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Abstract：
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.
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.
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.
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.
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.
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.
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.
摘要：
背景：化疗耐药是导致胰腺癌患者预后不良的主要因素，而癌症的干性是与化疗耐药相关的最关键因素之一，也是癌症治疗的一个非常有前途的方向。然而,癌症干性的确切分子机制尚未完全阐明。
方法：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。球样形成,菌落形成,进行蛋白质印迹和流式细胞术测定以分析PDAC细胞的癌症干细胞性和化学敏感性。进行异种移植实验以分析体内肿瘤形成率和生长。RNA测序，使用蛋白质印迹和生物信息学分析来鉴定DDIT4-AS1的下游途径。IP,进行RIP和RNA下拉测定以测试DDIT4-AS1、DDIT4和UPF1之间的相互作用。产生患者来源的异种移植(PDX)小鼠模型以评估对GEM的化学敏感性。
结果：DDIT4-AS1被确定为ALKBH5的下游靶标之一，将HuR募集到m6A修饰的位点对于DDIT4-AS1的稳定至关重要。DDIT4-AS1在PDAC中上调，与不良预后呈正相关。DDIT4-AS1沉默抑制干性并增强对GEM(吉西他滨)的化学敏感性。机械上,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DDIT4mRNA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。
结论：ALKBH5介导的m6A修饰导致PDAC中DDIT4-AS1过表达，和DDIT-AS1通过使DDIT4不稳定和激活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化学耐药的有效策略。
方法：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。球样形成,菌落形成,进行蛋白质印迹和流式细胞术测定以分析PDAC细胞的癌症干细胞性和化学敏感性。进行异种移植实验以分析体内肿瘤形成率和生长。RNA测序，使用蛋白质印迹和生物信息学分析来鉴定DDIT4-AS1的下游途径。IP,进行RIP和RNA下拉测定以测试DDIT4-AS1、DDIT4和UPF1之间的相互作用。产生患者来源的异种移植(PDX)小鼠模型以评估对GEM的化学敏感性。
结果：DDIT4-AS1被确定为ALKBH5的下游靶标之一，将HuR募集到m6A修饰的位点对于DDIT4-AS1的稳定至关重要。DDIT4-AS1在PDAC中上调，与不良预后呈正相关。DDIT4-AS1沉默抑制干性并增强对GEM(吉西他滨)的化学敏感性。机械上,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DDIT4mRNA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。
结论：ALKBH5介导的m6A修饰导致PDAC中DDIT4-AS1过表达，和DDIT-AS1通过使DDIT4不稳定和激活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化学耐药的有效策略。
