| Background:Cutaneous melanoma is the most aggressive of skin cancer with a worst prognosis,and it represents one of the fastest rising cancers which has the strong ability to invade.There is no effective treatment for melanoma,therefore,it is necessary to screen the gene marker related to melanoma prognosis,and establish a prognostic risk model for melanoma.YTHDF1 is one of the m6 A reader proteins,and plays an important role in tumor growth and development.Micro RNAs are a class of endogenous small noncoding RNAs with 22 nucleotides,which exist in eukaryotes and participate a lot of biological activities of the organism.miR-195-5p is abnormally expressed in variety of tumors and acts as tumor-suppressor gene.But there is no literature reported how miR-195-5p and YTHDF1 influence tumor activities and the relationship between them.Objective:1.Identification of the genetic markers and prognostic value of N6-methyladenosine(m6A)regulators in Cutaneous melanoma.2.Explore the changes of melanoma biological behavior caused by YTHDF1,and we aimed at whether miR-195-5p inhibits the proliferation,cell cycle and apoptosis via regulating YTHDF1.Method:1.Gene expression profiles,copy number variation(CNV)and simple nucleotide variation(SNV)data of patients were obtained from The Cancer Genome Atlas(TCGA)database,m RNA expression profiles were obtained from The GEO database.The variation of m6A-regulated genes in melanoma and its relationship with patient prognosis were analyzed by bioinformatics methods.A risk score model(m6Ascore)was established based on a multivariate regression method,and the prognostic value of m6 AScore was determined by univariate and multivariate Cox survival analyses.Unsupervised cluster analysis of the expression profiles of m6A-regulated genes identified different clinically distinct molecular subtypes,and according to m6A-related characteristic genes established a novel prognostic signature.Moreover,the effectiveness was verified in a test set and external validation set.2.The samples in the GSE112509 dataset were divided into YTHDF1 lowexpression group and YTHDF1 high-expression group,and differentially expressed genes between the two groups were identified by differential analysis,using GO and KEGG pathway enrichment analysis on them.The genes closely related to YTHDF1 expression were obtained by Pearson correlation analysis.After the intersection of the two genes,the differentially expressed genes related to YTHDF1 were obtained,constructing a protein interaction network,and screening hub genes to identify potential downstream target genes of YTHDF1.GEPIA and TCGA databases were used to analyze the difference of YTHDF1 and AURKA expression between normal skin and melanoma tissues;Kaplan-Meier survival curve was used to analyze the difference of overall survival rate of melanoma patients with high and low expression of YTHDF1 and AURKA.A375,A875 and HEM-L cells were detected the expression levels of YTHDF1 by q RT-PCR and Western blot.Establishment of YTHDF1 overexpressed and low-expressed A375 cell line,cell proliferation was detected by CCK-8,colony formation was detected by colony formation assay,cell cycle progression and apoptosis were detected by flow cytometry,and cell migration and invasion abilities were detected by plate scratch assay and transwell assay.q RT-PCR and Western blot validation of the effect of YTHDF1 on AURKA.3.Searching for upstream regulators of YTHDF1,the correlation between miR-195-5p and YTHDF1 were analyzed using Pearson correlation test.The interaction between miR-195-5p and YTHDF1 was explored through dual luciferase assay.A375,A875 and HEM-L cells were detected the expression level of miR-195-5p was detected by q RT-PCR.A375 cells were transfected with miR-195-5p mimics and inhibitor,using q RT-PCR and western blot to detect the effect of miR-195-5p on the expression of YTHDF1.Finally,miRNA recovery assay was used to examine miR-195-5p affects cell proliferation,cell cycle progression,and apoptosis by targeting YTHDF1.Result:1.There is a high degree of genomic variation in m6A-regulated genes in melanoma,and patients with higher frequency of genomic variation have poorer prognosis(p<0.01).Through univariate Cox regression analysis to analyze the expression and prognosis of m6A-regulated genes,we found that four m6A-regulated genes were closely related to the prognosis of patients: YTHDF1,METTL14,WTAP,RBM15.The multivariate regression method was used to establish a risk scoring model(m6AScore).The results of univariate and multivariate Cox analysis showed that m6 AScore was significantly correlated with the prognosis of melanoma patients.Unsupervised cluster analysis of the expression profiles of m6A-regulated genes identified three clinically distinct molecular subtypes,including degradation Enhanced subgroup,immune-enhanced subgroup,they had significant prognostic differences(p=0.046).m6A-related characteristic genes were identified through different genes expression spectrum,and a new prognostic signature was established,which could effectively identify samples with poor prognosis with enhanced immune infiltration,and was verified in the data set GSE65904 and GSE22153 of the chip.2.Bioinformatics technology was used to sort AURKA as the downstream of YTHDF1.GO and KEGG pathway enrichment analysis shows that AURKA is widely involved in processes related to cell proliferation and cell cycle.The expression of YTHDF1 and AURKA was significantly increased in melanoma tissues by TCGA and GEPIA database analysis.Kaplan-Meier survival curve analysis showed that patients with high expression of YTHDF1 and AURKA had a lower overall survival rate than patients with low expression.Compared with human epidermal melanin HEM-L cells,the m RNA and protein levels of YTHDF1 in A375 and A875 were significantly increased.Overexpressing YTHDF1 in A375,the proliferation ability of A375 is enhanced,and cell cycle process is accelerated,apoptosis phenomenon is reduced,and the migration and invasion ability are enhanced.Reducing expression of YTHDF1 in A375 induces inhibiting the cell proliferation arresting cell cycle in G0/G1 phase,increasing the phenomenon of apoptosis,and inhibiting the ability of migrate and invade.Overexpression of YTHDF1 increases AURKA m RNA and protein expression levels,while inhibition of YTHDF1 expression reduces AURKA m RNA and protein expression levels.3.miR-195-5p was an upstream regulatory gene of YTHDF1,Pearson correlation analysis showed that there was a significant negative correlation between the two genes,and luciferase reporter assay showed that miRNA-195-5p could directly target and bind to YTHDF1.The expression of miR-195-5p was low in A375 and A875.Inhibition of miR-195-5p can upregulate the expression of YTHDF1,while overexpression of miR-195-5p significantly inhibited the expression of YTHDF1.Reverse experiments confirmed that miR-195-5p inhibited cell proliferation,cell cycle progression,and promote cell apoptosis by targeting YTHDF1 in A375 cells.conclusions:1.There is a high degree of variation in m6 A regulatory genes in melanoma patients,and its expression and variation level are associated with poor clinical prognosis.Unsupervised clustering of samples based on m6 A regulated gene expression profiles identified molecular subgroups with enhanced degradation and enhanced immunity.Based on differential gene expression profiles,m6 A related signature genes were identified,and a prognostic model related to immune infiltration was established.2.The expression of YTHDF1 and AURKA in skin melanoma tissue are increased,and the high expression is closely related to the poor clinical prognosis of melanoma patients.YTHDF1 may promote the proliferation of melanoma cells,accelerate the cell cycle process,inhibit cell apoptosis,promote cell migration and invasion,and increase the malignancy of melanoma cells through AURKA.3.miR-195-5p inhibits melanoma cell proliferation and cell cycle progression and promotes apoptosis by regulating the expression of YTHDF1 in melanoma cells. |
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