IREB2, CHRNA5, CHRNA3, CHRNB4, HYKK, and PSMA4 mutations in LAC based on the TCGA database
A total of 576 patients with a histologically confirmed diagnosis of LAC were collected by TCGA in the present study, the main data of which contained whole exome sequencing, RNA-seq, copy number, gene expression, and CNV data. The individual mutation frequencies of the Provisional cohort are illustrated in Figure 1. IREB2 was mutated in 7% of tumors and 41 of the 576 sequenced patients had IREB2 mutations; CHRNA5 was mutated in 5% of tumors and 30 of the 576 sequenced patients had CHRNA5 mutations; CHRNA3 was mutated in 5% of tumors and 30 of the 576 sequenced patients had CHRNA3 mutations; CHRNB4 was mutated in 7% of tumors and 41 of the 576 sequenced patients had CHRNB4 mutations. Also, HYKK was mutated in 6% and PSMA4 had a mutation rate of 9%, with mutations in 35 and 53 of the 576 sequenced patients, respectively.
Association between IREB2, CHRNA5, CHRNA3, CHRNB4, HYKK, and PSMA4 expression and clinical pathological features based on the TCGA database
To understand the role of IREB2, CHRNA5, CHRNA3, CHRNB4, HYKK, and PSMA4 in the tumorigenesis and progression of LAC, this study analyzed the association between IREB2, CHRNA5, CHRNA3, CHRNB4, HYKK, and PSMA4 expression and the clinicopathological features. LAC patients were divided into two groups according to IREB2, CHRNA5, CHRNA3, CHRNB4, HYKK, and PSMA4 expression, based on median value, namely, a low expression group and a high-expression group. In this investigation, there was a significant association between high CHRNA5 and PSMA4 expression and gender (P=0.000, P=0.005), overall survival status (P=0.050, P=0.006), and recurrence-free survival status (P=0.019, P=0.049). There was also a significant association between high CHRNA5 expression and smoking (P=0.002), as shown in Table 1. However, CHRNA5 and PSMA4 expression was not associated with age (P=0.855), race (P=0.752), TNM stage (P=0.725), or disease stage (P=0.971) in the cohort (P>0.05).
Assessment of IREB2, CHRNA5, CHRNA3, CHRNB4, HYKK, and PSMA4 gene expression in LAC tissues based on the TCGA database
We examined lung expression of IREB2, CHRNA5, CHRNA3, CHRNB4, HYKK, and PSMA4 in normal and carcinoma tissues, in two experiment groups, based on the TCGA database. The results showed that CHRNA5, CHRNB4, HYKK, and PSMA4 were highly expressed in lung adenocarcinoma tissues, and IREB2 and CHRNA3 showed low expression in cancerous tissues (Figure 2). Furthermore, IREB2, CHRNA5, CHRNA3, CHRNB4, HYKK, and PSMA4 expression were significantly different between the normal and lung adenocarcinoma tissues (P<0.001, P<0.001, P=0.021, P<0.001, P<0.001, P<0.001) (Table 2).
Association between CHRNA5 and PSMA4 expression and survival based on the TCGA database
This investigation used TCGA datasets and Kaplan-Meier Plotter online data respectively for survival analysis. The results showed that the CHRNA5 and PSMA4 expression levels were significantly correlated with the prognosis. There was a significant correlation between CHRNA5 and PSMA4 expression level and prognosis. There were significant differences in overall survival (OS)/recurrence-free survival (RFS) between the high and low CHRNA5 and PSMA4 expression groups (P<0.05) and the higher the expression, the worse the prognosis. Further utilization of Kaplan-Meier Plotter online data analysis is shown in Fig. 3. However, there were no differences in OS/RFS between the high and low IREB2, CHRNA3, CHRNB4, and HYKK expression groups (shown in Supplemental Figure 2). The above results suggest that CHRNA5 and PSMA4 are adverse prognostic factors and high expression was significantly associated with shorter survival.
CHRNA5 and PSMA4 expression in Beas-2b, A549, and H1299 cells
To further demonstrate the role of CHRNA5 and PSMA4 expression in the development and progression of lung adenocarcinoma, we conducted a cell experiment to compare CHRNA5 and PSMA4 expression in normal and cancerous cells. The results showed that CHRNA5 and PSMA4 expression in A549 and H1299 cells was obviously higher than in Beas-2b cells (Figure 4).
CHRNA5 and PSMA4 expression in a tissue microarray of lung adenocarcinoma
Immunohistochemical staining revealed that CHRNA5 and PSMA4 levels in the lung tissues of the carcinoma group were considerably higher than those in the normal group. The positive expression products were brown-yellow granules, which were located in the nucleus and cytoplasm, respectively, as shown in Figure 5.
Relationship between CHRNA5 and PSMA4 expression levels and the clinical characteristics of patients with lung adenocarcinoma.
To further explore the effects of CHRNA5 and PSMA4 on the clinical pathology of lung adenocarcinoma, this study analyzed the association between CHRNA5 and PSMA4 expression and clinicopathological features. The LAC patients were divided into two groups according to CHRNA5 and PSMA4 expression, based on the median value, namely a low expression group and a high-expression group. In this investigation, there was a significant association between high CHRNA5 expression and T stage (P=0.026), OS status (P=0.007), and progression-free survival status (P=0.013). There was also a significant association between high PSMA4 expression and OS status (P=0.006) and progression-free survival status (P=0.007), as shown in Table 3. However, CHRNA5 and PSMA4 expression was not associated with age, race, TM stage, or disease stage within the cohort (P>0.05).
Correlation between the expression levels of seven PSMAs and OS/RFS
We used the Kaplan-Meier curve for survival analysis of our clinical data. The results showed that CHRNA5 and PSMA4 expression levels were significantly correlated with prognosis. There was a significant correlation between CHRNA5 and PSMA4 expression levels and prognosis. There were significant differences in OS/RFS between the high and low CHRNA5 and PSMA4 expression groups (P<0.05). It was determined that the higher the expression, the worse the prognosis (Figure 6).
Functional enriched analysis of CHRNA5, HYKK, and PSMA4
The results of the GSEA method suggested that the CHRNA5, HYKK, and PSMA4 high-expression samples were respectively enriched to cell cycle, base excision repair, oxidative phosphorylation, protein export, and aminoacyl tRNA biosynthesis, among others (see Figure 7).