Mechanism of METTL14 regulates HBV-HCC malignant progression by mediating m6A modification of FOXP3 and thus transcriptional activation of ALDOB

Abstract

Hepatocellular carcinoma (HCC) is a severe form of liver malignancy characterized by high incidence and mortality rates, complex etiology, and significant variability in prognosis. Forkhead box P3 (FOXP3), an essential transcription factor, plays a pivotal role in tumorigenesis, progression, and prognosis. This study aims to explore the function and underlying mechanism of FOXP3 in the malignant progression of HCC. The expression level of FOXP3 was predicted using the TNMplot database. The Kaplan-Meier Plotter for Survival Analysis (Kaplan-Meier Plotter) website was utilized to analyze the correlation between gene expression and prognosis. Gene expression levels were determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. The viability, proliferation, apoptosis, and invasion capacities of the cells were detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, flow cytometry, and transwell assay, respectively. The qPCR assay was used to detect the replication of hepatitis B virus (HBV). The enzyme-linked immunosorbent assay (ELISA) was utilized to detect hepatitis B surface antigen (HBsAg). The glucose consumption and lactate production of the cells were measured by special kits. The presence of m6A modification on FOXP3 was jointly predicted using the RNA Modification Base (RMbase) and the sequence-based RNA adenosine methylation site predictor (SRAMP) databases. Relevant predictions were conducted using the Gene Expression Profiling Interactive Analysis (GEPIA) database, the Encyclopedia of RNA Interactomes (ENCORI) database, and The Cancer Genome Atlas (TCGA) database to explore the expression correlation between genes. The N6-methyladenosine (m6A) methylation modification level of the FOXP3 was determined using the methylated RNA immunoprecipitation (MeRIP) assay. RNA immunoprecipitation (RIP) was used to detect the interaction of methyltransferase-like 14 (METTL14) or insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) with FOXP3. After cells were treated with actinomycin D (Act D), mRNA stability was measured by RT-qPCR. The dual luciferase reporter assay and chromatin immunoprecipitation (ChIP) were used to detect the interaction between FOXP3 and aldolase B (ALDOB). Mouse xenograft assays were used for in vivo validation. Gene expression in HCC tumor tissue was measured by immunohistochemistry (IHC) assay. FOXP3 was lowly expressed in HBV-related HCC and was associated with the poor prognosis of patients. Overexpression of FOXP3 inhibited the proliferation, metastasis, and glycolysis of HBV-related HCC cells. METTL14 stabilized FOXP3 mRNA through the m6A-IGF2BP1-dependent manner, and METTL14 inhibited the malignant behaviors of HBV-related HCC cells by targeting FOXP3. As a transcription factor, FOXP3 triggered the activation of transcription of ALDOB, thereby inhibiting the malignant behaviors of HBV-related HCC cells. METTL14 inhibited the tumor growth in vivo by activating FOXP3 expression. METTL14 regulates the malignant progression of HBV-HCC by mediating the m6A modification of FOXP3 in an IGF2BP1-dependent manner, thereby activating ALDOB transcription. This provides new insights into the targeted therapy for HCC.

Keywords: ALDOB; FOXP3; Hepatocellular carcinoma; IGF2BP1; METTL14.