胆管癌（CCA）是指从胆管上皮发展而来的恶性肿瘤的集合。大量临床证据和流行病学观察表明，CCA 的发病率和死亡率均呈上升趋势。手术切除是目前 CCA 唯一可用的治疗方法。然而不幸的是，只有一小部分患者在诊断时能够接受手术。而且，癌症切除后复发率很高，全身治疗的疗效有限。因此，鉴定用于 CCA 靶向分子治疗的新型生物标志物仍然是肿瘤学研究中的一项关键任务。我们的研究表明，RSPO3 的低表达与 CCA 患者较差的生存率相关。我们发现CCA中RSPO3启动子DNA高度甲基化，这与RSPO3的低表达相关。 CCA中RSPO3的表达受到DNA甲基转移酶DNMT3a和DNA去甲基化酶TET1之间的平衡的影响。体外和体内实验表明，使用dCas9DNMT3a靶向RSPO3启动子DNA甲基化促进CCA的致瘤性，而使用dCas9TET1CD靶向RSPO3启动子DNA去甲基化抑制CCA致瘤性。此外，在我们的主要CCA模型中，Rspo3的敲低促进了CCA的进展，而Rspo3的过度表达则抑制了CCA的进展。我们的研究结果表明，RSPO3的甲基化增加和表达减少可能表明CCA的预后不良。通过靶向启动子 DNA 去甲基化恢复 RSPO3 表达可以为 CCA 的精确治疗提供见解。© 2023。作者。

Cholangiocarcinoma (CCA) refers to a collection of malignant tumors that develop from the biliary epithelium. Extensive clinical evidence and epidemiological observations indicate a concerning increase in both the incidence and mortality rates of CCA. Surgical resection is currently the sole available cure for CCA. However, it is unfortunate that only a fraction of patients has access to surgery at the time of diagnosis. Moreover, there is a high incidence of cancer recurrence after resection, and systemic treatments have limited efficacy. Therefore, the identification of novel biomarkers for CCA-targeted molecular therapy remains a crucial task in oncology research.Our study demonstrated that low expression of RSPO3 was associated with poorer survival rates in patients with CCA. We found that the RSPO3 promoter DNA was hypermethylated in CCA, which was correlated with the low expression of RSPO3. The expression of RSPO3 was influenced by the balance between the DNA methyltransferase DNMT3a and the DNA demethylase TET1 in CCA. In vitro and in vivo experiments showed that targeting RSPO3 promoter DNA methylation using dCas9DNMT3a promoted tumorigenicity of CCA, while targeted RSPO3 promoter DNA demethylation using dCas9TET1CD inhibited CCA tumorigenicity. Additionally, in our primary CCA model, knockdown of Rspo3 promoted CCA progression, whereas overexpression of Rspo3 inhibited CCA progression.Our findings suggest that increased methylation and decreased expression of RSPO3 may indicate a poor prognosis in CCA. Restoring RSPO3 expression by targeting promoter DNA demethylation could offer insights for precise treatment of CCA.© 2023. The Author(s).