目的：目前，针对局部晚期直肠癌（LARC: locally advanced rectal cancer）患者的标准治疗方案是新辅助放化疗（NCRT: neoadjuvant chemoradiotherapy）联合手术和巩固化疗，或者是全新辅助治疗模式。尽管NCRT使得多数患者肿瘤得到降期，提高了肿瘤控制率及生存预后，然而，仍有部分患者对NCRT抵抗，对于这部分治疗抵抗的患者，NCRT增加了治疗毒副反应和手术并发症的风险。因此，如果能够了解患者治疗抵抗的机制，并提前预测患者治疗疗效，将更好的实现个体化治疗。近年来，环状RNA（circRNA : circular RNA）作为一种特殊的非编码RNA，在癌症的发生、发展及治疗中扮演着重要的角色。CircRNA因其环状结构使得它们较线性RNA更加稳定性，并且具有miRNA分子海绵吸附、与蛋白相互作用以及编码小肽等多种功能，被认为在肿瘤治疗中有着重要的应用价值。然而，尽管我们已经认识到circRNA在癌症中的重要作用，但它们在调控LARC对放疗敏感性的具体机制方面，仍存在着许多未知。因此，深入研究circRNA在LARC放疗敏感性调控中的作用机制，将为改善治疗策略和提高患者疗效提供重要的理论依据。

方法：本研究将NCRT敏感及抵抗的两组患者治疗前组织样本进行全转录组RNA测序，筛选两组间差异显著circRNA分子，并结合实时定量PCR的实验进行验证。通过细胞增殖实验、克隆形成实验、凋亡流式检测、同源重组（HR：Homologous Recombination）和非同源末端连接（NHEJ：Non-Homologous End Joining）报告质粒检测，探究circRNA的功能。在机制研究方面，通过RNA pulldown实验、RIP（RNA Immunoprecipitation）实验、细胞免疫荧光实验、Western Blot实验、银染、质谱等实验确定circRNA相互作用的蛋白分子及作用机制；此外，通过甲基化RIP-qPCR实验、双荧光素酶报告基因实验等，深入探究circRNA的甲基化修饰状态，以及维持circRNA稳定性的分子机制。最后，通过裸鼠皮下成瘤实验，在体内探索靶向该circRNA对于放疗的增敏作用。

结果：CircDNAJC3 (has_circ_0101041)与LARC患者放疗抵抗及不良生存预后密切相关。在体内和体外实验中，我们观察到circDNAJC3显著促进了结直肠癌细胞的增殖能力及克隆形成能力，同时抑制肿瘤细胞凋亡、并促进了NHEJ途径的DNA损伤修复。深入机制研究表明，circDNAJC3与MSH6蛋白相互作用，并且它们的表达量呈正相关。CircDNAJC3干扰了MSH6蛋白与HSP90B1蛋白和Rab11b蛋白的结合，进而抑制了溶酶体对MSH6蛋白的降解。此外，circDNAJC3还促进了MSH6蛋白与NHEJ途径的关键启动蛋白Ku70的相互作用，从而强化了NHEJ途径的修复功能。值得注意的是，circDNAJC3具有m6A甲基化位点，该位点受到去甲基化酶ALKBH5的去甲基化修饰，并可以和甲基阅读蛋白IGF2BP2结合，后者在调控circDNAJC3的稳定性中发挥了关键作用。

结论：本研究首次报道了受ALKBH5/IGF2BP2调控的m6A甲基化修饰circDNAJC3分子在结直肠癌放疗抵抗中的关键作用。circDNAJC3通过干扰MSH6蛋白的溶酶体降解途径，促进了MSH6蛋白与NHEJ途径关键蛋白Ku70的结合，增强了NHEJ途径的修复能力，提高了结直肠癌细胞的放疗抵抗能力。这一发现为我们深入理解LARC治疗抵抗机制提供了新的理论基础。

Purpose: Currently, the standard treatment for locally advanced rectal cancer (LARC) involves neoadjuvant chemoradiotherapy (NCRT) combined with surgery and adjuvant chemotherapy, or totally neoadjuvant treatment. Although NCRT leads to tumor downstaging, improving tumor control rates and survival outcomes for patients, some patients still exhibit resistance to NCRT. For these patients, NCRT increases the risks of therapeutic toxicity and surgical complications. Therefore, understanding the mechanisms of treatment resistance and predicting treatment efficacy in advance would enhance personalized treatment strategies. In recent years, circular RNA (circRNA), a special type of non-coding RNA, has been recognized for its crucial role in cancer initiation, progression, and treatment. Due to their circular structure, circRNAs exhibit increased stability compared to linear RNAs and possess various functions, such as acting as miRNA sponges, interacting with proteins, and encoding peptides, making them potential targets for cancer therapy. Although the significant roles of circRNAs in cancer have been reported, specific mechanisms in regulating the sensitivity of LARC to radiotherapy remain largely unknown, Therefore, exploring the mechanism of circRNAs in modulating the sensitivity of LARC to radiotherapy is crucial for improving treatment strategies and enhancing patient outcomes.

Methods: This study performed whole transcriptome RNA sequencing on pre-treatment tissue samples from two groups of patients with NCRT sensitivity or resistance. It was combined with real-time quantitative PCR experiments to identify significantly expressed circRNAs between the two groups. The functions of circRNA were explored through cell proliferation assays, clonogenic assays, apoptosis flow cytometry, homologous recombination (HR) and non-homologous end joining (NHEJ) reporter assays. In terms of mechanistic investigations, circRNA-protein interactions and their underlying mechanisms were elucidated through experiments such as RNA pulldown, RIP (RNA Immunoprecipitation), cell immunofluorescence, Western Blot, silver staining, mass spectrometry, etc. Moreover, the exploration of circRNA methylation modification status and the molecular mechanisms sustaining circRNA stability was conducted through Methylated RIP-qPCR and dual-luciferase reporter gene assays, etc. Finally, we explored the potential role of targeting circRNA to enhance radiosensitivity in vivo through subcutaneous tumor xenograft experiments in nude mice.

Results: CircDNAJC3 (has_circ_0101041) was found to be closely associated with radiotherapy resistance and poor survival prognosis in LARC patients. CircDNAJC3 significantly promoted the proliferative and clonogenic capabilities of colorectal cancer cells, inhibited tumor cell apoptosis, and facilitated DNA damage repair via the NHEJ pathway both in vitro and in vivo. Mechanistic investigations revealed that circDNAJC3 interacted with MSH6, and their expression levels were positively correlated. CircDNAJC3 interfered the binding of MSH6 to HSP90B1 and Rab11b, resulting in the inhibition of MSH6 degradation through the lysosome degradation pathway. Furthermore, circDNAJC3 enhanced the interaction between MSH6 and the crucial initiator protein Ku70 in the NHEJ pathway, thereby strengthening the repair function of the NHEJ pathway. Notably, m6A methylation modification of circDNAJC3 was identified, and this site is demethylated by demethylase ALKBH5 and can bind to methyl-binding protein IGF2BP2, which plays a key role in regulating the stability of circDNAJC3.

Conclusion: This study firstly reported the crucial role of ALKBH5/IGF2BP2-mediated methylation regulation of circDNAJC3 molecule. CircDNAJC3 stabilized MSH6 by disrupting the lysosome degradation pathway, and facilitated the interaction between MSH6 and the key protein Ku70 in the NHEJ pathway. This process strengthened the repair capacity of the NHEJ pathway, thereby increasing the radiotherapy resistance of colorectal cancer cells. This study provides a novel theoretical basis for enhancing our understanding of the mechanisms underlying treatment resistance in LARC.