RBM15 facilitates laryngeal squamous cell carcinoma progression by regulating TMBIM6 stability through IGF2BP3 dependent

Abstract

Background 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. Yet, the m6A regulatory mechanism underlying the carcinogenesis or progression of LSCC remains poorly understood. Methods The 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. Results The 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. Conclusion Our results revealed the essential roles of RBM15 and IGF2BP3 in m6A methylation modification in LSCC, thus identifying a novel RNA regulatory mechanism.

A comprehensive investigation of m6A regulators prognostic value and associated molecular pathways in breast invasive cancer

“…Our study indicated that high expression of VIRMA, METTL14, and RBM15B, and low expression of EIF3B, YTHDF1, and YTHDF3 is correlated with longer OS, and these genes could serve as markers to predict the prognosis of BIC (Figure 4). There are common and unique genes in the final set of identified genes as prognostic markers in our and the other cancer studies [63,75,79] which highlight the diverse roles of the m 6 A pathway genes in the progression of different human malignancies. To get more insights into the underlying mechanisms, the association of mutation frequency of the genes with prognostic value was examined and nine of the m 6 A pathway genes (METTL14, RBM15, WTAP, HNRNPA2B1, SRSF2, YTHDF1, YTHDF2, YTHDF3, and ALKBH5) were correlated with the prognosis of BIC (Figure S4).…”

The m6A RNA Modification Quantity and mRNA Expression Level of RNA Methylation-Related Genes in Head and Neck Squamous Cell Carcinoma Cell Lines and Patients

“…Accumulating data indicates the significant role of m 6 A RNA modification in the post-transcriptional regulation of gene expression that determines the proper function of crucial physiological processes. Therefore, disturbances in m 6 A RNA methylation may lead to the development of pathological changes, including cancers [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ]. HNSCC initiation, progression, and growth are complex molecular processes encompassing genetic and epigenetic alterations.…”

“…In the case of pancreatic cancer, the higher level of m 6 A modification measured using HPLC/MS was also correlated with patients’ overall survival and with more lymphatic metastases [ 22 ]. Among head and neck cancers, the higher level of m 6 A RNA modification in tumor tissues was found in 34 laryngeal squamous cell carcinoma (LSCC) and paired-matched adjacent normal tissues [ 14 ]. However, the measurements were done with a colorimetric method that is less sensitive than LC-ESI-MS/MS.…”

“…To date, only a few studies indicated the essential regulatory role of m 6 A post-transcriptional modification and RNA methylation-related genes in head and neck cancers [ 14 , 15 , 16 , 17 , 18 ]. In brief, our study quantifies the global m 6 A level in HNSCC cell lines and cancerous and paired-matched histopathologically unchanged tissues and correlates its content with expression of selected RNA methylation methyltransferase (METTL3), demethylase (FTO), and m 6 A binding proteins (YTHDF2, YTHDC2).…”

METTL3 Facilitates Colorectal Carcinoma Progression via Regulating m6A-CRB3-Hippo Axis

“…M6A modi cation is mainly mediated by the m6A methyltransferases, demethylases and reader proteins. The m6A methyltransferases mainly included methyltransferase-like 3 (METTL3) [4], methyltransferaselike 14 (METTL14) [5], and Wilms tumor 1 associated protein (WTAP) [6], RNA binding motif protein 15 [7], and vir like m6A methyltransferase associated [8]. The m6A demethylases mainly included fat-mass and obesity-associated protein (FTO) [9], alkylation repair homolog protein 5 (ALKBH5) [10].…”