化物(4NQO)的沃特,建立了模拟ESCC多阶段发展的小鼠模型。此外,我们建立了一个没有4NQO治疗小鼠的对照组。然后,对不同病理状态患者的食管组织进行转录组测序,包括低等级IN(LGIN),高级IN(HGIN),CA,和对照正常组织(NOR)样品。在LGIN中鉴定出差异表达基因(DEGs),HGIN,和CA组,并通过基因本体论和京都基因百科全书和基因组富集分析分析了DEGs的生物学功能。使用CIBERSORT算法检测免疫细胞浸润的模式。还进行了免疫组织化学(IHC)以验证我们的结果。最后,Luminex多重细胞因子分析用于测量小鼠的血清细胞因子水平。
结果:与NOR组相比,在LGIN中总共获得681541和840个DEG,HGIN,和CA组,分别。使用三组DEG的交点,我们确定了86个基因是参与ESCC发生发展的关键基因。富集分析显示,这些基因主要富集在角质化,表皮细胞分化,和白细胞介素(IL)-17信号通路。CIBERSORT分析显示,与NOR组相比,4NQO组M0和M1巨噬细胞显示更强的浸润,经IHC验证。血清细胞因子分析显示,与NOR组相比,IL-1β和IL-6上调,而IL-10在LGIN中下调,HGIN,和CA组。此外,代表性关键基因的表达,例如S100a8和Krt6b,在外部人体样本中得到验证,免疫组织化学染色结果与小鼠的结果一致。
结论:我们鉴定了一组以S100a8和Krt6b为代表的关键基因,并研究了它们潜在的生物学功能。此外,我们发现巨噬细胞浸润和炎症相关细胞因子水平的异常改变,如IL-1β,外周血中的IL-6和IL-10可能与ESCC的发生发展密切相关。
BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide, and its development comprises a multistep process from intraepithelial neoplasia (IN) to carcinoma (CA). However, the critical regulators and underlying molecular mechanisms remain largely unknown.
OBJECTIVE: To explore the genes and infiltrating immune cells in the microenvironment that are associated with the multistage progression of ESCC to facilitate diagnosis and early intervention.
METHODS: A mouse model mimicking the multistage development of ESCC was established by providing warter containing 4-nitroquinoline 1-oxide (4NQO) to C57BL/6 mice. Moreover, we established a control group without 4NQO treatment of mice. Then, transcriptome sequencing was performed for esophageal tissues from patients with different pathological statuses, including low-grade IN (LGIN), high-grade IN (HGIN), and CA, and controlled normal tissue (NOR) samples. Differentially expressed genes (DEGs) were identified in the LGIN, HGIN, and CA groups, and the biological functions of the DEGs were analyzed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The CIBERSORT algorithm was used to detect the pattern of immune cell infiltration. Immunohistochemistry (IHC) was also conducted to validate our results. Finally, the Luminex multiplex cytokine analysis was utilized to measure the serum cytokine levels in the mice.
RESULTS: Compared with those in the NOR group, a total of 681541, and 840 DEGs were obtained in the LGIN, HGIN, and CA groups, respectively. Using the intersection of the three sets of DEGs, we identified 86 genes as key genes involved in the development of ESCC. Enrichment analysis revealed that these genes were enriched mainly in the keratinization, epidermal cell differentiation, and interleukin (IL)-17 signaling pathways. CIBERSORT analysis revealed that, compared with those in the NOR group, M0 and M1 macrophages in the 4NQO group showed stronger infiltration, which was validated by IHC. Serum cytokine analysis revealed that, compared with those in the NOR group, IL-1β and IL-6 were upregulated, while IL-10 was downregulated in the LGIN, HGIN, and CA groups. Moreover, the expression of the representative key genes, such as S100a8 and Krt6b, was verified in external human samples, and the results of immunohistochemical staining were consistent with the findings in mice.
CONCLUSIONS: We identified a set of key genes represented by S100a8 and Krt6b and investigated their potential biological functions. In addition, we found that macrophage infiltration and abnormal alterations in the levels of inflammation-associated cytokines, such as IL-1β, IL-6, and IL-10, in the peripheral blood may be closely associated with the development of ESCC.