Identification and Validation of the N6-Methyladenosine RNA Methylation Regulator YTHDF1 as a Novel Prognostic Marker and Potential Target for Hepatocellular Carcinoma

Abstract

Purpose: N6-methyladenosine (m6A) RNA methylation has been implicated in various malignancies. This study aimed to identify the m6A methylation regulator-based prognostic signature for hepatocellular carcinoma (HCC) as well as provide candidate targets for HCC treatment.Methods: The least absolute shrinkage and selection operator (LASSO) analyses were performed to identify a risk signature in The Cancer Genome Atlas (TCGA) datasets. The risk signature was further validated in International Cancer Genome Consortium (ICGC) and Pan-Cancer Analysis of Whole Genomes (PCAWG) datasets. Following transfection of short hairpin RNA (shRNA) targeting YTHDF1, the biological activities of HCC cells were evaluated by Cell Counting Kit-8 (CCK-8), wound-healing, Transwell, flow cytometry, and xenograft tumor assays, respectively. The potential mechanisms mediated by YTHDF1 were predicted by overrepresentation enrichment analysis (ORA)/gene set enrichment analysis (GSEA) and validated by Western blotting.Results: Overexpression of m6A RNA methylation regulators was correlated with malignant clinicopathological characteristics of HCC patients. The Cox regression and LASSO analyses identified a risk signature with five m6A methylation regulators (KIAA1429, ZC3H13, YTHDF1, YTHDF2, and METTL3). In accordance with HCC cases in TCGA, the prognostic value of risk signature was also determined in ICGC and PCAWG datasets. Following analyzing the expression and clinical implications in TCGA and Gene Expression Omnibus (GEO), YTHDF1 was chosen for further experimental validation. Knockdown of YTHDF1 significantly inhibited the proliferation, migration, and invasion of HCC cells, as well as enhanced the apoptosis in vitro. Moreover, silencing YTHDF1 repressed the growth of xenograft tumors in vivo. Mechanism investigation indicated that YTHDF1 might promote the aggressive phenotypes by facilitating epithelial–mesenchymal transition (EMT) and activating AKT/glycogen synthase kinase (GSK)-3β/β-catenin signaling.Conclusion: The current study identified a robust risk signature consisting of m6A RNA methylation regulators for HCC prognosis. In addition, YTHDF1 was a potential molecular target for HCC treatment.

YTHDF1 promotes hepatocellular carcinoma progression via activating PI3K/AKT/mTOR signaling pathway and inducing epithelial-mesenchymal transition

“…Our study confirmed this promotion effect of YTHDF1 in the other two HCC cell lines Huh7 and MHCC-97H, which to a certain extent ruled out the cell dependence of YTHDF1 on this carcinogenic role of HCC. Interestingly, our study also demonstrated that YTHDF1 facilitated HCC cell cycle progression, which was predicted and indicated by previous studies [28,29,36]. The PI3K/AKT/mTOR signaling pathway plays a broad spectrum role in physiology, and its dysregulation often leads to multiple diseases, including cancer [37][38][39][40].…”

“…The abnormal activation of this well-characterized pathway affects cell proliferation and metabolism, and leads to less tumor differentiation, poor prognosis, and earlier recurrence in HCC, but has nothing to do with the underlying etiology of HCC [43]. Based on the previous study [36], we found that the PI3K/AKT/mTOR signaling pathway might be associated with the carcinogenic role of YTHDF1 in HCC by using bioinformatic GSEA. Then, the results of western blot assay indicated that the downregulation of YTHDF1 significantly decreased the expression of key pathway proteins AKT, p-AKT, mTOR, and p-mTOR.…”

“…Similarly, multiple studies have shown that YTHDF1 was significantly up-regulated in HCC and was associated with poor survival of patients with HCC by using bioinformatics and tissue samples analysis [28][29][30][31][32][33][34][35]. Previous studies demonstrated that YTHDF1 facilitated the proliferation, migration, and invasion of MHCC-LM3 and HepG2 two HCC cells [16,36]. Our study confirmed this promotion effect of YTHDF1 in the other two HCC cell lines Huh7 and MHCC-97H, which to a certain extent ruled out the cell dependence of YTHDF1 on this carcinogenic role of HCC.…”

Analysis and Validation of circRNA-miRNA Network in Regulating m6A RNA Methylation Modulators Reveals CircMAP2K4/miR-139-5p/YTHDF1 Axis Involving the Proliferation of Hepatocellular Carcinoma

“…YTHDF1 high expression was correlated with high pathological grade and advanced stage, which indicates YTHDF1 may be involved in the migration and metastasis of HCC. Indeed, previous studies have confirmed that upregulation of YTHDF1 improve the migratory and invasive capabilities of HCC cells ( 30 , 31 ), which provide clues for the investigation of circMAP2K4/miR-139-5p/YTHDF1 axis in the migration and metastasis of HCC in our future study.…”

“…As an m 6 A reader, YTHDF is also involved in the occurrence of HCC. YTHDF1 promotes HCC progression by enhancing FZD5 mRNA translation or AKT/GSK-3β/β-catenin signaling activation ( 30 , 31 ). YTHDF2 is involved in the decay of IL11 and Serpine2 mRNA, which are important genes that regulate the normalization and inflammation of vessels ( 32 ).…”