1. Pan-cancer analysis of AHSA1 mRNA expression
The HPA database shows that among the 63 types of human tissues, the top three tissues and organs with high AHSA1mRNA expression are the fallopian tube (pTPM = 222.2), testis (pTPM = 207.8), and smooth muscle (pTPM = 145.0). In the liver (pTPM = 33.8) Only ranked 42nd (Fig. 1A), suggesting the low expression of AHSA1 in human normal liver tissue. Further analysis of the expression and location of AHSA1 in a variety of tumor cells, the results suggest that AHSA1 is expressed in the cytosol(Fig. 1B).
We further analyzed the expression of AHSA1 mRNA in human tumors using TIMER database. The results showed that the difference of AHSA1 in different tumor tissues and normal tissues. Compared with normal tissues, the expression level of AHSA1 was significantly increased in BLCA(bladder urothelial carcinoma), BRCA (breast invasive carcinoma), CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA (esophageal carcinoma), HNSC (head and neck squamous cell carcinoma), KIRC (kidney renal clear cell carcinoma), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), PRAD (prostate adenocarcinoma), READ (rectum adenocarcinoma) and STAD (stomach adenocarcinoma), while it was significantly decreased in GBM (glioblastoma multiforme) (Fig. 2).
2. Expression levels of AHSA1 in HCC patients.
Nine HCC gene sets were analyzed in the HCCDB database: HCCDB1 (p = 0.000005880), HCCDB3 (p = 2.860e-9), HCCDB4 (p = 1.990e-76), HCCDB6 (p = 8.830e-40) ), HCCDB7 (p = 0.00001210), HCCDB11 (p = 0.07038), HCCDB12 (p = 0.04648), HCCDB13 (p = 7.240e-7), HCCDB15 (p = 3.840e-17), HCCDB16 (p = 0.00001120), HCCDB17 (p = 1.110e-17), HCCDB16 (p = 3.730e-32), this study found that AHSA1 mRNA is highly expressed in HCC compared with neighboring normal tissues (Fig. 3A).
The GEPIA analysis of the expression level of AHSA1 in a total of 369 liver cancer samples and 160 normal liver tissue samples in the TCGA database and GTEx database showed that the expression level of AHSA1 in liver cancer tissue was higher than that in normal liver tissue, and the difference was statistically significant (Log2FC Cutoff = 0.8, P < 0.001) (Fig. 3B). Further verified by the Oncomine database, there are 4 sub-studies in the database that meet the screening criteria. The 4 sub-studies, namely Roessler Liver2, and Wurmbach Liver2, contained 299 liver cancer tissue samples and 261 normal liver tissue samples. A meta-analysis on it showed that the expression level of AHSA1 in liver cancer tissue was higher than that in normal liver tissue (P = 0.005) (Fig. 3C).
3. Prognostic value of AHSA1 for HCC patients.
We next investigated the prognostic value of AHSA1 for HCC using the Kaplan-Meier plotter. High AHSA1 was related to worse prognosis in HCC (OS: HR = 1.51(1.07–2.31), P = 0.019; DSS: HR = 1.67༈1.07–2.68༉, P = 0.032) (Fig. 4). Moreover, the univariate showed that high AHSA1 expression is significantly associated with poor overall survival༈HR = 2.115༈1.498–2.988༉, P༜0.001༉. Multivariate analysis showed that AHSA1 was an independent prognostic factor for OS of HCC using TCGA database༈HR = 1.970༈1.395–2.782༉, P༜0.001༉(Fig. 5).
A nomogram based on AHSA1 was established to estimate the 1-, 3-, and 5-year survival by using clinicopathological factors and Gene expression. Herein, as showed in Fig. 4F, the actual 1-, 3-, and 5-year survival times were consistent with the predicted ones by calibration plots of the nomogram. In the ROC curve, the AUC used to predict 1-year, 3-year, and 5-year survival rates are 0.721, 0.711, and 0.725, respectively (Fig. 6).
4. Biological process and pathway enrichment analysis.
For a more comprehensive understanding to the functional characteristics of AHSA1, we applied GO and KEGG analysis to DEGs using the clusterProfiler package. GO CC analysis showed that these DEGs were significantly enriched in secretory granule lumen, cytoplasmic vesicle lumen, vesicle lumen, tertiary granule and microvillus. For GO BP analysis, it is mainly related to mediating the activation of neutrophils to participate in the immune response. In terms of GO MF, it is mainly related to monosaccharide binding, glucose binding, vitamin binding, etc (Fig. 7A). KEGG pathway indicated that these differentially expressed genes primarily took part in glycolysis/gluconeogenesis, central carbon metabolism in cancer, tyrosine metabolism, and phenylalanine metabolism pathways (Fig. 7B). KEGG signaling pathway analysis showed that the expression of AHSAl is involved in the signal transduction process related to tumor cell energy metabolism.
5. AHSA1 expression is correlated with immune infiltration in LIHC
TIMER2.0 was used to analyze the correlation between AHSA1 expression and hepatocellular carcinoma immune infiltration, and the results showed: AHSA1 expression and B cells (r = 0.256, p = 1.47e-06), CD4 + T cells (r = 0.166, p = 1.95e-03), regulatory T cells (r = 0.152, p = 4.83e-03), macrophages (r = 0.171, p = 1.43e-03), neutrophils (r = 0.167, p = 1.87e-03), the degree of immune infiltration of dendritic cells (r = 0.405, p = 4.37e-15) has a significant correlation. This suggests that AHSA1 may be involved in the immune infiltration process of liver cancer cells (Fig. 8).
To further study the relationship between different immune infiltrating cells and AHSA1, GEPIA database was analyzed to analyze the association between AHSA1 and immune marker genes of several immune cells. The results showed that compared with normal liver tissues, the expression level of AHSA1 was significantly correlated with the immune marker set of some immune cells in liver cancer tissues(cor > 0, p < 0.05) (Table 1). These results further suggest that the expression of AHSA1 may be related to Part of the tumor's immune cell infiltration is related, and the tumor progression is further promoted through immune infiltration.
6. Correlations of AHSA1 Expression with Glycolysis in HCC
Glycolysis of tumor cells plays an important role in the progression of HCC. By GEPIA database to investigate the correlation between AHSA1 and the expression of 8 glycolysis related genes in HCC. The results showed that the expression of AHSA1 was significantly positively correlated with PAM, NUP155, KDELR3, NSDHL, ENO1, SRD5A3, GOT2, and PKM (Fig. 9).
7. AHSA1 Related Potential Drug in HCC.
To further explore AHSA1 as a potential therapeutic target for HCC, CMap assays were employed. Based on the rationale of CMap analysis, we loaded the genes with positive cor-relation to AHSA1 as upregulated genes and the genes with negative correlation as downregulated genes into the CMap database to obtain drugs with inhibitory effect on AHSA1. the two compounds with the highest negative enrichment score (etacrynic acid and blebbistatin) were identified as potential therapeutic agents for HCC (Fig. 10).