Cholangiocarcinoma represents the second most common primary liver malignancy. The incidence rate has constantly increased over the last decades. Cholangiocarcinoma silent nature limits early diagnosis and prevents efficient treatment. Immunoblotting and immunohistochemistry were used to assess the expression profiling of USP9X and EGLN3 in cholangiocarcinoma patients. ShRNA was used to silence gene expression. Cell apoptosis, cell cycle, CCK8, clone formation, shRNA interference and xenograft mouse model were used to explore biological function of USP9X and EGLN3. The underlying molecular mechanism of USP9X in cholangiocarcinoma was determined by immunoblotting, co-immunoprecipitation and quantitative real time PCR (qPCR). Here we demonstrated that USP9X is downregulated in cholangiocarcinoma which contributes to tumorigenesis. The expression of USP9X in cholangiocarcinoma inhibited cell proliferation and colony formation in vitro as well as xenograft tumorigenicity in vivo. Clinical data demonstrated that expression levels of USP9X were positively correlated with favorable clinical outcomes. Mechanistic investigations further indicated that USP9X was involved in the deubiquitination of EGLN3, a member of 2-oxoglutarate and iron-dependent dioxygenases. USP9X elicited tumor suppressor role by preventing degradation of EGLN3. Importantly, knockdown of EGLN3 impaired USP9X-mediated suppression of proliferation. USP9X positively regulated the expression level of apoptosis pathway genes de through EGLN3 thus involved in apoptosis of cholangiocarcinoma. These findings help to understand that USP9X alleviates the malignant potential of cholangiocarcinoma through upregulation of EGLN3. Consequently, we provide novel insight into that USP9X is a potential biomarker or serves as a therapeutic or diagnostic target for cholangiocarcinoma.

中文翻译：

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USP9X通过去泛素化EGLN3调节KIF1Bβ的表达促进胆管癌细胞凋亡

胆管癌是第二常见的原发性肝脏恶性肿瘤。在过去的几十年中，发病率不断增加。胆管癌的沉默性质限制了早期诊断并阻碍了有效治疗。免疫印迹和免疫组织化学用于评估 USP9X 和 EGLN3 在胆管癌患者中的表达谱。ShRNA 用于沉默基因表达。通过细胞凋亡、细胞周期、CCK8、克隆形成、shRNA干扰和异种移植小鼠模型探索USP9X和EGLN3的生物学功能。USP9X 在胆管癌中的潜在分子机制是通过免疫印迹、免疫共沉淀和定量实时 PCR (qPCR) 确定的。在这里，我们证明 USP9X 在胆管癌中下调，这有助于肿瘤发生。USP9X 在胆管癌中的表达抑制了体外细胞增殖和集落形成以及体内异种移植物的致瘤性。临床数据表明 USP9X 的表达水平与良好的临床结果呈正相关。机制研究进一步表明 USP9X 参与了 EGLN3 的去泛素化，EGLN3 是 2-酮戊二酸和铁依赖性双加氧酶的成员。USP9X 通过阻止 EGLN3 的降解引发肿瘤抑制作用。重要的是，EGLN3 的敲低损害了 USP9X 介导的增殖抑制。USP9X通过EGLN3正向调节凋亡通路基因de的表达水平，从而参与胆管癌的凋亡。这些发现有助于理解 USP9X 通过上调 EGLN3 减轻胆管癌的恶性潜能。因此，我们对 USP9X 是一种潜在的生物标志物或作为胆管癌的治疗或诊断靶点提供了新的见解。