mRNA and protein expression levels of KPNAs in HCC
Analyzing the transcription levels of KPNAs in HCC and normal tissues in the ULCAN database, the results showed that the transcription levels of all members of the KPNAs family were significantly up-regulated in HCC tissues (Figure.1). Analyzing the data in the TIMER database got the same result (Figure.2a). In the GEPIA database, the expression levels of KPNAs family members in HCC are all increased, but compared with normal tissues, except for the statistically significant increase in KPNA2, the up-regulation of other members is not statistically significant (Figure.2b-h). Next, we used the HPA database to analyze the protein expression levels of KPNAs family members in HCC. As shown in Fig. 3, the protein expression level of KPNA2-6 in HCC tissues is significantly higher than that in normal liver tissues. KPNA1 is moderately expressed or not expressed in normal hepatocytes and cholangiocytes but is moderately expressed in cholangiocytes of cancer tissues. In conclusion, KPNAs family members are up-regulated in HCC.
The relationship between the expression level of KPNAs in HCC and clinicopathological characteristics
We used the UALCAN database to analyze the relationship between KPNAs and tumor stage, tumor grade and TP53 mutation status in HCC. As shown in Fig. 4, compared with normal tissues, KPNAs were significantly up-regulated in stage 1, 2, and 3 tumors. Compared with normal tissues and stage 4 tumors, except for the statistical significance of KPNA2 expression, the expression of other KPNAs family members is not statistically significant, which may be related to the limited number of stage 4 cases (n = 6). There are significant differences in the expression of KPNA2 in tumor stages 1 and 2, stage 1 and 3, KPNA4 in stage 1 and 2, stage 2 and 3, and KPNA6 expression between stage 1 and stage 3. As shown in Fig. 5, there is a close relationship between KPNAs and tumor grade. Compared with normal tissues, the higher the expression of KPNAs, the higher the tumor grade. In addition, the expression levels of some KPNAs members are also related to different grades of tumors. For example, the expression of KPNA2, KPNA4, KPNA5 and KPNA7 is significantly different between grade 1 and grade 3, grade 2 and grade 3. Moreover, the expression level of KPNA7 between grade 1 and grade 2, grade 2 and grade 4 of tumors is also statistically significant. We also analyzed whether the expression of KPNAs is related to the mutation of TP53. Compared with normal tissues and tumor tissues without mutations, the expression level of all members except KPNA3 in TP53 mutant tissues was significantly up-regulated (Fig. 6). In short, the expression of KPNAs family members is significantly related to the staging and grade of HCC tumors and whether TP53 is mutated.
The relationship between KPNAs and the prognosis of HCC patients
Kaplan-Meier Plotter was used to evaluate the relationship between the expression level of KPNAs and the OS and RFS of patients. In general, the expression level of KPNAs is negatively correlated with the OS and RFS of HCC patients. The expression levels of KPNA2, KPNA4, KPNA5, and KPNA6 are statistically significant with the patient's OS, while the expression levels of KPNA2, KPNA5, and KPNA6 are significantly related to the patient's RFS (Fig. 7, Fig. 8). Therefore, KPNAs may be associated with the prognosis of HCC patients, have the potential to evaluate the prognosis of patients, and may be used as a biomarker to predict the prognosis of HCC patients in the future.
The relationship between the mutation of KPNAs and the prognosis of HCC patients
We used the cBioPortal tool to analyze the alteration in KPNAs in HCC patients and their relationship with the patient's prognosis. We found that 159 (44%) of 360 cases had changed. The alteration types mainly included Mutation, Amplification, Deep Deletion, mRNA High, mRNA Low and Multiple Alterations (Fig. 9b). The mutation rates of KPNA1-7 were 4%, 13%, 6%, 11%, 7%, 13%, and 8%, respectively (Figure.9a). In addition, as shown in Fig. 9c and d, the OS of patients with KPNAs mutations was significantly shortened (p = 0.0166), while the alteration of KPNAs had no significant effect on disease-free survival (DFS) (P = 0.0571)
The relationship between the expression of KPNAs in HCC and tumor immunity
Is the expression of KPNAs related to tumor immunity in HCC? We used the TIMER database to explore. As shown in Fig. 10, there is no significant correlation between the expression of a few KPNAs and tumor immune infiltration (e.g. KPNA1 and CD4 + T, KPNA2 and CD8 + T, KPNA6 and CD4 + T, KPNA7 and CD8 + T, KPNA7 and neutral Granulocytes). But overall the expression level of KPNAs is positively correlated with the immune infiltration of B cells, CD4 + T, CD8 + T, dendritic cells, macrophages and neutrophils. And this correlation is statistically significant. It proves that KPNAs participate in the immune infiltration process in HCC.
Co-expression and enrichment analysis of KPNAs related genes
The related genes of KPNAs are obtained from the cBioPortal database. We first sort out the top 20 genes that are most closely related to each member of the KPNAs family, and then summarize the above genes and remove duplicate genes. Use STRING online software to obtain the PPI of the above genes (Fig. 11). Metascape is used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The results are shown in Fig. 12a. The biological processes involved in KPNAs mainly include "sister chromatid segregation, NLS-bearing protein import into nucleus, peptidyl-serine phosphorylation, protein localization to chromosome, centromeric region, organelle localization," and so on. Cellular components such as "spindle, condensed chromosome, centromeric region, SWI/SNF complex, apicolateral plasma membrane, PML body" are related to KPNAs (Fig. 12b). KPNAs also regulate molecular functions, such as "nuclear import signal receptor activity, protein kinase activity, kinase binding, microtubule-binding, insulin receptor binding," and so on (Fig. 12c). The KEGG enrichment found that KPNAs-related pathways mainly include "Cell cycle, Tight junction, MAPK signaling pathway, Autophagy-animal, Hippo signaling pathway, etc." (Fig. 12d).