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.

Keywords: IGF2BP3; Laryngeal squamous cell cancer (LSCC); N6-methyladenosine (m6A); RNA binding motif protein 15 (RBM15); TMBIM6.

Fig. 1

RBM15 was overexpressed in LSCC and associated with poor prognosis. a Global RNA m6A methylation was evaluated by the m6A RNA Methylation Quantification Kit. b Heatmap of differentially expressed genes in LSCC identified by mRNA-seq. c Protein Cluster Analysis heatmap of significantly differentially expressed proteins in LSCC tissues and adjacent nontumor tissue. d qRT-PCR assay verified the expression of RBM15 in 34 pairs of LSCC specimens. e The expression level of RBM15 was related to the clinicopathological characteristics of LSCC patients. f Expression levels of RBM15 in NHBEC and LSCC cell lines were examined using RT-qPCR. g Kaplan–Meier analysis of Overall Survival in HNSC patients. h Kaplan-Meier survival analysis of 122 LSCC tumour specimens showed that high RBM15 expression levels were significantly associated with poor OS. i Representative image of IHC staining of RBM15 in 122 LSCC tumour specimens

Fig. 2

Interference of the expression of RBM15 negatively affected migration and invasion in LSCC cells. a The expression efficiency of RBM15 after transfection with shCtrl or shRBM15 in LSCC cells. b The expression efficiency of RBM15 after transfection with Vector or RBM15 in LSCC cells. c Western blot analysis to measure RBM15 protein levels in LSCC cells transfected with shCtrl and/or shRBM15 and/or RBM15 vectors. d-g Wound healing assays were performed to measure the cell migration ability in LSCC cells after transfection with shCtrl and/or shRBM15 and/or RBM15 vectors. h-k Transwell assays were conducted to examine the cell migration and invasion ability in LSCC cells after transfection with shCtrl and/or shRBM15 and/or RBM15 vectors

Fig. 3

RBM15 regulates LSCC growth and apoptosis in mice. a Inhibition of RBM15 impaired the growth of xenografted tumors. b Overexpression of RBM15 stimulated the growth of xenografted tumors. c and d Representative images of TUNEL staining quantification of RBM15 positive staining in xenografted tumours after transfection with shCtrl and/or shRBM15 and/or RBM15 vectors

Fig. 4

RBM15 participated in the methylation of TMBIM6 in the form of m6A modification. a The methylated RNA (m6A) level was determined after RBM15 knockdown in the LSCC cell lines. b Hierarchical clustering for mRNAs with differential “m6A quantity”. c Coalition analysis of significant changes in RNA expression level and m6A level in pairs of LSCC specimens. d The expression of selected mRNAs was investigated by qRT-PCR in AMC-HN-8 cells transfected with shCtrl or shRBM15. e The expression of selected mRNAs was investigated by qRT-PCR in TU-212 cells transfected with vector or RBM15-expressing plasmid. f MeRIP assay was performed to identify whether m6A modification was enriched in the TMBIM6 sequence. g and h MeRIP assays were performed to identify variation in m6A modification enrichment in TMBIM6 after silencing or overexpressing RBM15 in LSCC cells. i and j qRT-PCR analysis of TMBIM6 mRNA after RBM15 inhibition or overexpression. k Diagram summarizing the mechanism of MazF RNA endonuclease. MazF cleaves only nonmethylated RNA, sites with m6A methylation remained uncleaved. l A schematic diagram showing the methylation site on TMBIM6 and mutates this site. m The luciferase activities in AMC-HN-8 cells co-transfected with TMBIM6-WT or TMBIM6-Mut together with shCtrl or shRBM15. n The luciferase activities in TU-212 cells co-transfected with TMBIM6-WT or TMBIM6-Mut together with Vector or RBM15. o and p qRT-PCR verified the expression of TMBIM6 in 34 pairs of LSCC tissues and cell lines. q Correlation analysis was conducted on the expression levels of RBM15 and TMBIM6 in 34 cases of LSCC tissues. r Correlation analysis was performed on the expression levels of RBM15 and TMBIM6 in the TCGA database. s The analysis results of the TCGA database showed the relationship between the expression level of TMBIM6 in HNSC patients and the overall survival rate. t The expression efficiency of TMBIM6 after transfection with shCtrl or shTMBIM6 in LSCC cells. u The expression efficiency of TMBIM6 after transfection with Vector or TMBIM6 in LSCC cells

Fig. 5

RBM15 accelerates LSCC malignant progression by upregulating TMBIM6. a Western blot was performed to investigate the expression of RBM15 and TMBIM6 in AMC-HN-8 cells after transfection with shRBM15–2 and/or TMBIM6. b Western blot was conducted to examine the expression of RBM15 and TMBIM6 proteins in TU-212 cells after transfection with shTMBIM6–2 and/or RBM15. c Transwell assay was performed to measure the cell invasion ability of AMC-HN-8 cells after transfection with shRBM15–2 and/or TMBIM6. Hoechst staining assay was performed to measure cell apoptosis of AMC-HN-8 cells after transfection with shRBM15–2 and/or TMBIM6. d Transwell assay was performed to measure the cell invasion ability of TU-212 cells after transfection with shTMBIM6–2 and/or RBM15. Hoechst staining assay was performed to measure the cell apoptosis of TU-212 cells after transfection with shTMBIM6–2 and/or RBM15. e Knockdown of RBM15 inhibited TMBIM6-induced AMC-HN-8 cells subcutaneously tumor growth in nude mice. f Knockdown of TMBIM6 inhibited RBM15-induced TU-212 cells subcutaneously tumor growth in nude mice

Fig. 6

IGF2BP3 was involved in m6A methylation modification in LSCC. a RIP-qPCR was showing the enrichment of IGF2BP3 binding to TMBIM6 m6A modification sites. b and c After knocked down or overexpressed IGF2BP3, qRT-PCR evaluated the expression of IGF2BP3 in LSCC cells. d and e MeRIP assays were performed to identify variation in m6A modification enrichment in TMBIM6 after silencing or overexpressing IGF2BP3 in LSCC cells. f and g qRT-PCR of TMBIM6 mRNA after IGF2BP3 inhibition or overexpression in LSCC cells. h RIP-qPCR unveiled the interaction within IGF2BP3 and TMBIM6 mRNA after RBM15 inhibition. i The luciferase activities in AMC-HN-8 cells co-transfected with TMBIM6-WT or TMBIM6-Mut together with shCtrl or shIGF2BP3. j The luciferase activities in TU-212 cells co-transfected with TMBIM6-WT or TMBIM6-Mut together with Vector or IGF2BP3. k qRT-PCR was used to detect IGF2BP3 expression in 34 pairs of LSCC tissues. l Results based on the TCGA and GEPIA databases showed the expression level of IGF2BP3 in HNSC. m IHC staining of IGF2BP3 in LSCC tumour specimens. n Kaplan-Meier survival analysis showed that the expression level of IGF2BP3 in LSCC patients was significantly related to the OS. o and p Data from the TCGA database showed that the expression level of IGF2BP3 in HNSC patients was significantly related to OS and RFS. q Correlation analysis was conducted on the expression levels of TMBIM6 and IGF2BP3 in 34 cases of LSCC tissues. r Correlation analysis was performed on the expression levels of TMBIM6 and IGF2BP3 in the TCGA database

Fig. 7

m6A modification of TMBIM6 mRNA enhances TMBIM6 stability through IGF2BP3-dependent. a Western blot was performed to investigate the expression of TMBIM6 and IGF2BP3 in AMC-HN-8 cells after transfection with shTMBIM6–2 and/or IGF2BP3. b Western blot was conducted to examine the expression of TMBIM6 and IGF2BP3 in TU-212 cells after transfection with shIGF2BP3–1 and/or TMBIM6. c Transwell assay was performed to measure the cell invasion ability of AMC-HN-8 cells after transfection with shTMBIM6–2 and/or IGF2BP3. Hoechst staining assay was performed to measure cell apoptosis of AMC-HN-8 cells after transfection with shTMBIM6–2 and/or IGF2BP3. d Transwell assay was performed to measure the cell invasion ability of TU-212 cells after transfection with shIGF2BP3–1 and/or TMBIM6. Hoechst staining assay was performed to measure cell apoptosis of TU-212 cells after transfection with shIGF2BP3–1 and/or TMBIM6. e-h RNA stability assay showed that different transfection groups have a different effect on the half-life of TMBIM6 mRNA

Fig. 8

Western blot results suggested that after both RBM15 and IGF2BP3 were knocked down in AMC-HN-8 cells, the TMBIM6 protein level was significantly reduced, and the TMBIM6 protein expression increased after both RBM15 and IGF2BP3 were overexpressed, but was restored after the knockdown of RBM15 or IGF2BP3