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MolCancer:m6a去甲基化酶alkbh5介导的DDIT4-AS1上调通过激活mTOR途径维持胰腺癌的干性并抑制化疗敏感性

文献解读

2022-09-26      

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化疗耐药是导致胰腺癌患者预后不良的主要因素,而癌症干性是与化疗耐药相关的最关键因素之一,是非常有前景的癌症治疗方向。然而,癌症干性的确切分子机制尚未完全阐明。


m6A-RNA 免疫沉淀和测序用于筛选 m6A 相关的 mRNA 和 lncRNA。 qRT-PCR 和 FISH 用于分析 DDIT4-AS1 表达。进行球体形成、集落形成、蛋白质印迹和流式细胞术测定以分析 PDAC 细胞的癌症干性和化学敏感性。进行异种移植实验以分析体内肿瘤形成率和生长情况。 RNA测序、蛋白质印迹和生物信息学分析用于鉴定DDIT4-AS1的下游通路。进行 IP、RIP 和 RNA 下拉分析以测试 DDIT4-AS1、DDIT4 和 UPF1 之间的相互作用。生成了源自患者的异种移植物 (PDX) 小鼠模型以评估对 GEM 的化学敏感性。


DDIT4-AS1 被确定为 ALKBH5 的下游目标之一,将 HuR 募集到 m6A 修饰位点对于 DDIT4-AS1 稳定至关重要。 DDIT4-AS1 在 PDAC 中上调,与预后不良呈正相关。 DDIT4-AS1 沉默抑制干性并增强对 GEM(吉西他滨)的化学敏感性。从机制上讲,DDIT4-AS1 通过阻止 SMG5 和 PP2A 与 UPF1 的结合来促进 UPF1 的磷酸化,从而降低 DDIT4 mRNA 的稳定性并激活 mTOR 通路。此外,在 PDX 衍生模型中抑制 DDIT4-AS1 增强了 GEM 对 PDAC 的抗肿瘤作用。ALKBH5 介导的 m6A 修饰导致 PDAC 中 DDIT4-AS1 过表达,并且 DDIT-AS1 通过破坏 DDIT4 和激活 mTOR 通路增加癌症干性并抑制对 GEM 的化学敏感性。针对 DDIT4-AS1 及其通路的方法可能是治疗 PDAC 化疗耐药的有效策略。


Abstract

Background: Chemoresistance is a major factor contributing to the poor prognosis of patients with pancreatic cancer, and cancer stemness is one of the most crucial factors associated with chemoresistance and a very promising direction for cancer treatment. However, the exact molecular mechanisms of cancer stemness have not been completely elucidated.


Methods: m6A-RNA immunoprecipitation and sequencing were used to screen m6A-related mRNAs and lncRNAs. qRT-PCR and FISH were utilized to analyse DDIT4-AS1 expression. Spheroid formation, colony formation, Western blot and flow cytometry assays were performed to analyse the cancer stemness and chemosensitivity of PDAC cells. Xenograft experiments were conducted to analyse the tumour formation ratio and growth in vivo. RNA sequencing, Western blot and bioinformatics analyses were used to identify the downstream pathway of DDIT4-AS1. IP, RIP and RNA pulldown assays were performed to test the interaction between DDIT4-AS1, DDIT4 and UPF1. Patient-derived xenograft (PDX) mouse models were generated to evaluate chemosensitivities to GEM.


Results: DDIT4-AS1 was identified as one of the downstream targets of ALKBH5, and recruitment of HuR onto m6A-modified sites is essential for DDIT4-AS1 stabilization. DDIT4-AS1 was upregulated in PDAC and positively correlated with a poor prognosis. DDIT4-AS1 silencing inhibited stemness and enhanced chemosensitivity to GEM (Gemcitabine). Mechanistically, DDIT4-AS1 promoted the phosphorylation of UPF1 by preventing the binding of SMG5 and PP2A to UPF1, which decreased the stability of the DDIT4 mRNA and activated the mTOR pathway. Furthermore, suppression of DDIT4-AS1 in a PDX-derived model enhanced the antitumour effects of GEM on PDAC.


Conclusions: The ALKBH5-mediated m6A modification led to DDIT4-AS1 overexpression in PDAC, and DDIT-AS1 increased cancer stemness and suppressed chemosensitivity to GEM by destabilizing DDIT4 and activating the mTOR pathway. Approaches targeting DDIT4-AS1 and its pathway may be an effective strategy for the treatment of chemoresistance in PDAC.


原文链接

https://pubmed.ncbi.nlm.nih.gov/36056355/



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