基于结直肠印戒细胞癌类器官模型探索DDB2/ATF4功能复合体调控谷氨酰胺代谢影响化疗耐药的机制研究

由于体内外研究模型的缺乏，结直肠印戒细胞癌(SRCC)化疗抵抗的机制鲜有报道。经过探索，申请人成功建立了SRCC类器官研究模型。基于此模型，申请者进行一系列前期研究发现DDB2和ATF4形成功能复合体，调控介导谷氨酰胺摄取的转运体SLC1A5以及分解的关键酶GPT2，激活谷氨酰胺代谢，增强SRCC类器官的干性，可能是SRCC化疗抵抗的重要机制。为了进一步证明上述假设并阐明机制，我们将基于类器官模型采用免疫荧光、共沉淀和GST-pulldown方法验证DDB2和ATF4的相互作用；CHIP/RE-CHIP、Luciferase实验证实DDB2促进ATF4靶向富集于SLC1A5和GPT2启动子区域，激活SLC1A5和GPT2表达；并通过基因干预和临床组织标本重验证DDB2/ATF4-SLC1A5/GPT2通路在SRCC谷氨酰胺代谢和化疗抵抗中的作用机制，为干预SRCC化疗耐药提供新的靶点。

Due to the lack of in vivo and in vitro research models, the mechanism of chemo-resistance in colorectal signet-ring cell carcinoma (SRCC) has been rarely reported. After exploration, the applicant successfully established SRCC organoid research model. Based on this model, the applicant conducted a series of preliminary studies and found that the formation of functional complex of DDB2 and ATF4 can regulate transporter SLC1A5 mediating glutamine uptake and the key enzyme GPT2 for decomposition, activate glutamine metabolism and enhance stemness of SRCC organoids, which may be an important mechanism for SRCC chemo-resistance. In order to further prove the above hypothesis and elucidate the mechanism, the interaction between DDB2 and ATF4 was verified by immunofluorescence, co-precipitation and GST-pulldown techinques based on the organoid model. CHIP/RE-CHIP and Luciferase experiments confirmed that DDB2 promoted the enrichment of ATF4 targeting in SLC1A5 and GPT2 promoter regions, and activated the expression of SLC1A5 and GPT2. Moreover, the mechanism of DDB2/ATF4-SLC1A5/GPT2 pathway in SRCC glutamine metabolism and chemo-resistance was re-verified through gene intervention and clinical tissue samples, which provides a new target for the intervention of SRCC chemo-resistance.