Comprehensive analysis of circRNAs for N7-methylguanosine methylation modification in human oral squamous cell carcinoma.

N7-methylguanosine (m7G) modification is closely related to the occurrence of tumors. However, the m7G modification of circRNAs in oral squamous cell carcinoma (OSCC) remains to be investigated. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to measure the methylation levels of m7G and identify m7G sites in circRNAs in human OSCC and normal tissues. The host genes of differentially methylated and differentially expressed circRNAs were analyzed by Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, and circRNA-miRNA-mRNA networks were predicted using the miRanda and miRDB databases. The analysis identified 2348 m7G peaks in 624 circRNAs in OSCC tissues. In addition, the source of m7G-methylated circRNAs in OSCC was mainly the sense overlap region compared with normal tissues. The most conserved m7G motif in OSCC tissues was CCUGU, whereas the most conserved motif in normal tissues was RCCUG (R = G/A). Importantly, GO enrichment and KEGG pathway analysis showed that the host genes of differentially methylated and differentially expressed circRNAs were involved in many cellular biological functions. Furthermore, the significantly differentially expressed circRNAs were analyzed to predict the circRNA-miRNA-mRNA networks. This study revealed the whole profile of circRNAs of differential m7G methylation in OSCC and suggests that m7G-modified circRNAs may impact the development of OSCC.

Transcriptome Mapping of the Internal N7-Methylguanosine Methylome in Messenger RNAs in Human Oral Squamous Cell Carcinoma.

BACKGROUND: Internal N7-methylguanosine (m7G) methylation in mammalian messenger RNAs (mRNAs) is essential in disease development. However, the status of internally m7G-modified mRNAs in oral squamous cell carcinoma (OSCC) remains poorly understood. METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to identify the m7G modification level of mRNAs and the expression of mRNAs between OSCC and normal tissues. These differentially methylated and expressed genes were subjected to Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and construction of protein-protein interaction (PPI) networks. Quantitative real-time PCR (qPCR) assay was performed to detect the expression of Baculoviral IAP Repeat Containing 3 (BIRC3) in vitro. The biological function of BIRC3 in OSCC was clarified using CCK-8, Transwell migration and Western blot assays. RESULTS: The m7G-mRNA profile showed 9514 unique m7G peaks within 7455 genes in OSCC tissues. In addition, the most conserved m7G motif within mRNAs in OSCC was GGARG (R = G/A). The identified m7G peaks were mainly distributed in the coding sequence region within mRNAs in OSCC. GO enrichment and KEGG pathway analyses showed that m7G-modified genes were closely related to cancer progression. m7G-modified hub genes were screened from the constructed PPI networks. Furthermore, BIRC3 with high m7G methylation showed high expression in OSCC cell lines, as confirmed by qPCR assay. Functionally, the knockdown of BIRC3 significantly inhibited the proliferation and migration ability of CAL-27 cells in vitro functional assays. In addition, the relative expression of E-cadherin expression was elevated, while Vimentin and N-cadherin protein expression was decreased in CAL-27 cells transfected with si-BIRC3. This study suggests that BIRC3 could promote OSCC proliferation and migration, which may be associated with involvement in epithelial-mesenchymal transition (EMT) progression. CONCLUSIONS: This paper constructed a transcriptome map of internal m7G in mRNAs, which provides potential research value to study the role of m7G methylation in OSCC.

Long noncoding RNA lnc-H2AFV-1 promotes cell growth by regulating aberrant m6A RNA modification in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor in the oral and maxillofacial regions, and long noncoding RNAs (lncRNAs) play crucial roles in the occurrence and progression of HNSCC. The lncRNA lnc-H2AFV-1 was found to be upregulated in HNSCC tissues; however, the function of lnc-H2AFV-1 in regulating HNSCC proliferation and the potential molecular mechanism is unclear. The present study evaluated the expression of lnc-H2AFV-1 in HNSCC tissues using quantitative real-time PCR (qPCR) and associated abundant lnc-H2AFV-1 expression with tumor size. Functionally, lnc-H2AFV-1 significantly promoted the proliferation of HNSCC cells in vitro and in vivo. Quantified N6-methyladenosine (m6A) RNA methylation and dot blot assays revealed that total m6A methylation in HNSCC cells was accompanied by lnc-H2AFV-1 expression. Western blotting showed that the expression of methyltransferase-like (METTL) 3 and METTL14 was consistent with that of lnc-H2AFV-1, whereas the expression of demethylase fat mass and obesity-associated (FTO) was contrary to that of lnc-H2AFV-1. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-qPCR revealed that lnc-H2AFV-1 overexpression led to the elevated expression and maximal m6A methylation of intraflagellar transport (IFT) 80 in HNSCC. In addition, METTL3/14 knockdown decreased IFT80 expression. Thus, our findings suggested that lnc-H2AFV-1 might be a biomarker that alters m6A modification by regulating the m6A methylases METTL3/14 and FTO and then mediating the downstream target IFT80 to promote HNSCC progression.