WTAP/IGF2BP3 介导的 GBE1 表达通过上调 c-Myc 加速胰腺癌细胞的增殖并增强其干性。

WTAP/IGF2BP3-mediated GBE1 expression accelerates the proliferation and enhances stemness in pancreatic cancer cells via upregulating c-Myc.

机构信息

Zhejiang Key Laboratory of Tumor Molecular Diagnosis and Individualized Medicine, Zhejiang Provincial People's Hospital, Affiliated People's Hospital, Hangzhou Medical College, Hangzhou, China.

General Surgery, Cancer Center, Department of Gastrointestinal and Pancreatic Surgery, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China.

出版信息

Cell Mol Biol Lett. 2024 Jul 3;29(1):97. doi: 10.1186/s11658-024-00611-8.

DOI:10.1186/s11658-024-00611-8

PMID:38961325

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11223412/

Abstract

BACKGROUND

Pancreatic cancer (PC) is one of the most malignant cancers with highly aggressiveness and poor prognosis. N6-methyladenosine (m6A) have been indicated to be involved in PC development. Glucan Branching Enzyme 1 (GBE1) is mainly involved in cell glycogen metabolism. However, the function of GBE1 and Whether GBE1 occurs m6A modification in PC progression remains to be illustrated.

METHODS

The clinical prognosis of GBE1 was analyzed through online platform. The expression of GBE1 was obtained from online platform and then verified in normal and PC cell lines. Lentivirus was used to generated GBE1 stable-overexpression or knockdown PC cells. Cell Counting Kit (CCK-8), colony formation assay, sphere formation assay and flow cytometry assay were conducted to analyze cell proliferation and stemness ability in vitro. Subcutaneous and orthotopic mouse models were used to verify the function of GBE1 in vivo. RNA immunoprecipitation (RIP) assay, RNA stability experiment and western blots were conducted to explore the molecular regulation of GBE1 in PC.

RESULTS

GBE1 was significantly upregulated in PC and associated with poor prognosis of PC patients. Functionally, GBE1 overexpression facilitated PC cell proliferation and stemness-like properties, while knockdown of GBE1 attenuated the malignancy of PC cells. Importantly, we found the m6A modification of GBE1 RNA, and WTAP and IGF2BP3 was revealed as the m6A regulators to increase GBE1 mRNA stability and expression. Furthermore, c-Myc was discovered as a downstream gene of GBE1 and functional rescue experiments showed that overexpression of c-Myc could rescue GBE1 knockdown-induced PC cell growth inhibition.

CONCLUSIONS

Our study uncovered the oncogenic role of GBE1/c-Myc axis in PC progression and revealed WTAP/IGF2BP3-mediated m6A modification of GBE1, which highlight the potential application of GBE1 in the targeted therapy of PC.

摘要

背景

胰腺癌（PC）是最具侵袭性和预后不良的恶性肿瘤之一。N6-甲基腺苷（m6A）已被证明参与 PC 的发展。葡聚糖分支酶 1（GBE1）主要参与细胞糖原代谢。然而，GBE1 的功能以及 GBE1 是否在 PC 进展中发生 m6A 修饰仍有待说明。

方法

通过在线平台分析 GBE1 的临床预后。从在线平台获取 GBE1 的表达，并在正常和 PC 细胞系中进行验证。使用慢病毒生成 GBE1 稳定过表达或敲低 PC 细胞。细胞计数试剂盒（CCK-8）、集落形成实验、球体形成实验和流式细胞术分析体外细胞增殖和干性能力。皮下和原位小鼠模型用于体内验证 GBE1 的功能。RNA 免疫沉淀（RIP）实验、RNA 稳定性实验和 Western blot 用于探索 GBE1 在 PC 中的分子调控。

结果

GBE1 在 PC 中显著上调，与 PC 患者的预后不良相关。功能上，GBE1 过表达促进 PC 细胞增殖和干性样特性，而 GBE1 敲低则减弱 PC 细胞的恶性程度。重要的是，我们发现了 GBE1 RNA 的 m6A 修饰，WTAP 和 IGF2BP3 被揭示为增加 GBE1 mRNA 稳定性和表达的 m6A 调节剂。此外，c-Myc 被发现是 GBE1 的下游基因，功能挽救实验表明，过表达 c-Myc 可以挽救 GBE1 敲低诱导的 PC 细胞生长抑制。