The genetic abnormalities of NEIL3, a DNA glycosylase that initiates base excision repair by hydrolyzing the N-glycosidic bond and releasing the damaged base, are reported to be correlated with hepatocarcinogenesis[28]. Recent study also demonstrates that the Neil3 gene expression levels are highly related to HCC recurrence in patients receiving hepatectomy, which suggests that NEIL3 may serve as a prognostic marker for HCC [11]. To the best of our knowledge, NEIL3 expression, together with its potential prognostic impact on HCC, has not yet been explored, and the potential role of NEIL3 in HCC is the focus in this study.
As found in this study, NEIL3 was highly expressed in HCC, as well as bladder, breast, biliary, head and neck, colorectal, esophageal, gastric, kidney, lung, uterus, prostatic, and rectal cancers, compared with that in normal tissues. The differential NEIL3 expression was further observed in numerous independent datasets with regard to HCC based on the HCCDB database. Emphatically, bioinformatic analysis using the high throughput RNA-sequencing data from TCGA demonstrated that, the up-regulated NEIL3 expression in HCC was related to the advanced clinicopathological characteristics (such as high grade, clinical stage and T stage), short survival time, and dismal prognosis, which were supported by the results acquired from UALCAN. Furthermore, the Kaplan-Meier Plotter data suggested that high NEIL3 expression was correlated with a high hazard ratio (HR) for poor OS and PFS of HCC. To be specific, the up-regulated NEIL3 expression was correlated with the poor prognosis for HCC at stage 2–4, grade 1–3 and T1-T3. Moreover, patients with micro-vascular invasion, alcohol consumption, hepatitis virus infection and Sorafenib treatment also had worse OS and PFS in the presence of high NEIL3 expression. In addition, as far as HR was concerned, the prognosis for male and Asian patients with up-regulated NEIL3 expression was worse than that for female and white patients. Taken together, these findings strongly suggested that NEIL3 was a prognostic biomarker for HCC.
To further investigate the functions of NEIL3 in HCC, GSEA was implemented using TCGA data, and then the differentially enriched pathways in the high NEIL3 expression phenotype as well as those linked to cancer and immunity, were selected. Among them, Base excision repair scored 2.17 on NES, and the polymorphisms of base-excision repair genes are reported to shape the development of hepatitis C virus (HCV) infection, liver cirrhosis and HCC, which predicts the OS[29, 30]. Besides, plenty of studies have elaborated that, the Notch signaling that may induce epithelial-mesenchymal transition (EMT) and mutation or loss of P53, the ErbB pathway that regulates the fundamental cellular processes, and the mTOR signaling that determines cell fate at different levels, play crucial roles in hepatocarcinogenesis[31–33]. Nevertheless, this study was the first to propose that, NEIL3 might be involved in these pathways in the HCC setting, suggesting that NEIL3 might serve as a potential prognostic marker and therapeutic target of HCC.
To further probe the corrections between NEIL3 and other genes in HCC, the interactions between NEIL3 and other functional partners were investigated in the LinkedOmics database. Our results revealed that, there were plenty of genes co-expressed with NEIL3 in LIHC, among which, CENPE, a spindle checkpoint protein, exhibited the highest Spearman’s correlation of 0.774. Of interest, CENPE has been identified as a tumor suppressor gene, and its decreased expression may contribute to HCC development[34]. However, recent study documents that CENPE is remarkably up-regulated in HCC[35], consistent with our results on CENPE in TCGA. Therefore, CENPE was speculated to play a dual role in HCC progression. NCAPG, which ranks the second in our list, has shown its effect on HCC cell proliferation and migration, and it is evidently correlated with HCC recurrence, metastasis, differentiation and TNM stage[36]. Additionally, Hu et al. demonstrated that the over-expression of KIF4A, which was also highly correlated with NEIL3, was regulated by forkhead box M1 (FOXM1); besides, it had a bearing on HCC recurrence and progression, and might serve as a promising prognostic marker[37]. Furthermore, enrichment analyses on target gene sets using GSEA helps to reveal the important networks of target kinases and transcription factors (TFs). Our results suggested that the functional network of NEIL3 mainly participated in chromosome segregation, spindle organization, cell cycle regulation, and DNA replication. Interestingly, NEIL3 is also proposed in previous study to be involved in the cell signaling pathway that senses oxidative stress (OS), and contributes to the recruitment of TFs as well as the introduction of chromatin modifications, thereby resulting in the fine tuning of cellular processes for promoting cell survival and successful cell differentiation[38]. Moreover, among the highly NEIL3-related kinases investigated in this study, PLK1, one of the conserved serine/threonine kinase family members involved in multiple mitotic processes (including functional maturation of centrosomes, establishment of the bipolar spindle, chromosome segregation and response to DNA damage), was suggested in previous study to be a factor that determined the SN38 sensitivity of tumor cells, and its over-expression was inversely correlated with the survival rate of HCC[39]. Besides, other kinases, such as CDK1 and AURKB, which are involved in our network, also regulate genomic stability, mitosis and the cell cycle[40, 41]. Among the TFs targeted by NEIL3, E2F1 represents a key link in the cell cycle regulation network, and its aberrant expression participates in HCC occurrence and development, which also predicts the unfavorable patient prognosis[42]. As reported, E2F1, which is tightly linked to cell division cycle associated 5 (CDCA5), Sirtuin 5 (SIRT5) and other important molecules, may serve as a vital anti-apoptotic factor in liver cancer due to its ability to offset c-Myc-driven apoptosis[43, 44]. As suggested in previous literature, the repressive complex, which includes the component of E2F4, is relieved by CDK upon re-entry into the cell cycle; thereafter, E2F1-Sp1, which is in the form of complex E2F1-NF-Y, can be recruited to the promoter[45]. Therefore, it was deduced in this study that NEIL3 might regulate this process. Therefore, our analyses suggested that PLK, CDK, E2F1, and E2E4 were all the concernful targets of NEIL3, and that NEIL3 acted through these factors to regulate the cell cycle and proliferation capacity of HCC. Nonetheless, further studies are warranted to test this hypothesis.
Another important discovery of this study was that, NEIL3 expression was correlated with the diverse immune infiltration levels in HCC. As demonstrated by our results, the infiltration levels of macrophages, DCs, B cells, CD8 + T cells, CD4 + T cells and neutrophils showed significantly positively correlations with NEIL3 expression in HCC. Moreover, the correlations of NEIL3 expression with the marker genes of immune cells implicated that NEIL3 might be involved in some immunocyte functions in HCC. In this study, cholangiocarcinoma (CHOL), in which NEIL3 was not associated with immune cell infiltration, was applied as the control. In general, after being adjusted by purity, marker genes related to T cells such as CCR8, CCR3, CTLA4, ICOS and TIM-3, had higher correlations with NEIL3 expression than those of other immunocyte types, while no significant correlation was observed in the control group. What’s more, results obtained from GEPIA database analysis indicated that, NEIL3 might potentially affect T cell exhaustion, as well as T cell receptor (TCR) and Treg functions, as supported by the evidence that the up-regulated NEIL3 expression was positively correlated with the expression of most Treg, TCR and T cell exhaustion markers (like SHNP2, CCR8, TIM-3, CTLA4, CD28, CD45 and PD-1). PD-1, a negative costimulatory receptor that is critical for suppressing T cell activation both in vitro and in vivo, becomes clustered with TCR upon binding to its ligand PD-L1, which is transiently associated with the phosphatase SHP2[46]. Typically, NEIL3 may exert its own strength to participate in this system. CCR8, together with CCR10, is the chemokine receptor responsible for Treg cell migration to the tumor microenvironment (TME) [47], and high NEIL3 expression is also proved to be linked with this molecule. On the other hand, TIM-3, a critical surface protein on the exhausted T cells[48], shows positive correlation with NEIL3 expression in HCC. On the other hand, NEIL3 expression is also markedly correlated with the regulation of several T helper cells (Th1, Th2, Tfh, and Th17) markers in HCC. These correlations may indicate a potential mechanism by which NEIL3 regulates T cell functions in HCC. An accumulating amount of data indicate that, DNA glycosylases in general are involved in the adaptive immunity. However, few previous studies are carried out to examine the relationship of NEIL3 with immunity. For instance, Torisu et al. had proposed a putative role of NEIL3 in lymphocytes and/or other immune cells[49]. Meanwhile, other study demonstrates that Neil3-deficient mice are linked with signs of inflammation and autoimmunity when they are challenged by inflammatory stimuli, and that individuals with NEIL3 mutation show certain subclinical signs of autoimmunity[50]. Nevertheless, it remains to be verified about the role of NEIL3 in humans with immunodeficiency and autoimmune disorders. In a word, although the relationship of NEIL3 over-expression with immune cell infiltration is generally not strong, this study has opened up a new field of vision regarding the influence of NEIL3 on HCC and its underlying role in recruiting and regulating the immune infiltrating cells in HCC.
Nonetheless, some limitations should be noted in this study, which should be taken into consideration when interpreting our results. Firstly, transcriptomic analysis only reflected some aspects of genes, rather than the global alterations. Secondly, there were few stage 4 patients in the LIHC samples, since most HCC patients were first diagnosed at the advanced stage with dismal prognosis. Finally, our results were not validated via another independent cohort, which was also a limitation in this study, and the reliability of our molecular results was still challenged due to the lack of experiments in vitro or in vivo.
In conclusion, NEIL3, which may be positively co-expressed with CEPNE, NCAPG and KIF14, can serve as a potential prognostic molecular marker for the poor survival of HCC. Moreover, the base excision repair, the P53 pathway, the T cell receptor pathway, the mTOR pathway, the ERBB pathway and the Notch signaling may be the key pathways that are regulated by NEIL3; whereas PLK, CDK, E2F1_Q6 and E2F1_Q4 in EC may be targeted by NEIL3, and are involved in cell cycle regulation and DNA replication. Additionally, NEIL3 is also found to be associated with the infiltration of immune cells, especially for the functions of certain T cells related to HCC. Further experimental validation should be performed to confirm the biological impact of NEIL3.