PSMA1, a Poor Prognostic Factor, Promotes Tumor Growth in Lung Squamous Cell Carcinoma

Abstract

Lung squamous cell carcinoma (LUSC) has a poor clinical prognosis and lacks effective targeted therapy. This study is aimed at investigating the role of PSMA1 (proteasome subunit alpha type-1) in LUSC. The differential expression genes (DEGs) in LUSC were retrieved from The Cancer Genome Atlas (TCGA) by "edgR" algorithm and by "limma" R package. Then, the relationship between genes and overall survival (OS) was explored by the least absolute shrinkage and selection operator (LASSO) and multivariate Cox (multi-Cox) regression. Next, the PSMA1 expression in tissues of LUSC was detected by IHC, qRT-PCR, and western blot (WB). Moreover, the effects of PSMA1 on the proliferation and viability of LUSC cell were explored by cell counting kit 8 (CCK-8) assays, colony formation assays, and flow cytometry (FCM) analysis. All 4421 DEGs were screened by TCGA database, and 26 genes associated with OS were selected by multi-Cox. Based on TCGA database, PSMA1 was highly expressed in tissues of LUSC patients, and OS and FP of patients with PSMA1 overexpression were significantly lower than those of patients with low PSMA1 expression. Furthermore, PSMA1 knockdown significantly decreased the proliferation of LUSC cells and promoted the apoptosis of LUSC cells, and these effects were reversed by PSMA1 overexpression. The results of this project supported that PSMA1 might be a critical gene regulating the development of LUSC and has the potential to be explored as a prognostic biomarker of LUSC.

Figure 1

Differentially expressed gene in LUSC based on TCGA database. (a) Volcano plots of differentially expressed gene in the limma package based on an adj.P.Val < 0.05 and log |log FC| > 1; (b) volcano plots of differentially expressed gene in the edgR package based on FDR < 0.05 and |log FC| > 1; the red dots represent the upregulated genes; the green dots represent the downregulated genes based; the gray spots represent genes with no significant difference in expression. (c) The Venn diagram demonstrates the intersections of genes between the limma package and edgR package.

Figure 2

The prognostic genes in LUSC. (a) The prognostic genes extracted by univariate Cox regression analysis. (b) The trajectory of each prognosis-related candidate gene's coefficient in diffuse-type LUSC was observed in the LASSO coefficient profiles with the changing of the lambda in LASSO algorithm. (c) After the 10-fold cross-validation, a confidence interval was obtained for partial likelihood deviance as the lambda changed. (d) The prognostic genes related with OS extracted by multi-Cox regression analysis.

Figure 3

The survival analysis of PSMA1 with LUSC. (a, b) The expression of PSMA1 in tissues from patients with LUSC: (a) TIMER database and (b) UALCAN database. (c, d) The survival analysis of PSMA1 in tissues from patients with LUSC: (c) OS and (d) FP.

Figure 4

Analysis of PSMA1 level in cells. (a) The mRNA expression of PSMA1 in tissues of LUSC patients was detected by qRT-PCR. (b) Protein expression of PSMA1 was detected by WB in tissues from patients with LUSC. (c) IHC staining for PSMA1 in tissues of LUSC patients. (d) Quantitative RT-PCR analysis of PSMA1 in LTEP-s, NCI-H596, and NCI-H520 cells. (e) Western blot analysis of PSMA1 in LTEP-s, NCI-H596, and NCI-H520 cells. PSMA1 molecular weight: 84 kDa.

Figure 5

The study of the mechanism of PSMA1 in the cell. (a) The protein expression of PSMA1 was analyzed in PSMA1-depleted cells and PSMA1-overexpressed cells by western blot (right) analyses. (b) The viability cell was detected by CCK-8 when PSMA1 was sliced and PSMA1 was overexpressed. The cells were measured at 24, 48, 72, and 96 h. (c) Clone formation assay showing the proliferation ability of the LTEP-s and NCI-H596 cells with the vector-PSMA1 group and shPSMA1-1# and shPSMA1-2# compared with the vector-empty group and shNC group, respectively. (d) Cell apoptosis distribution was determined by flow cytometric analysis in LTEP-s and NCI-H596 cells in the vector-PSMA1 group and shPSMA1-1# and shPSMA1-2# compared with the vector-empty group and shNC group.