Clin Exp Otorhinolaryngol.  2023 Feb;16(1):75-86. 10.21053/ceo.2022.01480.

SERPINE1 as an Independent Prognostic Marker and Therapeutic Target for Nicotine-Related Oral Carcinoma

• Guo X
• Sun Z
• Chen H
• Ling J
• Zhao H
• Chang A
• Zhuo X

• KMID: 2539771
• DOI: http://doi.org/10.21053/ceo.2022.01480

Abstract

Objectives
. Nicotine is an ingredient of tobacco, and exposure to nicotine increases the risks of various cancers, including oral cancer. Previous studies have focused on the addictive properties of nicotine, but its carcinogenic mechanism has rarely been studied. We aimed to explore the key genes in the process through which nicotine promotes the occurrence and development of oral cancer via data mining and experimental verification.
Methods
. This study involved three parts. First, key genes related to nicotine-related oral cancer were screened through data mining; second, the expression and clinical significance of a key gene in oral cancer tissues were verified by bioinformatics. Finally, the expression and clinical significance of the key gene in oral cancer were histologically investigated, and the effects of its expression on cell proliferation, invasion, and drug resistance were cytologically assessed.
Results
. SERPINE1 was identified as the key gene, which was upregulated in nicotine-treated oral cells and may be an independent prognostic factor for oral cancer. SERPINE1 was enriched in various pathways, such as the tumor necrosis factor and apelin pathways, and was related to the infiltration of macrophages, CD4+T cells, and CD8+T cells. Overexpression of SERPINE1 was associated with N staging and may be involved in hypoxia, angiogenesis, and metastasis. Knockdown of SERPINE1 in oral cancer cells resulted in weakened cell proliferation and invasion ability and increased sensitivity to bleomycin and docetaxel.
Conclusion
. This study revealed SERPINE1 as a key gene for nicotine-related oral cancer, indicating that SERPINE1 may be a novel prognostic indicator and therapeutic target for oral carcinoma.

Keyword

Nicotine; Mouth Neoplasm; Computational Biology; Neoplasm Metastasis; Prognosis

Figure

• 

  Fig. 1. (A) Heatmap of the differentially expressed genes (DEGs) screened from the GSE89923 dataset. The horizontal axis represents the names of the genes, and the right vertical axis represents the samples. Red represents the upregulated genes and blue represents the downregulated ones. (B, C) Enrichment analysis of the DEGs by Gene Ontology (GO; B) and Kyoto Encyclopedia of Genes and Genomes (KEGG; C). (D, E) The top 15 genes in a protein-protein interaction network of the DEGs are sorted by degree (D) and betweenness (E) values. (F) Venn analysis of the top 15 hub genes ranked by the degree and betweenness values, respectively. The intersection contained eight genes. IL, interleukin; TNF, tumor necrosis factor; TGF, transforming growth factor; NF, nuclear factor.

• 

  Fig. 2. (A) The expression of SERPINE1 in pan-cancer based on The Cancer Genome Atlas (TCGA) database. SERPINE1 expression was significantly higher in head and neck cancer (HNC) tissues than in normal tissues (P<0.001). (B, C) The survival curves showed that HNC patients with high SERPINE1 expression had a shorter overall survival time than those with low SERPINE1 expression (P<0.05). (B) TCGA. (C) GSE65858. (D) Multivariate Cox regression analysis with different variables indicated that SERPINE1 was an independent prognostic factor for HNC (P<0.05). (E) TNMplot presented that high expression of SERPINE1 was associated with metastasis (P<0.05). (F) The predicted targets of SERPINE1. The interaction plot showed 57 target genes that may be closely related to SERPINE1. (G, H) Gene Ontology (GO; G) and Kyoto Encyclopedia of Genes and Genomes (KEGG; H) enrichment analyses of the 57 genes. TPM, transcripts per million; HR, hazard ratio; CI, confidence interval; pT, pathological tumor; pN, pathological N; pTNM, pathological tumor-node-metastasis; TGF, transforming growth factor. *P<0.05; **P<0.01; ***P<0.001.

• 

  Fig. 3. (A) The association between SERPINE1 expression and tumor purity, as well as the infiltration levels of several immune cells (TIMER algorithm). (B) Analysis of the relationship between the expression of SERPINE1 and the infiltration levels of 22 types of immune cells by the Cibersort algorithm. The darker color indicates a higher correlation (*P<0.05). (C) The correlation of SERPINE1 expression with malignant phenotypes in head and neck cancer tissues. Scatter plots showed positive correlations between SERPINE1 expression and malignant phenotypes, such as (C1) metastasis, (C2) hypoxia, (C3) the epithelial-mesenchymal transition, and (C4) angiogenesis. (D) The relationship between SERPINE1 expression and the drug sensitivity of cancer cells. Red represents a positive correlation, while blue stands for a negative correlation. TPM, transcripts per million; TCGA, The Cancer Genome Atlas; HNSC, Head and Neck squamous cell carcinoma; NK, natural killer; EMT, epithelial-mesenchymal transition; GDSC, Genomics of Drug Sensitivity in Cancer; mRNA, messenger RNA; FDR, false discovery rate.

• 

  Fig. 4. (A) The messenger RNA (mRNA) expression of SERPINE1 was higher in nicotine-treated oral cells (DOK/NIC) and oral cancer cell lines (Cal27, SAS, HSC-3) than that in DOK cells, respectively. (B) The immunohistochemistry scores of SERPINE1 protein expression in oral cancer tissues were markedly higher than in the normal controls. (C) The expression scores for SERPINE1 protein were higher in cancer samples with lymph node metastasis (LNM) than in those without LNM. (D) The scores of SERPINE1 expression were higher in the samples with high pathological stages than in those with low stages. No associations were presented concerning age (E), sex (F), and Tstage (G). IHC, immunohistochemistry; NS, not significant (P>0.05). *P<0.05.

• 

  Fig. 5. (A) The mRNA expression of SERPINE1 was significantly downregulated in the SERPINE1-silenced oral cancer cells (sh-SERPINE1-Cal27 or sh-SERPINE1-SAS) compared with that of the control cells (sh-NC-Cal27 and sh-NC-SAS). (B) The trend of SERPINE1 protein expression was in line with that of mRNA expression. (C) The cell proliferation abilities of the SERPINE1-silenced cancer cells were significantly lower than those of the control cells. (D) The number of colonies formed in the SERPINE1-silenced cells was significantly lower than that in the control cells. (E) The invasive abilities in the SERPINE1-silenced cells were significantly inhibited compared with those in the control cells. (F) The administration of Bleomycin or docetaxel resulted in a significant decrease in cell viability in SERPINE1-silenced cells compared with the control cells. mRNA, messenger RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; OD, optical density. *P<0.05.

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