Abstract
BackgroundLaryngeal cancer has the highest mortality rate among head and neck tumours. RNA N6-methyladenosine (m6A) is the most plentiful and variable in mammalian mRNA. Yet, the m6A regulatory mechanism underlying the carcinogenesis or progression of LSCC remains poorly understood.MethodsThe m6A RNA methylation quantification kit was used to detect tissue methylation levels. m6A microarray analysis, mRNA transcriptomic sequencing (mRNA-seq), and proteomics were used to determine RBM15, TMBIM6, and IGF2BP3. Immunohistochemical (IHC), quantitative real-time PCR (qRT-PCR) and Western blot were used to investigate RBM15, TMBIM6, and IGF2BP3 expression in tissue samples and cell lines. The biological effects of RBM15 were detected both in vitro and in vivo. The combination relationship between RBM15/IGF2BP3 and TMBIM6 was verified by RNA immunoprecipitation (RIP) assay, Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNase Mazf, and luciferase report assay. RNase Mazf was used to determine the methylation site on TMBIM6 mRNA. Hoechst staining assay was used to confirm the apoptotic changes. The actinomycin D verified TMBIM6 stability.ResultsThe global mRNA m6A methylation level significantly increased in LSCC patients. RBM15, as a “writer” of methyltransferase, was significantly increased in LSCC and was associated with unfavorable prognosis. The knockdown of RBM15 reduced the proliferation, invasion, migration, and apoptosis of LSCC both in vitro and in vivo. The results were reversed after overexpressing RBM15. Mechanically, TMBIM6 acted as a downstream target of RBM15-mediated m6A modification. Furthermore, RBM15-mediated m6A modification of TMBIM6 mRNA enhanced TMBIM6 stability through IGF2BP3-dependent.ConclusionOur results revealed the essential roles of RBM15 and IGF2BP3 in m6A methylation modification in LSCC, thus identifying a novel RNA regulatory mechanism.
Highlights
Laryngeal cancer has the highest mortality rate among head and neck tumours
RNA binding motif protein 15 (RBM15) is overexpressed in Laryngeal squamous cell carcinoma (LSCC) and associated with poor prognosis To investigate the roles of the m6A-associated genes in LSCC development, we first analyzed the methylation level in 34 LSCC and paired standard samples
The results showed that compared with adjacent non-tumor tissues, there were 841 differentially expressed mRNAs in LSCC (Fig. 1b)
Summary
Laryngeal cancer has the highest mortality rate among head and neck tumours. RNA N6-methyladenosine (m6A) is the most plentiful and variable in mammalian mRNA. It is urgent to find effective molecular markers and reveal the mechanisms underlying LSCC tumorigenesis or development so as to explore more effective strategies for early diagnosis, prognostic evaluation, and treatment. N6-methyladenosine (m6A) is the most abundant RNA modification, which was the first revealed in mRNA and has been recently found in precursor mRNAs, circRNAs, and lncRNAs [5,6,7]. Recent researches have demonstrated that m6A RNA methylation has an essential role in various human diseases such as hypertension [10], cardiac hypertrophy [11], viral infection [12], diabetes [13] and cancers [14]. The RNA m6A expression patterns and their relevant mechanisms in LSCC remain largely unknown
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