O-连接的N-乙酰葡糖胺化的YTHDF2通过m⁶A修饰调控肿瘤抑制基因，从而促进膀胱癌进展。

Wang Li, Ren Da, Cai Zeqiang, Hu Wentao, Chen Yuting, Zhu Xuan

Department of Thoracic Surgery, Second Xiangya Hospital, Central South University, Changsha 410011.

Thoracic Surgery Research Laboratory, Second Xiangya Hospital, Central South University, Changsha 410011.

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2025 May 28;50(5):827-839. doi: 10.11817/j.issn.1672-7347.2025.250202.

OBJECTIVES

Bladder cancer is a common malignancy with high incidence and poor prognosis. N-methyladenosine (mA) modification is widely involved in diverse physiological processes, among which the mA recognition protein YTH N-methyladenosine RNA binding protein F2 (YTHDF2) plays a crucial role in bladder cancer progression. This study aims to elucidate the molecular mechanism by which O-linked -acetylglucosamine (O-GlcNAc) modification of YTHDF2 regulates its downstream target, period circadian regulator 1 (), thereby promoting bladder cancer cell proliferation.

METHODS

Expression of YTHDF2 in bladder cancer was predicted using The Cancer Genome Atlas (TCGA). Twenty paired bladder cancer and adjacent normal tissues were collected at the clinical level. Normal bladder epithelial cells (SV-HUC-1) and bladder cancer cell lines (T24, 5637, EJ-1, SW780, BIU-87) were examined by quantitative real-time PCR (RT-qPCR), Western blotting, and immunohistochemistry for expression of YTHDF2, PER1, and proliferation-related proteins [proliferating cell nuclear antigen (PCNA), minichromosome maintenance complex component 2 (MCM2), Cyclin D1]. was silenced in 5637 and SW780 cells, and cell proliferation was assessed by Cell Counting Kit-8 (CCK-8), colony formation, and EdU assays. Bioinformatics was used to predict glycosylation sites of YTHDF2, and immunoprecipitation (IP) was performed to detect O-GlcNAc modification levels of YTHDF2 in tissues and cells. Bladder cancer cells were treated with DMSO, OSMI-1 (O-GlcNAc inhibitor), or Thiamet G (O-GlcNAc activator), followed by cycloheximide (CHX), to assess YTHDF2 ubiquitination by IP. knockdown and Thiamet G treatment were further used to evaluate mRNA stability, mA modification, and cell proliferation. TCGA was used to predict PER1 expression in tissues; SRAMP predicted potential PER1 mA sites. Methylated RNA immunoprecipitation (MeRIP) assays measured PER1 mA modification. Finally, the effects of knocking down and on 5637 and SW780 cell proliferation were assessed.

RESULTS

expression was significantly upregulated in bladder cancer tissues compared with adjacent tissues (mRNA: 2.5-fold; protein: 2-fold), which O-GlcNAc modification levels increased 3.5-fold (<0.001). YTHDF2 was upregulated in bladder cancer cell lines, and its knockdown suppressed cell viability (<0.001), downregulated PCNA, MCM2, and CyclinD1 (all <0.05), reduced colony numbers 3-fold (<0.01), and inhibited proliferation. YTHDF2 exhibited elevated O-GlcNAc modification in cancer cells. OSMI-1 reduced YTHDF2 protein stability (<0.01) and enhanced ubiquitination, while Thiamet G exerted opposite effects (<0.001). Thiamet G reversed the proliferation-suppressive effects of knockdown, promoting cell proliferation (<0.01) and upregulating PCNA, MCM2, and CyclinD1 (all <0.05). Mechanistically, YTHDF2 targeted PER1 via mA recognition, promoting mRNA degradation. Rescue experiments showed that knockdown reversed the inhibitory effect of knockdown on cell proliferation, upregulated PCNA, MCM2, and Cyclin D1 (all <0.05), and promoted bladder cancer cell proliferation (<0.001).

CONCLUSIONS

O-GlcNAc modification YTHDF2 promotes bladder cancer development by downregulating the tumor suppressor gene through mA-mediated post-transcriptional regulation.

目的

膀胱癌是一种常见的恶性肿瘤，发病率高且预后差。N-甲基腺苷（m⁶A）修饰广泛参与多种生理过程，其中m⁶A识别蛋白YTH N-甲基腺苷RNA结合蛋白F2（YTHDF2）在膀胱癌进展中起关键作用。本研究旨在阐明YTHDF2的O-连接β- N -乙酰葡糖胺（O-GlcNAc）修饰调节其下游靶点周期昼夜节律调节因子1（PER1），从而促进膀胱癌细胞增殖的分子机制。

方法

利用癌症基因组图谱（TCGA）预测YTHDF2在膀胱癌中的表达。临床收集20对膀胱癌及癌旁正常组织。通过定量实时PCR（RT-qPCR）、蛋白质印迹法和免疫组织化学检测正常膀胱上皮细胞（SV-HUC-1）和膀胱癌细胞系（T24、5637、EJ-1、SW780、BIU-87）中YTHDF2、PER1及增殖相关蛋白[增殖细胞核抗原（PCNA）、微小染色体维持蛋白复合体组分2（MCM2）、细胞周期蛋白D1]的表达。在5637和SW780细胞中沉默YTHDF2，通过细胞计数试剂盒-8（CCK-8）、集落形成实验和EdU实验评估细胞增殖。利用生物信息学预测YTHDF2的糖基化位点，并通过免疫沉淀（IP）检测组织和细胞中YTHDF2的O-GlcNAc修饰水平。用二甲基亚砜（DMSO）、OSMI-1（O-GlcNAc抑制剂）或噻美汀G（O-GlcNAc激活剂）处理膀胱癌细胞，随后用环己酰亚胺（CHX）处理，通过IP评估YTHDF2的泛素化。进一步采用YTHDF2敲低和噻美汀G处理评估PER1 mRNA稳定性、PER1 m⁶A修饰及细胞增殖。利用TCGA预测组织中PER1的表达；SRAMP预测潜在的PER1 m⁶A位点。甲基化RNA免疫沉淀（MeRIP）实验检测PER1的m⁶A修饰。最后，评估敲低YTHDF2和PER1对5637和SW780细胞增殖的影响。

结果

与癌旁组织相比，膀胱癌组织中YTHDF2表达显著上调（mRNA：2.5倍；蛋白：2倍），其O-GlcNAc修饰水平增加3.5倍（P<0.001）。YTHDF2在膀胱癌细胞系中上调，敲低YTHDF2可抑制细胞活力（P<0.001），下调PCNA、MCM2和细胞周期蛋白D1（均P<0.05），使集落数减少3倍（P<0.01），并抑制细胞增殖。YTHDF2在癌细胞中的O-GlcNAc修饰升高。OSMI-1降低YTHDF2蛋白稳定性（P<0.01）并增强泛素化，而噻美汀G则产生相反作用（P<0.001）。噻美汀G逆转了YTHDF2敲低对增殖的抑制作用，促进细胞增殖（P<0.01），并上调PCNA、MCM2和细胞周期蛋白D1（均P<0.05）。机制上，YTHDF2通过m⁶A识别靶向PER1，促进PER1 mRNA降解。挽救实验表明，PER1敲低可逆转YTHDF2敲低对细胞增殖的抑制作用，上调PCNA、MCM2和细胞周期蛋白D1（均P<0.05），并促进膀胱癌细胞增殖（P<0.001）。