已知环状 RNA (circRNA) 通过调节 microRNA (miRNA) 在各种癌症中发挥竞争性内源 RNA (ceRNA) 的作用。然而，在结直肠癌 (CRC) 中，circ000240/miRNA/mRNA 的确切病理作用仍不确定。我们实验室使用 qRT-PCR 在匹配的 CRC 肿瘤和邻近正常组织样本对中评估 hsa_circ_000240 的表达水平。然后，为了确定 hsa_circ_000240 是否在 CRC 中充当 ceRNA，检索了关联的 miRNA 和基因靶标。使用网络方法对候选基因进行拓扑分析，确定了与 CRC 疾病相关的最关键的中心基因和子网络。利用微阵列和bulk RNA测序分析来综合评估CRC中miRNA和mRNA的表达水平。单细胞 RNA-seq 分析也用于评估细胞水平上显着的总生存 (OS) 基因。 ATAC-seq 数据提供了对候选基因可及染色质区域的深入了解。研究发现 CRC 组织中 hsa_circ_000240 显着上调。三个 miRNA 与目标 circRNA 相互作用。进一步鉴定了由三种 miRNA 调控的 1680 个交叉基因，并且所鉴定的邻近基因的相关功能突出了它们与癌症的相关性。对所构建网络的拓扑分析发现了 33 个在 CRC 中表达显着高的枢纽基因。在这些基因中，CHEK1、CDC6、FANCI、GINS2、MAD2L1、ORC1、RACGAP1 和 SMC4 等 8 个基因已被证明对总体生存有显着影响。单细胞 RNA 测序的利用明确证实了 CRC 患者中 CDC6 和 ORC1 表达水平的增强，以及它们与免疫细胞浸润的显着联系。 ATAC-seq 分析显示，CDC6 和 ORC1 高表达的 Chr2、4 和 12 中的可及性区域发生了改变。 CDC6和ORC1的相关性分析进一步强调了候选基因表达与CTLA4、CD247、TIGIT和CD244等耗竭标记物的关联。候选基因与染色质重塑和组蛋白乙酰化呈正相关。这些表观遗传修饰在影响结直肠癌中 CDC6 和 ORC1 表达后的癌症进展中发挥着重要作用。此外，结果表明，CDC6 启动子区域的甲基化率在 CRC 疾病中升高，证实了 CDC6 的功能重要性及其与 hsa_circ_000240 和相关 ceRNA 在 CRC 中的相互作用。总之，本研究强调了 hsa_circ_000240 作为 CRC 中 ceRNA 的作用。它为进一步剖析 CDC6、ORC1 以及 CRC 治疗的潜在新型表观遗传学和免疫治疗靶点开辟了新途径。© 2023。作者。

Circular RNAs (circRNA) are known to function as competing endogenous RNA (ceRNA) in various cancers by regulating microRNAs (miRNA). However, in colorectal cancer (CRC), the precise pathological role of circ000240/miRNA/mRNA remains indeterminate. The expression level of hsa_circ_000240 was evaluated using qRT-PCR in matching pairs of CRC tumor and adjacent normal tissue samples in our laboratory. Then, to determine whether hsa_circ_000240 acted as a ceRNA in CRC, the linked miRNAs and gene targets were retrieved. Topological analysis of candidate genes using a network approach identified the most critical hub genes and subnetworks related to CRC disease. Microarray and bulk RNA sequencing analyses were utilized to comprehensively evaluate the expression levels of both miRNA and mRNA in CRC. Single-cell RNA-seq analysis was also used to evaluate the significant overall survival (OS) genes at the cellular level. ATAC-seq data provided insights into candidate genes' accessible chromatin regions. The research uncovered a considerable upregulation of hsa_circ_000240 in CRC tissues. Three miRNAs interacted with the target circRNA. One thousand six hundred eighty intersected genes regulated by three miRNAs were further identified, and the relevant functionality of identified neighbor genes highlighted their relevance to cancer. The topological analysis of the constructed network has identified 33 hub genes with notably high expression in CRC. Among these genes, eight, including CHEK1, CDC6, FANCI, GINS2, MAD2L1, ORC1, RACGAP1, and SMC4, have demonstrated a significant impact on overall survival. The utilization of single-cell RNA sequencing unequivocally corroborated the augmented expression levels of CDC6 and ORC1 in individuals with CRC, alongside their noteworthy connection with the infiltration of immune cells. ATAC-seq analyses revealed altered accessibility regions in Chr2, 4, and 12 for CDC6 and ORC1 high-expression. Correlation analysis of CDC6 and ORC1 further highlighted the association of candidate gene expression with exhaustion markers such as CTLA4, CD247, TIGIT, and CD244. The candidate genes exhibit a positive correlation with chromatin remodeling and histone acetylation. These epigenetic modifications play a significant role in influencing the cancer progression following expression of CDC6 and ORC1 in CRC. Additionally, results showed that the methylation rate of the promoter region of CDC6 was elevated in CRC disease, confirming the functional importance of CDC6 and their interaction with hsa_circ_000240 and associated ceRNA in CRC. In conclusion, this study highlights hsa_circ_000240's role as a ceRNA in CRC. It opens new avenues for further dissection of CDC6, ORC1, and underlying novel epigenetics and immunotherapy targets for CRC therapy.© 2023. The Author(s).