N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the tumorigenesis of hepatocellular carcinoma (HCC), providing novel insights into the molecular pathogenesis of this disease. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been well studied in HCC. Here we investigated the biological role and underlying mechanism of WTAP in liver cancer. We determined the expression of WTAP and its correlation with clinicopathological features using tissue microarrays and the Cancer Genome Atlas (TCGA) dataset. And we clarified the effects of WTAP on HCC cells using cell proliferation assay, colony formation, Edu assay and subcutaneous xenograft experiments. We then applied RNA sequencing combined with gene expression omnibus (GEO) data to screen candidate targets of WTAP. Finally, we investigated the regulatory mechanism of WTAP in HCC by m6A dot blot assay, methylated RNA immunoprecipitation (MeRIP) assay, dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay and Chromatin immunoprecipitation (ChIP) assay. We demonstrated that WTAP was highly expressed in HCC which indicated the poor prognosis, and that WTAP expression served as an independent predictor of HCC survival. Functionally, WTAP promoted the proliferation capability and tumor growth of HCC cells in vitro and in vivo. Furthermore, ETS proto-oncogene 1 (ETS1) was identified as the downstream effector of WTAP. The m6A modification regulated by WTAP led to post-transcriptional suppression of ETS1, with the implication of Hu-Antigen R (HuR) as an RNA stabilizer. Then ETS1 was found to inhibit the progression of HCC and could rescue the phenotype induced by WTAP deficiency. Moreover, WTAP modulated the G2/M phase of HCC cells through a p21/p27-dependent pattern mediated by ETS1. We have identified that WTAP is significantly up-regulated in HCC and promotes liver cancer development. WTAP-guided m6A modification contributes to the progression of HCC via the HuR-ETS1-p21/p27 axis. Our study is the first to report that WTAP-mediated m6A methylation has a crucial role in HCC oncogenesis, and highlights WTAP as a potential therapeutic target of HCC treatment.

Tumor‐associated macrophages (TAMs) are recognized as antitumor suppressors, but how TAMs behave in the hypoxic environment of hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrated that hypoxia inducible factor 1α induced increased expression of triggering receptor expressed on myeloid cells‐1 (TREM‐1) in TAMs, resulting in immunosuppression. Specifically, TREM‐1‐positive (TREM‐1⁺) TAMs abundant at advanced stages of HCC progression indirectly impaired the cytotoxic functions of CD8⁺ T cells and induced CD8⁺ T‐cells apoptosis. Biological and functional assays showed that TREM‐1⁺ TAMs had higher expression of programmed cell death ligand 1 (PD‐L1) under hypoxic environment. However, TREM‐1⁺ TAMs could abrogate spontaneous and PD‐L1‐blockade‐mediated antitumor effects in vivo, suggesting that TREM‐1⁺ TAM‐induced immunosuppression was dependent on a pathway separate from PD‐L1/programmed cell death 1 axis. Moreover, TREM‐1⁺ TAM‐associated regulatory T cells (Tregs) were crucial for HCC resistance to anti‐PD‐L1 therapy. Mechanistically, TREM‐1⁺ TAMs elevated chemokine (C‐C motif) ligand 20 expression through the extracellular signal‐regulated kinase/NF‐κβ pathway in response to hypoxia and tumor metabolites leading to CCR6⁺Foxp3⁺ Treg accumulation. Blocking the TREM‐1 pathway could significantly inhibit tumor progression, reduce CCR6⁺Foxp3⁺ Treg recruitment, and improve the therapeutic efficacy of PD‐L1 blockade. Thus, these data demonstrated that CCR6⁺Foxp3⁺ Treg recruitment was crucial for TREM‐1⁺ TAM‐mediated anti‐PD‐L1 resistance and immunosuppression in hypoxic tumor environment. Conclusion: This study highlighted that the hypoxic environment initiated the onset of tumor immunosuppression through TREM‐1⁺ TAMs attracting CCR6⁺Foxp3⁺ Tregs, and TREM‐1⁺ TAMs endowed HCC with anti‐PD‐L1 therapy resistance.

Background: N6-methyladenosine (m⁶A) modification is an emerging layer of epigenetic regulation which is widely implicated in the tumorigenicity of hepatocellular carcinoma (HCC), offering a novel perspective for investigating molecular pathogenesis of this disease. The role of AlkB homolog 5 (ALKBH5), one of the m⁶A demethylases, has not been fully explored in HCC. Here we clarify the biological profile and potential mechanisms of ALKBH5 in HCC. Methods: Expression of ALKBH5 and its correlation with clinicopathological characteristics of HCC were evaluated using tissue microarrays and online datasets. And biological effects of ALKBH5 in HCC were determined in vitro and in vivo. Subsequently, methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA sequencing (RNA-seq), and following m⁶A dot blot, MeRIP-qPCR, RIP-qPCR or dual luciferase reporter assays were employed to screen and validate the candidate targets of ALKBH5. Results: We demonstrated that ALKBH5 was down-regulated in HCC, and decreased ALKBH5 expression was an independent prognostic factor of worse survival in HCC patients. Functionally, ALKBH5 suppressed the proliferation and invasion capabilities of HCC cells in vitro and in vivo. Mechanistically, ALKBH5-mediated m⁶A demethylation led to a post-transcriptional inhibition of LY6/PLAUR Domain Containing 1 (LYPD1), which could be recognized and stabilized by the m⁶A effector IGF2BP1. In addition, we identified that LYPD1 induced oncogenic behaviors of tumors in contrast to ALKBH5. Dysregulation of ALKBH5/LYPD1 axis impelled the progression of HCC. Conclusion: Our study reveals that ALKBH5, characterized as a tumor suppressor, attenuates the expression of LYPD1 via an m⁶A-dependent manner in HCC cells. Our findings enrich the landscape of m⁶A-modulated tumor malignancy, and provide new insights into potential biomarkers and therapeutic targets of HCC treatment.

Multi-drug resistance is the major cause of chemotherapy failure in hepatocellular carcinoma (HCC). YAP, a critical effector of the Hippo pathway, has been shown to contribute to the progression, metastasis and invasion of cancers. However, the potential role of YAP in mediating drug resistance remains obscure. RT-qPCR and western blot were used to assess YAP expression in HCC cell lines. CCK-8 assays, flow cytometry, a xenograft tumour model, immunochemistry and GFP-mRFP-LC3 fusion proteins were utilized to evaluate the effect of YAP on multi-drug resistance, intracellular ROS production and the autophagy of HCC cells in vitro and in vivo. Autophagy inhibitor and rescue experiments were carried out to elucidate the mechanism by which YAP promotes chemoresistance in HCC cells. We found that BEL/FU, a typical HCC cell line with chemoresistance, exhibited overexpression of YAP. Moreover, the inhibition of YAP by shRNA or verteporfin conferred the sensitivity of BEL/FU cells to chemotherapeutic agents through autophagy-related cell death in vitro and in vivo. Mechanistically, YAP silencing significantly enhanced autophagic flux by increasing RAC1-driven ROS, which contributed to the inactivation of mTOR in HCC cells. In addition, the antagonist of autophagy reversed the enhanced effect of YAP silencing on cell death under treatment with chemotherapeutic agents. Our findings suggested that YAP upregulation endowed HCC cells with multi-drug resistance via the RAC1-ROS-mTOR pathway, resulting in the repression of autophagy-related cell death. The blockade of YAP may serve as a promising novel therapeutic strategy for overcoming chemoresistance in HCC.

Hepatitis B virus (HBV) is one of the major risk factors of hepatocellular carcinoma (HCC). Increasing evidence indicates that microRNA (miRNA)–mRNA axis is involved in HCC. However, a comprehensive miRNA–mRNA regulatory network in HBV-related HCC is still absent. This study aims to identify potential miRNA–mRNA regulatory pathways contributing to pathogenesis of HBV-related HCC. Microarray GSE69580 was downloaded from Gene Expression Omnibus (GEO) database. GEO2R and ‘R-limma’ were used to conduct differential expression analysis. The common miRNAs appeared in the two analytic sets were screened as potential differentially expressed miRNAs (DE-miRNAs). The prognostic roles of screened DE-miRNAs in HCC were further evaluated using Kaplan–Meier plotter database. Target genes of DE-miRNAs were predicted by miRNet. Then, protein–protein interaction (PPI) networks were established for these targets via the STRING database, after which hub genes in the networks were identified by Cytoscape. Functional annotation and pathway enrichment analyses for the target genes were performed through DAVID database. Three enriched pathways related to HBV-related HCC were selected for further analysis and potential target genes commonly appeared in all three pathways were screened. Cytoscape was employed to construct miRNA-hub gene network. The expression and correlation of potential miRNAs and targets were further detected in clinical HBV-related HCC samples by qRT-PCR. 7 upregulated and 9 downregulated DE-miRNAs were accessed. 5 of 7 upregulated DE-miRNAs and 5 of 7 downregulated DE-miRNAs indicated significant prognostic roles in HCC. 2312 and 1175 target genes were predicted for the upregulated and downregulated DE-miRNAs, respectively. TP53 was identified as the hub gene in the PPI networks. Pathway enrichment analysis suggested that these predicted targets were linked to hepatitis B, pathways in cancer, microRNAs in cancer and viral carcinogenesis. Further analysis of these pathways screened 20 and 16 target genes for upregulated and downregulated DE-miRNAs, respectively. By detecting the expression of 36 target genes, six candidate target genes were identified. Finally, a potential miRNA–mRNA regulatory network was established based on the results of qRT-PCR and expression correlation analysis. In the study, potential miRNA–mRNA regulatory pathways were identified, exploring the underlying pathogenesis and effective therapy strategy of HBV-related HCC.