SERPINB5 is a prognostic biomarker and promotes proliferation, metastasis and epithelial-mesenchymal transition (EMT) in lung adenocarcinoma

Abstract

Background: Serine protease inhibitors clade B serpins (SERPINBs) are the largest subclass of protease inhibitors, once thought of as a tumor suppressor gene family. However, some SERPINBs exhibit functions unrelated to the inhibition of catalytic activity.

Methods: The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Gene Set Cancer Analysis (GSCA), and cBioPortal databases were utilized to investigate SERPINBs expression, prognostic correlation, and genomic variation in 33 cancer types. We also conducted a comprehensive transcriptome analysis in multiple lung adenocarcinoma (LUAD) cohorts to reveal the molecular mechanism of SERPINB5 in LUAD. Then, qPCR and immunohistochemistry were used to verify the expression and prognostic value of SERPINB5 in LUAD patients. Furthermore, knockdown and overexpression of SERPINB5 in LUAD cell lines were performed to evaluate cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT).

Results: The expression of SERPINB5 was upregulated and demethylated in LUAD, and its abnormally high expression was significantly correlated with poor overall survival (OS). In addition, the expression of SERPINB5 was analyzed to determine its prognostic value in LUAD and confirmed that SERPINB5 was an independent predictor of LUAD in TCGA and GEO cohorts and qPCR validation with 106 clinical samples. At last, A knockdown of SERPINB5 in LUAD cells reduced proliferation, migration, and EMT. Proliferation, migration, and invasion are promoted by the overexpression of SERPINB5.

Conclusion: Therefore, SERPINB5 has shown potential as a prognostic biomarker for LUAD, and it may become a potential therapeutic target for lung adenocarcinoma.

Keywords: EMT; SERPINB5; biomarker; lung adenocarcinoma; metastasis.

FIGURE 1

Expression and clinical correlation analysis of SERPINBs in different types of cancers. (a) The expression level of SERPINBs in cancer tissues and adjacent normal tissues. (b) Different expression SERPINBs between cancer tissues and adjacent normal tissues in lung adenocarcinoma (LUAD). (c) Association of SERPINB expression with patient overall survival in pancancer. (d) Kaplan–Meier survival curves of overall survival (OS) and progression‐free survival (PFS) comparing the high and low expression of SERPINB5, SERPINB6 and SERPINB7 in LUAD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Mutation status of SERPINB gene family. (a) Single nucleotide variation (SNV) frequency of SERPINBs in pancancer. (b) Association between SNV and overall survival (OS) of SERPINBs in pancancer. (c–e) The alteration frequency of SERPINB2, SERPINB3 and SERPINB4 in different cancers obtained from the cBioPortal. (f–h) Association between copy number variation (CNV) frequency and OS of SERPINB5, SERPINB6 and SERPINB7 in LUAD. (i–k) Methylation of SERPINB5, SERPINB6 and SERPINB7 in lung adenocarcinoma (LUAD). (l) Forest plot of genes mutating differentially in TCGA‐LUAD cohort of the low‐ and the high‐SERPINB5 groups. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Clinical correlation analysis of SERPINB5 in lung adenocarcinoma (LUAD). (a) SERPINB5 expression of paired normal and tumor tissue in TCGA‐LUAD. (b, c) Immunohistochemical analysis of SERPINB5 in 44 paired samples of tumor and normal lung tissues. (d) Real‐time polymerase chain reaction (RT‐PCR) validation of the relationship between SERPINB5 mRNA expression and overall survival in the SYSUCC cohort. (e) Overexpression mRNA of SERPINB was an independent prognostic marker of LUAD patients in the SYSUCC cohort. (f–h) Kaplan–Meier overall survival (OS) results of SERPINB5 in three GEO lung adenocarcinoma cohorts. ***p < 0.001.

FIGURE 4

Multiple cohort transcriptome analysis of SERPINB5 in lung adenocarcinoma (LUAD). (a) The differentially expressed genes analyzed by volcano plots between high and low SERPINB5 expression. (b) Venn plot shows the number of unique and overlapping upregulated genes among the three cohorts. (c, d) KEGG pathway analysis of co‐upregulated genes in high SERPINB5 subgroup. (e) KEGG pathway enrichment analysis of 272 hub genes in protein–protein interaction (PPI) network. (f) PPI network shows the 272 hub genes screened by the STRING database and Cytoscape. (g) Top15 hub genes are identified by the “Cytohubba” plug‐in. (h) The node gene cluster with the highest score constructed by the “MCODE” plug‐in.

FIGURE 5

Effects of SERPINB5 knockdown on A549 and PC9 cell invasion, migration and proliferation. (a) Real‐time polymerase chain reaction (RT‐PCR) and western blot were used to determine the efficiency of SERPRINB5 knockdown in A549 and PC9 cell. (b) A scratch test was used to determine the cell migration. Cell migration (c) and invasion (d) were detected using a transwell assay. (e) Cell counting kit‐8 (CCK‐8) assays were performed to detect the cell proliferation of A549 and PC9. (f) The colony‐forming assay was used to reflect the proliferation of PC9 and A549. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6

SERPINB5 knockdown suppresses the epithelial‐mesenchymal transition (EMT) of lung adenocarcinoma (LUAD) cells (a) Immunofluorescence staining showed the changes in the expression of EMT‐associated genes E‐cadherin (green) and vimentin (red) in LUAD cells. Nuclei were counterstained with DAPI (blue). (b) Western blot assay of the expression of EMT markers in LUAD cells transfected with si‐SERPINB5.

FIGURE 7

Effects of SERPINB5 overexpression on A549 and PC9 cell invasion, migration and proliferation. (a) Real‐time polymerase chain reaction (RT‐PCR) and western blot were used to determine the efficiency of SERPRINB5 overexpression in A549 and PC9 cell. (b) A scratch test was used to determine the cell migration. (c) Cell migration and (d) invasion were detected by transwell assay. (e) Cell counting kit‐8 (CCK‐8) assays were performed to detect the cell proliferation of A549 and PC9. (f) The colony‐forming assay was used to reflect the proliferation of PC9 and A549. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 8

Gene set enrichment analysis (GSEA) for samples with high and low SERPINB5 expression in the TCGA and GEO cohorts. (a) Enrichment of epithelial‐mesenchymal transition in high SERPINB5 group versus low SERPINB5 group. (b) The top 15 enriched gene sets in the HALLMARK collection. FDR, false discovery rate; NES, normalized enrichment score.