Knockdown of YAP1 Reduces YTHDF3 to Stabilize SMAD7 and thus Inhibit Bladder Cancer Stem Cell Stemness

Background: The previous study has proved the oncogenic role of Yes-associated protein 1 (YAP1) in bladder cancer (BLCA), thus this study focused on its impact on bladder cancer stem cells (BCSCs) and underlying mechanism. Method: BCSCs were obtained by treating human BLCA cells UMUC3 with cisplatin and identified by measuring CD133 + in UMUC3/BCSCs via flow cytometry. YAP1 interaction proteins and mothers against decapentaplegic homolog 7 ( SMAD7 ) N6-methyladenosine (m6A) site were analyzed by bioinformatics. BCSCs were transfected. SMAD7 m6A level, YTH domain-containing family proteins 3 (YTHDF3)- SMAD7 interaction, YAP1/YTHDF3 expression in BCSCs were assessed by methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP) or quantitative reverse transcription PCR (qRT-PCR), respectively. BCSC proliferation was detected by 5-Bromo-2-deoxyuridine (BrdU) staining. UMUC3/BCSC migration/invasion and tumour sphere formation were determined by Transwell or tumour sphere formation assays. YAP1/YTHDF3/ SMAD7 /transforming growth factor (TGF)- beta 1/stemness marker expressions in UMUC3/BCSCs were measured by Western blot assay. Result: BCSCs showed higher CD133 + ratio, expressions of stemness marker/YAP1/YTHDF3/TGF- beta 1, lower SMAD7 expression and greater invasion/migration/tumour sphere formation capabilities than UMUC3 cells. YAP1 knockdown decreased SMAD7 m6A level and impaired YTHDF3- SMAD7 interaction in BCSCs. YAP1 silencing inhibited cell growth/invasiveness/migration/tumour sphere formation and stemness-associated protein/YTHDF3/TGF- beta 1 expressions while upregulating SMAD7 expression in BCSCs, which was offset by YTHDF3 overexpression. Conclusion: The silencing of YAP1 in BCSCs impedes the YTHDF3-mediated degradation of m6A-modified SMAD7 , culminating in diminished cell stemness.