## 4 Discussion  
In this study, we re-annotated the lncRNA chip of GSE53624 in GEO, which contained 119 normal tissues and matched cancer tissues and completed the follow-up information. Then, we obtained the expression profile of 28,885 genes and a total of 210 down-regulated and 80 up-regulated DEGs were obtained by R package \"limma\". To reveal the underlying molecular mechanisms of the DEGs, functional enrichment analysis, pathway enrichment analysis, and PPI analysis were performed. The PPI network analysis showed that the 10 DEGs with top degrees (ELN, MMP13, SPP1, MMP10, COL1A1, CSF2, MMP1, SERPINE1, CXCL8, and MMP3) may play central roles in ESCC. GO analysis showed that DEGs were mainly related to the extracellular matrix, such as extracellular matrix organization, extracellular constraint organization, extracellular matrix disassembly, and cytokine activity, which indicated that it is valuable for further study the DEGs and the key genes may become new diagnostic and therapeutic targets for ESCC. Pathway enrichment analysis revealed that DEGs enrichment of ESCC involved cytokine-cytokine receptor interaction, chemical carcinogenesis, ECM-receptor interaction, IL17 signaling pathway, and other related pathways. On the one hand, this finding confirmed that the DEGs play an essential role in ESCC's occurrence and development. On the other hand, it also suggested that the abnormal changes in the IL-17 signaling pathway might be of great significance in developing ESCC. The IL-17 signaling pathway is involved in the body's immune response [20] and inflammatory response [21]. Hence, we used GEPIA2 to explore the relationship between DEGs expression and immune markers and found that the DEG expression level was significantly correlated with immune cell markers in ESCC. Specifically, MMP10 expression level was significantly correlated with 12 out of 42 immune cell markers, MMP13 expression level was significantly correlated with 30 out of 42 immune cell markers, SPP1 expression level was significantly correlated with 26 out of 42 immune cell markers, and COL1A1 ex  
Figure 8: Validation of the DEGs by GEPIA2 and Western blot. (**a**) The top 10 genes expression in ESCA from GEPIA2 database (* \(p<0.05\)); (**b**) Expression of four key genes (SPP1, MMP10, MMP13 and COL1A1) evaluated by Western blot; (**c**) Western blot quantification (each protein expression was normalized against expression level of \(\beta\)-actin and Het-1A cell line, * \(p<0.05\), ** \(p<0.005\), *** \(p<0.0001\)). pression level was significantly correlated with 27 out of 42 immune cell markers (Table S5). Our results indicated that the immune response and inflammatory response may promote the development of ESCC. We further validated the expression of the DEGs through the online database GEPIA2 and Western blot. In the GEPIA2 database, COL1A1, CXCL8, SPP1, MMP13, SERPNE1, MMP1, MMP3, and MMP10 were significantly higher expressed. We also, based on the TCGA data, analyzed the expression levels of the MMP13, MMP10, COL1A1, and SPP1 in the main pathological stages of ESCA. Violin plots showed that stage II and stage III have higher gene expression levels than stage I, which suggested an association of increased key genes expression with tumor progression (Figure S3). The results of the Western blot further confirmed that the expression levels of SPP1, COL1A1, MMP13, and MMP10 were consistent with the expression trends in our bioinformatics analysis. SPP1, also known as osteopontin (OPN), is an ECM protein that has been involved in a range of physiological and pathological processes [22]. Studies have shown that SPP1 is overexpressed in a variety of tumors and can be involved in tumor cell proliferation, angiogenesis, chemotherapeutic resistance, migration, and invasion [22; 23; 24]. It is valuable in tumor diagnosis and prognosis prediction of breast [23; 24], lung [25], liver [26], ovarian [27], and bladder cancers [28]. In breast cancer, SPP1 activates the NF-kB signaling pathway [29].