| Bladder cancer is the second most common malignancy of the urinary system.The occurrence of bladder cancer is a complicated,multi-factor,and multi-step pathological process and is affected by both internal genetic factors and external environmental factors.Although surgical resection is a conventional and effective clinical treatment for non-muscle-invasive bladder cancer(NMIBC),the latter still recurs and metastasises easily.Therefore,the study of the underlying molecular mechanisms of bladder cancer and discovery of new therapeutic targets thereof has great significance.N6-methyladenine(m6A)is one of the most common modifications on the mRNA and ncRNA levels.The process of m6A modification requires the participation of methylation transferases(Writers),demethylases(Erasers),and methylation reading proteins(Readers).The aberrant regulation of this modification is closely associated with the occurrence and development of many malignancies.The fat mass and obesityassociated(FTO)gene is a common m6A eraser that has been proven to have cancerpromoting activity.Some studies have shown that FTO affects tumour progression via regulating microRNA(miRNA)expression.MicroRNAs(miRNAs)are responsible for the regulation of gene expression at the post-transcriptional modification manner by targeting target mRNAs and forming RNA-induced silencing complexes(RISC),thereby regulating cell growth,proliferation,differentiation,and apoptosis.miR-576 is involved in the progression of many cancers.Many researches have verified that m6A could regulate the formation of mRNA and non-coding RNA in a post-transcriptional modification manner.Cyclin-dependent kinase 6(CDK6),a serine/threonine protein kinase,is a member of the cyclin-dependent kinase family.It is involved in G1 progression and the G1/S transition and accelerates progression through the G1/S-phase checkpoint of the cell cycle.CDK6 overexpression increases cell proliferation and reduces DNA repair capacity.The aberrant expression of CDK6 is closely associated with the occurrence and development of many cancers.Based on the above background,this study firstly investigated the expression of FTO in bladder tumor tissue and its relationship with clinical indicators,as well as the effect of FTO expression on the proliferation,migration,invasion,cell cycle and tumorigenic ability;By identifying the molecular signaling pathways,it was verified that FTO regulates bladder cancer cells’ malignant behaviors in an m6A-dependent manner.This study verified the role of FTO in bladder urothelial carcinoma and the related molecular signaling pathway,which could provide a new method to predict the prognosis of patients as well as a potential diagnostic biomarker for bladder cancer.Part one:The expression level and role of FTO in bladder urothelial carcinomaThe purpose of this part:1.To clarify the expression of FTO in bladder urothelial carcinoma;2,To study the relationship between the expression level of FTO and related clinical indicators;3.To explore the effect of FTO on the proliferation,invasion and migration of bladder urothelial carcinoma cells.The results list as follows:1.FTO was obviously upregulated in bladder cancer tissuesThe expression levels of FTO were significantly higher in bladder cancer tissues than in adj acent normal tissues.2.FTO was closely associated with clinicopathological parametersThe tissue microarray analysis revealed that the expression of FTO was correlated with the TNM stage.The T stage was higher in the FTO group with high expression(P=0.035).We predicted the prognosis of FTO in bladder cancer using the Gepia database and found that patients with bladder cancer exhibiting high FTO levels may have a worse prognosis.Moreover,the same conclusion was drawn from the tissue microarray data.3.The expression level of FTO regulated bladder cancer cell proliferation and invasion in vitro and in vivoCCK-8 assay showed that overexpression of FTO could promote cell proliferation,while knockdown of FTO inhibited cell proliferation.Transwell migration and invasion assays showed that overexpression of FTO enhanced cell migration and invasion abilities,while knockdown of FTO reduced the above abilities Flow cytometry analysis showed that FTO overexpression could promote cell cycle transfer into S stage;A subcutaneous xenograft tumour model showed that overexpression of FTO could promote the growth of tumor in mice,while knockdown of FTO inhibited the growth of tumor in mice.Through the in vitro and in vivo experiments above,it was verified that FTO could enhance the abilities of proliferation,migration,invasion and tumorigenesis of bladder urothelial carcinoma cells,as well as regulated the cell cycle.Part two:FTO targeted CDK6 via the FTO/miR-576/CDK6 pathways in an m6A-dependent mannerThe purpose of this part:1.To explore the mechanism by which FTO regulated the synthesis of miR-576;2.To verify that miR-576 could partially rescue the effects of FTO on cell proliferation and clone formation;3.To verify that FTO regulated the expression of CDK6 through the FTO/miR-576/CDK6 axis;4.To explore the relationship between the expression level of CDK6 in bladder urothelial carcinoma and FTO.1.FTO regulated the processing of miR-576Using the LinkedOmics database,we found that 7 miRNAs were predicted to be negatively correlated with FTO in bladder cancer tissues.We verified that only miR576 was altered in T24 and UM-UC-3 cells after FTO overexpression or knockdown by RT-qPCR.The results of RIP assay indicated that FTO regulates the processing of miR-576 in an m6A-depend manner.2.miR-576 rescued the proliferating function induced by FTOWe upregulated miR-576 using miR-576 mimics in stable FTO-overexpressing T24 and UM-UC-3 cells and performed a series of restoration assays.Our results showed that miR-576 could partly reversed the effects of FTO on cell proliferation and colony formation ability.3.FTO targeted CDK6 via the FTO/miR-576/CDK6 pathways in bladder cancerPotential target genes of miR-576 were predicted using databases such as TargetScan and miRDB,we discovered binding sites for miR-576 in the 3’untranslated regions(UTRs)of CDK6 mRNA.The result of dual luciferase reporter assay suggested that CDK6 is a direct downstream target of miR-576.RT-qPCR and western blot assays verified that FTO could regulate the expression of CDK6.Moreover,miR-576 could partly reversed the effects of FTO on the expression of CDK6.4.The expression of CDK6 was positively correlated with FTO in bladder cancer tissuesThe expression of CDK6 increased significantly in bladder cancer tissues compared to corresponding adjacent tissues.A negative correlation was observed between miR-576 and CDK6 expressions,whereas a positive correlation was observed between FTO and CDK6 expressionsin 20 tumour tissues.The IHC of subcutaneous xenograft tumours showed a negative correction between the expression of CDK6,ki67 and FTO.In summary,our research verified that FTO was highly expressed in bladder cancer and associated with poor prognosis in bladder cancer patients.FTO promoted bladder cancer cell proliferation,migration,and invasion via the FTO/miR-576/CDK6 pathways in an m6A-dependent manner.Our work revealed that FTO plays a critical role in bladder cancer and could be a potential diagnostic or prognostic biomarker for this disease. |
