敲低 YAP1 减少 YTHDF3 的表达，从而稳定并抑制膀胱癌干细胞的干性。

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

机构信息

Department of Urology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Chinese Medicine), 310000 Hangzhou, Zhejiang, China.

Department of Urology, The First Affiliated Hospital of Zhejiang University, 310009 Hangzhou, Zhejiang, China.

出版信息

Discov Med. 2024 Jul;36(186):1486-1498. doi: 10.24976/Discov.Med.202436186.138.

DOI:10.24976/Discov.Med.202436186.138

PMID:39054719

Abstract

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 () N6-methyladenosine (m6A) site were analyzed by bioinformatics. BCSCs were transfected. m6A level, YTH domain-containing family proteins 3 (YTHDF3)- 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//transforming growth factor (TGF)-β1/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-β1, lower expression and greater invasion/migration/tumour sphere formation capabilities than UMUC3 cells. YAP1 knockdown decreased m6A level and impaired YTHDF3- interaction in BCSCs. YAP1 silencing inhibited cell growth/invasiveness/migration/tumour sphere formation and stemness-associated protein/YTHDF3/TGF-β1 expressions while upregulating expression in BCSCs, which was offset by YTHDF3 overexpression.

CONCLUSION

The silencing of YAP1 in BCSCs impedes the YTHDF3-mediated degradation of m6A-modified , culminating in diminished cell stemness.

摘要

背景

之前的研究已经证明了 Yes 相关蛋白 1（YAP1）在膀胱癌（BLCA）中的致癌作用，因此本研究侧重于其对膀胱癌干细胞（BCSCs）的影响及其潜在机制。

方法

通过用顺铂处理人 BLCA 细胞 UMUC3 获得 BCSC，并通过流式细胞术测量 UMUC3/BCSCs 中的 CD133 来鉴定。通过生物信息学分析 YAP1 相互作用蛋白和母系对抗 decapentaplegic 同源物 7（）N6-甲基腺苷（m6A）位点。转染 BCSC。通过甲基化 RNA 免疫沉淀（MeRIP）、RNA 免疫沉淀（RIP）或定量逆转录 PCR（qRT-PCR）分别评估 BCSC 中的 m6A 水平、YTH 结构域家族蛋白 3（YTHDF3）相互作用、YAP1/YTHDF3 表达。通过 5-溴-2-脱氧尿苷（BrdU）染色检测 BCSC 增殖。通过 Transwell 或肿瘤球体形成测定法测定 UMUC3/BCSC 迁移/侵袭和肿瘤球体形成。通过 Western blot 测定法测量 UMUC3/BCSC 中的 YAP1/YTHDF3//转化生长因子（TGF）-β1/干细胞标记物表达。

结果

BCSC 比 UMUC3 细胞具有更高的 CD133 比例、干细胞标记物/YAP1/YTHDF3/TGF-β1 表达、更低的表达和更强的侵袭/迁移/肿瘤球体形成能力。YAP1 敲低降低了 BCSC 中的 m6A 水平并损害了 YTHDF3-相互作用。YAP1 沉默抑制了 BCSC 中的细胞生长/侵袭/迁移/肿瘤球体形成和干细胞相关蛋白/YTHDF3/TGF-β1 表达，同时上调了表达，而 YTHDF3 过表达则抵消了这一作用。