Overexpression of hnRNP K mRNA in HCC based on bioinformatics databases
In order to study the mRNA expressing level of hnRNP K in various tumors and their healthy samples across all TCGA tumors, we used the TIMER database. From TIMER analysis, the expressing of hnRNP K mRNAs was remarkably greater in HCC specimens (Fig1 A). To identify the expressing of hnRNP K in HCC, our team analyzed datasets from the Oncomine database. In the Oncomine data base, the expressing of hnRNP K mRNA was remarkably greater in HCC in contrast to healthy hepatic samples in the Wurmbach liver (Fig1 B) and Roessler Liver 2 (Fig1 C) datasets. Increased expressing of hnRNP K in HCC was confirmed in the TCGA dataset via the UALCAN (Fig1 D). Our team also explored the relationship between the expressing of hnRNP K and the prognoses of HCC sufferers via GEPIA, the result revealed that the high expressing of hnRNP K mRNA was significantly related to poor clinic outcome (Fig1 E). Meanwhile, similar results were verified in GEO data base as well (Fig2 A-B). Additionally, the expressing of hnRNP K protein was upregulated in HCC samples in contrast to healthy samples in HPA (Fig2 C). Taken together, the data in multiple databases suggested that the expressing of hnRNP K in HCC tumors was remarkably greater in contrast to healthy hepatic samples, and high expressing of hnRNP K may correlate with poor prognosis in HCC.
Association between hnRNP K mRNA expression and clinicopathologic features of HCC
To further assess the relationship between the expressing of hnRNP K mRNAs and clinicopathologic features of HCC via TCGA data via UALCAN web tool. As to cancer gradation, the expressing of hnRNP K was remarkably regulated upward in every cancer gradation, we also discovered that the expressing of hnRNP K was remarkably greater in grade 3 in contrast to grade 1 and 2 (Fig3 A). Interestingly, the expressing of hnRNP K was remarkably elevated in stage 3 compared with stage 1 and 2 and we also observed an increase in hnRNP K expression in stage 1 thru 3 compared to normal samples, but no diversity was observed in the expressing of hnRNP K between stage 4 and normal samples (Fig3 B). Moreover, in contrast to healthy samples, the expressing of hnRNP K was significantly increased irrespective of gender, age, race and histological subtypes. (Fig3 C-F).
Overexpression of hnRNP K protein in HCC
For the sake of exploring the expressing feature of hnRNP K in HCC, the protein expressing of hnRNP K was analyzed in 80 pairs of HCC and neighboring specimens via TMA-based IHC (Fig4 A). The outcomes revealed that hnRNP K expressing level was remarkably greater in HCC samples in contrast to the healthy adjacent liver samples (Table 1, Fig4 B).
Relationship of hnRNP K expression and clinicopathological parameters in HCC tissues
The clinicopathologic parameters of HCC patients are provided (Table 2) to analyze the association between hnRNP K protein level and clinicopathologic parameters. Among the HCC tissue specimens, 40 cases (50%) were identified as having a high hnRNP K protein expression, and the remaining 40 samples had a low hnRNP K protein expression. Higher expressing level of hnRNP K was remarkably related to higher pathologic gradation (p=0.044), and the survival status of all patients (p = 0.011), In addition, our team discovered that higher expressing level of hnRNP K was remarkably related to post-operation recurrence (p = 0.007). However, hnRNP K expression did not correlate with gender, age, tumor size, AFP level, GGT level, TBIL level, TNM stage, tumor number, cirrhotic nodules and HBV infection.
Assessment of prognosis significance of hnRNP K in HCC
The Kaplan–Meier survival analyses were utilized to further assess the prognosis significance of hnRNP K in HCC. Our team observed that patients with high expressing level of hnRNP K was tightly related to shorter OS in contrast to sufferers with lower expressing level (Fig4 C).
A univariate Cox proportion risk model was then performed to investigate which factors affected the overall survival, the results showed that GGT level (p=0.034), Edmondson grade (p=0.015), and hnRNP K expression (p=0.012) correlated with overall survival. while only Edmondson grade (p=0.035) and the expressing level of hnRNP K were remarkable in multivariable analyses, revealing that the expressing of hnRNP K protein was an independent risky factor for poorer prognoses in HCC sufferers (Table 3, Fig4 D).
Association between hnRNP K and tumor immune infiltration
Next, our team assessed the relationship between hnRNP K expressing level and tumor immune infiltration using TCGA LIHC data through TIMER tool. Positive correlations which were statistically significant (p<0.05) were found between hnRNP K and the infiltration level of the five immunocytes, include B cells (r=0.321, p=1.13e-09), CD4+T cells (r=0.377, p=4.81e-13), CD8+T cells(r=0.287, p=6.41e-08), macrophagus (r=0.446, p=4.96e-18), neutrophilic cells (r=0.51, p=3.25e-24), and DCs (r=0.419, p=6.42e-16). In addition, our team discovered that the hnRNPK expressing level was related to cancer purity in a positive way (r=0.128, p=1.17e-02). (Fig5 A)
For the purpose of investigating the underlying association between hnRNPK and infiltration immunocytes, our team identified the association between hnRNPK and multiple immunocyte biomarkers in TIMER and GEPIA. Those biomarkers were extensively considered relevant symbols of diverse immunocytes, like B cells, CD8+ T cells, M1/M2 macrophagus, cancer-related macrophagus, mononuclear cells, NK, neutrophilic cells and DCs in LIHC. The diverse function T cells like Tfh, Th1, Th2, Th9, Th17, Th22, Treg and depleted T cells were studied as well herein. Our team discovered that the contents of the majority of immunity sets marking B cells, diverse T cells, TAMs, M1/ M2 macrophagus, mononuclear cells and DCs were related to the expressing of hnRNP K in HCC (Table 4). These results indicate that hnRNP K might influence the immune cell infiltration level affecting tumor occurrence and development, explaining the poor prognosis in HCC.
GO and KEGG pathway analysis with hnRNP K and correlated genes identifying signaling pathways in HCC
Our team intended to reveal the underlying signaling pathways participating in hnRNP K and correlated genes in LIHC. First, we analyzed transcriptome datasets from TCGA LIHC data sets via the R2: Genomics Analysis and Visualisation Platform. 3314 genes with cutoffs of |r| > 0.3 (r > 0.3 and r < − 0.3) and Bonferroni adjusted p<0.01 were considered as correlated genes (include 2384 positive correlation and 930 negative correlation). Next, GO and KEGG pathway analysis of hnRNP K and its positively and negatively co-expressed genes were performed though online databases DAVID and KOBAS.
According to the GO analysis results, hnRNP K and the positively associated genes were involved in cell cycle, DNA duplication and repair, p53 binding and hippo signaling etc (Fig6 A). Moreover, we also observed that hnRNP K and its negative correlated genes were relevant to cell autophagy (Fig6 B).
KEGG pathway analyses were also performed on those genes. Results of the KEGG analysis showed these genes were not only related to cell cycle, viral carcinogenesis, Jak-STAT signal path, the expressing of PD-L1 and PD-1 check point pathway in tumor, TGF-beta signal path, and p53 signal path but also related to HCC associated signal paths like MAPK, PI3K-Akt and Hedgehog signal paths (Fig6 C).