背景:人类羟基类固醇脱氢酶样2(HSDL2),调节癌症进展,参与脂质代谢。然而,HSDL2在胆管癌(CCA)中的作用及其通过调节铁凋亡调节CCA进展的机制尚不清楚。
方法:通过定量实时聚合酶链反应(qRT-PCR)测定CCA细胞和组织中HSDL2的表达水平,西方印迹,和免疫组织化学。高患者的总生存率和无病生存率与使用Kaplan-Meier曲线评估低HSDL2表达。扩散,迁移,使用细胞计数试剂盒-8,集落形成评估CCA细胞的侵袭,5-乙炔基-2'-脱氧尿苷DNA合成,和transwell分析。使用异种移植小鼠模型探索p53对肿瘤生长的影响。采用免疫荧光法分析CCA患者SLC7A11的表达。通过流式细胞术测量铁凋亡水平,丙二醛测定,和谷胱甘肽测定。通过转录组测序分析HSDL2调节的信号通路。使用生物信息学和荧光素酶报告基因测定评估p53和SLC7A11之间的相关性。
结果:HSDL2在原代人CCA组织中的表达低于匹配的邻近非肿瘤胆管组织。HSDL2下调是CCA患者总生存期和无病生存期缩短的重要危险因素。此外,HSDL2敲除增强了增殖,迁移,和CCA细胞的侵袭。对HSDL2敲低细胞的转录组分析表明,差异表达基因在p53信号通路中显著富集,和HSDL2下调增加SLC7A11水平。这些发现与qRT-PCR和蛋白质印迹结果一致。其他实验表明,p53表达在体内和体外调节HSDL2对CCA增殖的影响,并且p53与SLC7A11启动子结合以抑制铁凋亡。
结论:HSDL2敲低通过p53/SLC7A11轴抑制铁凋亡促进CCA进展。因此,HSDL2是CCA的潜在预后标志物和治疗靶标。
BACKGROUND: Human hydroxysteroid dehydrogenase-like 2 (HSDL2), which regulates cancer progression, is involved in lipid metabolism. However, the role of HSDL2 in cholangiocarcinoma (CCA) and the mechanism by which it regulates CCA progression by modulating ferroptosis are unclear.
METHODS: HSDL2 expression levels in CCA cells and tissues were determined by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. The overall survival and disease-free survival of patients with high vs. low HSDL2 expression were evaluated using Kaplan-Meier curves. The proliferation, migration, and invasion of CCA cells were assessed using Cell Counting Kit-8, colony formation, 5-ethynyl-2\'-deoxyuridine DNA synthesis, and transwell assays. The effect of p53 on tumor growth was explored using a xenograft mouse model. The expression of SLC7A11 in patients with CCA was analyzed using immunofluorescence. Ferroptosis levels were measured by flow cytometry, malondialdehyde assay, and glutathione assay. HSDL2-regulated signaling pathways were analyzed by transcriptome sequencing. The correlation between p53 and SLC7A11 was assessed using bioinformatics and luciferase reporter assays.
RESULTS: HSDL2 expression was lower in primary human CCA tissues than in matched adjacent non-tumorous bile duct tissues. HSDL2 downregulation was a significant risk factor for shorter overall survival and disease-free survival in patients with CCA. In addition, HSDL2 knockdown enhanced the proliferation, migration, and invasion of CCA cells. The transcriptome analysis of HSDL2 knockdown cells showed that differentially expressed genes were significantly enriched in the p53 signaling pathway, and HSDL2 downregulation increased SLC7A11 levels. These findings were consistent with the qRT-PCR and western blotting results. Other experiments showed that p53 expression modulated the effect of HSDL2 on CCA proliferation in vivo and in vitro and that p53 bound to the SLC7A11 promoter to inhibit ferroptosis.
CONCLUSIONS: HSDL2 knockdown promotes CCA progression by inhibiting ferroptosis through the p53/SLC7A11 axis. Thus, HSDL2 is a potential prognostic marker and therapeutic target for CCA.