LIHC is the second greatest cause of death from cancer around the world, and LIHC has a complex pathogenesis and a lack of effective treatment strategies, especially in advanced stages. LIHC patients treated with clinical radiotherapy and immunotherapy drugs still have a higher rates of mortality. Therefore, it is crucial to explore new LIHC biomarkers to assess and improve the prognosis of LIHC patients.
As a member of the hnRNPS family, studies on HNRNPLL have focused on the role of alternative splicing. HNRNPLL has been shown to be involved in immune infiltration related to alternative splicing in gliomas (32), and it has been shown to regulate stem cell renewal and replacement through alternative splicing (33). Other studies have suggested that HNRNPLL regulates the secretion of immunoglobulins in plasma cells by binding to proteins (34), and HNRNPLL has been demonstrated to regulate hepatic glucose metabolism through the lncLGR long non-coding RNA (35). However, the role and mechanism of HNRNPLL in hepatocellular carcinoma has not been systematically elucidated, making it necessary to further explore how HNRNPLL participates in the progression and immune infiltration of LIHC. Through bioinformatics analysis, we found that HNRNPLL was differentially expressed in LIHC and was significantly correlated with tumor staging and prognosis, suggesting that HNRNPLL may become a biological marker for LIHC prognosis. In LIHC, the high expression of HNRNPLL was associated with poorer prognosis compared to low expression of HNRNPLL. To further explore the possible reasons behind this finding, we explored several aspects, including gene mutations. Although HNRNPLL itself had a low mutation rate in LIHC, we found a significant increase in the proportion of TP53 gene mutations in patients with high HNRNPLL expression. TP53 is a common tumor suppressor gene that promotes tumor growth, and once TP53 is mutated, it loses its tumor suppressor activity (36). DNA methylation affects the function of the genome by regulating gene expression or silencing. The first intron and its negative correlation with gene expression are independent of tissue and species (37). In contrast, positive associations are often observed with those located in the open-sea region (38, 39). The present study found that there were high-methylation sites located in the body region and in the open-sea region, including cg14738493, cg01868347, cg06548118, and cg01422416, which were associated with an increased mortality rate in LIHC patients. Some studies have suggested that methylation levels in this region are positively correlated with gene expression. However, we subsequently confirmed that the methylation levels of the methylation sites, except for cg14738493, were not significantly correlated with HNRNPLL expression. These results may suggest that HNRNPLL methylation, independent of expression levels, affects prognosis. We then used GO analysis, KEGG analysis, and GSEA to predict the possible functions and pathways of HNRNPLL. GO analysis found that HNRNPLL, similar to other members of the HNRNP family, mainly participated in molecular functions, including RNA binding, transcription factor binding, and nucleotide binding. GSEA demonstrated that metabolic-related pathways, such as oxidative stress and fatty acid oxidation, were enriched. As reported in the literature, TP53 mutations mediate metabolic reprogramming of liver cancer cells. Therefore, it may be worthwhile to explore whether there is any correlation between HNRNPLL and TP53 mutations.
The immune microenvironment is a hot topic of current research. We utilized the TIMER webtool to demonstrate the correlation of HNRNPLL with six forms of immune cells, which include B cells, CD8 + cells, CD4 + T cells, macrophages, neutrophils, and Tregs, as well as tumor purity. We then used CIBERSORT to quantify the immune infiltration status of TCGA data and found that the enrichment score of Tregs was greater in the higher HNRNPLL expression group than those in the lower HNRNPLL expression group. These results were further confirmed by the quantification results of quanTIseq. Additionally, we used the TISCH webtool to visualize the expression of HNRNPLL in various immune cells, including Tregs, in multiple cancers, including LIHC. Furthermore, we demonstrated the distribution of HNRNPLL in Tregs using single-cell transcriptomics. The aforementioned methods all indicated a strong association between Tregs and HNRNPLL. Tregs have a vital function in inhibiting antitumor immune responses (40), which often leads to poor prognosis in cancer cases. The current investigation found that individuals with higher HNRNPLL expression had a higher Treg enrichment score with Tregs expressing more HNRNPLL on their surface. Both the high HNRNPLL expression group and the high Treg enrichment group exhibited significantly worse prognosis. Finally, we used the TIDE algorithm to predict the response to immune therapy in TCGA samples. The results showed a significant elevation in TIDE scores but a redction in response rates in the high HNRNPLL expression group, indicating that patients with higher HNRNPLL expression may not be applicable for immune therapy. As we revealed that no significant association between the methylation levels and expression of HNRNPLL, we further investigated the correlation between methylation sites and immune infiltration, and we found that some methylation sites with increased levels, which usually lead to worse prognosis, were not significantly associated with Tregs. In contrast, these sites were accompanied by higher levels of M2 macrophage infiltration. As a subtype of macrophages, M2 macrophages typically promote cancer progression (41) as demonstrated in the current investigation. Indeed, this leads us to believe even more strongly that methylation and expression of HNRNPLL have independent effects on the development and progression of liver cancer. Understood. It seems that further experimental validation may be necessary to confirm these findings.
We confirmed the role of HNRNPLL in both types of liver cancer cells, and found that reducing the protein expression level of HNRNPLL significantly decreased the migration ability of HepG2 and Huh7 cells, while also causing more cells to be arrested in the G0 and G1 phases, thus affecting cell division and playing a role in the progression of the tumor.