The Expression And Function Of YT521-B Homeodomain-Containing Protein 1 In Muscle-Invasive Bladder Cancer

Objective RNA-modifying N6-methyladenosine(m6A)modulators are important for tumourigenesis or progression.YT521-B homeodomain-containing protein 1(YTHDC1)is one of the m6A nuclear readers,but its role in muscle-invasive bladder cancer(MIBC)has not been elucidated.In this study,we screened for differentially expressed and prognostically relevant m6A-regulated genes,verified the expression of YTHDC1 and analyzed the correlation with clinicopathological features,and explored the possible mechanisms of YTHDC1 in the occurrence and development of MIBC.Methods The Cancer Genome Atlas(TCGA)MIBC cohort(n = 408)was used to evaluate the expression and prognostic analysis of previously reported m6A regulatory genes.Real time quantitative polymerase chain reaction(qPCR)or immunohistochemical staining(IHC)was used to validate the YTHDC1 expression in fresh frozen specimens and formalin fixed paraffin embedded(FFPE)specimens from MIBC patients,as well as bladder cancer cell lines T24,UMUC3 and normal urinary tract epithelial cell SV-HUC-1.Combined with YTHDC1 expression and the clinicopathological characteristics of TCGA cohort,bioinformatics analysis was conducted to explore the potential biological function of YTHDC1 in MIBC.From the tumour cell aspect,the target genes in bladder cancer cells after YTHDC1 downregulation were screened by TCGA database RNA-seq data and validated by q PCR.The target gene lncRNA-CYTOR was knocked down in bladder cancer cells by the RNAi MAX transfection method,the proliferation ability of bladder cancer cells was detected by the CCK8 assay,and the change of migration ability of bladder cancer cells after knockdown was observed by the Transwell assay.In terms of immune microenvironment,YTHDC1 expression in the TCGA data database was analyzed with the corresponding tumor immune cell infiltration levels from the TIMER 2.0 database,and YTHDC1 expression was verified with M2 macrophage infiltration by analyzing CD68 and CD163 immunohistochemically stained positive M2 macrophages in clinical paraffin sections with YTHDC1 staining IHC scores in relation to M2 macrophage infiltration.In addition,as for MIBC treatment,we also examined the changes in sensitivities of bladder cancer cells to cisplatin,methotrexate,vincristine,gemcitabine,doxorubicin and paclitaxel after YTHDC1 knockdown by drug sensitivity assay.By analyzing the transcriptome of the anti-PD-L1 immunotherapy trial cohort IMvigor210(n=298),we explored the correlation between YTHDC1 expression and immunotherapy response and prognostic analysis.Results In the TCGA database MIBC cohort,only YTHDC1,one of the 32 known m6A-modified regulatory genes,was downregulated in tumour tissues and positively correlated with prognosis.In clinical samples,YTHDC1 expression was reduced in MIBC tumour tissues compared to paraneoplastic tissues.At the cellular level,both bladder cancer cell lines T24 and UMUC3 had reduced YTHDC1 expression compared to normal uroepithelial cells SV-HUC-1.In terms of clinicopathological features,YTHDC1 downregulation was also associated with Ⅲ/Ⅳ tumour stage,local lymph node metastasis,non-papillary histological type and mutations in key genes such as RB1,ERBB2 and KMT2A,independent of distant metastasis.From the tumor cell aspect,screening by analyzing TCGA database RNA-seq data and validation by q PCR in bladder cancer cells after YTHDC1 knockdown revealed that YTHDC1 negatively regulated the expression of lncRNA-CYTOR.Lnc RNA-CYTOR was highly expressed after YTHDC1 downregulation,while knockdown of lncRNA-CYTOR attenuated bladder cancer cell proliferation and migration ability.From the aspect of immune microenvironment,YTHDC1 expression was found to be negatively associated with M2 macrophage immune infiltration,which may be related to immunosuppressive regulation.Furthermore,in terms of treatment,knockdown of YTHDC1 resulted in increased resistance of bladder cancer cells to methotrexate,vincristine and paclitaxel,but had no effect on cisplatin,gemcitabine and doxorubicin killing.Analysis of transcriptomic data from anti-PD-L1 immunotherapy revealed that YTHDC1 expression did not correlate with anti-PD-L1 immunotherapy response,and anti-PD-L1 immunotherapy did not improve the poor prognosis of patients with low YTHDC1 expression.These results suggest that YTHDC1 downregulation in MIBC has an impact on the biological behaviors and chemotherapeutic drug resistance of tumor cells,as well as on the immune microenvironment;its expression level showed significant correlation with several important pathological features of MIBC.Therefore,YTHDC1 could be a potential prognostic predictor and therapeutic target for MIBC.Conclusion1.YTHDC1 expression is reduced in MIBC,especially in the basal/squamous cell subtype,and lower expression levels are associated with poor prognosis.2.YTHDC1 regulates lncRNA-CYTOR expression,which further affects the proliferation and migration ability of bladder cancer cells.3.YTHDC1 expression level was negatively correlated with M2 macrophage infiltration in MIBC.4.Low expression of YTHDC1 induced increased resistance of bladder cancer cells to chemotherapeutic drugs methotrexate,vincristine and paclitaxel.