Weiwei Jin, Yanru Yao, Yuhan Fu, Xiangxiang Lei, Wen Fu, Qiliang Lu, Xiangmin Tong, Qiuran Xu, Wei Su, Xiaoge Hu
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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.</p><p><strong>Results: </strong>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. 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引用次数: 0
摘要
背景：胰腺癌（PC）是侵袭性极强、预后极差的恶性肿瘤之一。有研究表明，N6-甲基腺苷（m6A）与胰腺癌的发病有关。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是否会在PC进展过程中发生m6A修饰仍有待说明：方法：通过在线平台对 GBE1 的临床预后进行分析。方法：通过在线平台分析了 GBE1 的临床预后，并在正常细胞系和 PC 细胞系中验证了 GBE1 的表达。使用慢病毒生成 GBE1 稳定表达或敲除的 PC 细胞。采用细胞计数试剂盒（CCK-8）、集落形成试验、球形成试验和流式细胞术分析体外细胞增殖和干性能力。采用皮下和正位小鼠模型来验证 GBE1 在体内的功能。通过RNA免疫沉淀（RIP）实验、RNA稳定性实验和Western印迹来探讨GBE1在PC中的分子调控：结果：GBE1在PC中明显上调，并与PC患者的不良预后相关。结果：GBE1在PC中明显上调，并与PC患者的不良预后相关。在功能上，GBE1的过表达促进了PC细胞的增殖和类干细胞特性，而敲除GBE1则会减轻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是增加GBE1 mRNA稳定性和表达的m6A调控因子。此外，研究还发现c-Myc是GBE1的下游基因，功能拯救实验表明，过表达c-Myc可拯救GBE1敲除诱导的PC细胞生长抑制：我们的研究发现了GBE1/c-Myc轴在PC进展中的致癌作用，并揭示了WTAP/IGF2BP3介导的GBE1的m6A修饰，这凸显了GBE1在PC靶向治疗中的潜在应用。
WTAP/IGF2BP3-mediated GBE1 expression accelerates the proliferation and enhances stemness in pancreatic cancer cells via upregulating c-Myc.
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.
期刊介绍：
Cellular & Molecular Biology Letters is an international journal dedicated to the dissemination of fundamental knowledge in all areas of cellular and molecular biology, cancer cell biology, and certain aspects of biochemistry, biophysics and biotechnology.
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