M6A Methyltransferase METTL3 Promotes The Progression Of Acral Melanoma Via Mediating TXNDC5 Methylation

Acral melanoma represents one of the most highly malignant cancers in dermatology clinical practice,is a rare subtype in Caucasians but accounts for more than 70%in Asian population.AM exhibits distinct genetic signatures and clinical outcomes compared with other types of cutaneous melanoma,potentially due to difficulty in early diagnosis and respond poorly to targeted therapy and immunotherapy.One of the important research directions to solve the clinical dilemma is to find powerful biomarkers and therapeutic targets for AM patients.Accumulating evidence confirmed that the mutational load in AM is low,its epigenetic instability occurs early and manifests more frequently than genetic changes.Therefore,epigenetic mechanisms are thought to play an important role in AM pathogenesis.The m6A modification is an important epigenetic mechanism regulating various biological processes.Recently,the rapid development and application of high-throughput sequencing technology further boosted research into the biological significance of m6A modification.Numerous studies have demonstrated that m6A modification plays an important role in tumorigenesis and drug resistance,given its prominence in human cancers.However,the role of m6A modification in AM remains poorly understood.In this study,we found that the overall m6A modification level and the expression of the core methyltransferase METTL3 were upregulated in AM tumor tissue.Both in vitro and in vivo experiments confirmed the pro-oncogenic effect of METTL3 on AM.Further investigation indicated that TXNDC5 was downregulated by decreasing m6A-levels after METTL3 knockdown and ultimately inhibiting the progression of AM.Chapter 1 The expression and significance of RNA m6A modification and METTL3 in AM tissues and cellsThe research in this chapter detected the RNA m6A modification level and the expression of the core methyltransferase METTL3 in AM tissues and cells.We further analyzed the relationship between METTL3 expression and the clinicopathological characteristic of AM patients.Total RNA and protein were extracted from tissues and melanoma cells.RNA m6A modification levels were detected by m6A methylation quantitative kit(colorimetric method)and dot blot assay.The expression of METTL3 at the mRNA and protein levels was detected by qRT-PCR,Western blot,and immunohistochemical.Paired t-test was used to analyze the differential expression of m6A modification and METTL3.Pearson χ2 or Fisher’s exact test was used to analyzing the relationship between METTL3 expression and clinicopathological characteristics.Our results confirmed that the m6A modification level was imbalanced in AM patients.The high expression of METTL3 was associated with tumor Breslow thickness and tumor stage and contributed to the elevated m6A modification level in AM tumor tissues.Thus,the tumor-promoting role of METTL3 in AM has been noted.Chapter 2 Effects of METTL3 knockdown on the biological function of melanoma cellsIn this chapter,we analyzed the effects of METTL3 on the biological functions of melanoma cells in vivo and in vitro.Stable knockdown of METTL3 cell(sh-METTL3)and the non-target control cell(NTC)were constructed by lentivirus transfection.The knockdown efficiency was verified by qRT-PCR and Western blot.Proliferation activity was examined by cell counting,CCK-8 assay,colony formation assay,and EdU staining.The migration ability was detected by wound healing assay and Transwell migration assay.The invasion capability was assessed by Transwell assay with Matrigel.The effect of METTL3 on xenograft tumor formation in vivo was detected by subcutaneous injection of transfected cells in nude mice.The results showed that the knockdown of METTL3 inhibited proliferation,invasion,and migration of A375,A875,and HMY-1 cells in vitro.The volume and weight of the xenografts in the sh-METTL3 group were significantly lower than in the NTC group,further revealing the inhibitory effect of METTL3 depletion on melanoma formation.Chapter 3 METTL3 Targeted TXNDC5 in Melanoma RegulationWe attempt to find the downstream targets of METTL3-m6A and further clarify the regulatory mechanism of METTL3 in this chapter.The RNA m6A modification levels in transfected cells were detected by m6A methylation quantification kit(colorimetric method)and dot blot assay;RNA-seq and MeRIP-seq were performed on NTC and shMETTL3 cells,and differentially expressed genes with decreased m6A peaks was obtained by Peak calling Analysis and gene differential Peak analysis;Gene Product Properties and enrichment pathways were collected by GO and KEGG analysis.The diminished m6A peak of target genes were visualized by IGV software.The expression relationship between METTL3 and TXNDC5 in the TCGA database was analyzed by Pearson correlation analysis.Actinomycin D(ActD)was used for testing RNA stability.The expression of TXNDC5 in AM tissues and melanoma cell lines was determined by qRT-PCR and Western blot.The results showed that the overall m6A level of transfected cells decreased synchronously with METTL3 knockdown.We found a series of genes whose transcripts were subjected to METTL3-mediated m6A modification.We identified that the TXNDC5 expression was decreased both in RNA level and m6A level after METTL3 knockdown.Similarly,the enhanced protein expression of TXNDC5 was also confirmed in melanoma tissues and cell lines.These results suggested that METTL3 may affect the tumorigenesis of melanoma by modulating the stability of TXNDC5 mRNA in an m6A-dependent manner.