Zhang Peizhi, Xu Yingkun, Chen Shaoan, Wang Zicheng, Zhao Leizuo, Chen Chen, Kang Weiting, Han Rongyu, Qiu Jiechuan, Wang Qingliang, Gao Han, Wu Guangzhen, Xia Qinghua
Department of Urology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250021, China.
Department of Endocrine and Breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400042, China.
J Oncol. 2022 Jun 21;2022:2724515. doi: 10.1155/2022/2724515. eCollection 2022.
To investigate the expression of the ADP-ribosylation factor (ARF)-like proteins (ARLs) and ARL4C in clear cell renal cell carcinoma (ccRCC) based on bioinformatics analysis and experimentally determine the effect and mechanism of ARL4C on cellular properties involved in ccRCC progression.
After downloading the data of cancer patients from the TCGA database, we used various bioinformatics analysis websites and methods to analyze the expression and function of ARLs and ARL4C. The differential expression of ARL4C in clinical renal cancer tissues versus adjacent normal tissues was further verified using immunohistochemistry and real-time quantitative reverse-transcription (qRT-PCR). qRT-PCR was used to explore the expression of ARL4C mRNA in normal renal cells versus different ccRCC cell lines, and the protein expression of ARL4C was further verified using western blotting. CCK-8, colony formation, and EdU assays were used to determine the effect of ARL4C knockdown on ccRCC cell proliferation. We also used wound healing and Transwell assays to analyze the changes in ccRCC cell migration and invasion following ARL4C knockdown. Finally, we used western blotting to probe the molecular mode of action of ARL4C in ccRCC cells after exposure to Wnt signaling pathway agonists.
Biological function analysis showed that methylation of ARL4C and changes in immune cell infiltration and targeted drug sensitivity caused by altered ARL4C expression affected the prognosis of ccRCC. Further bioinformatics analysis suggested that the expression of ARL4C mRNA was increased in ccRCC, and this was associated with a poor prognosis in ccRCC patients. Increased expression of ARL4C was further verified using qRT-PCR and western blotting of human ccRCC tissue samples. Downregulation of ARL4C significantly inhibited the proliferation, migration, and invasion of ccRCC cells, and activation of the Wnt/-catenin pathway promoted the expression of ARL4C. As an essential downstream effector of the Wnt signaling pathway, ARL4C increased the expression of cyclin D1 and c-myc, thereby increasing the ability of the cells to undergo epithelial-mesenchymal transition (EMT) and ccRCC progression.
As a critical factor in the Wnt/-catenin pathway, ARL4C regulates EMT and progression in ccRCC.
基于生物信息学分析，研究ADP核糖基化因子（ARF）样蛋白（ARLs）及ARL4C在透明细胞肾细胞癌（ccRCC）中的表达，并通过实验确定ARL4C对ccRCC进展相关细胞特性的影响及机制。
从TCGA数据库下载癌症患者数据后，我们使用各种生物信息学分析网站和方法来分析ARLs及ARL4C的表达和功能。采用免疫组织化学和实时定量逆转录（qRT-PCR）进一步验证ARL4C在临床肾癌组织与癌旁正常组织中的差异表达。运用qRT-PCR探究ARL4C mRNA在正常肾细胞与不同ccRCC细胞系中的表达情况，并用蛋白质印迹法进一步验证ARL4C的蛋白表达。采用CCK-8、集落形成和EdU实验确定敲低ARL4C对ccRCC细胞增殖的影响。我们还运用伤口愈合实验和Transwell实验分析敲低ARL4C后ccRCC细胞迁移和侵袭的变化。最后，我们使用蛋白质印迹法探究在暴露于Wnt信号通路激动剂后ARL4C在ccRCC细胞中的分子作用模式。
生物学功能分析表明，ARL4C的甲基化以及ARL4C表达改变引起的免疫细胞浸润和靶向药物敏感性变化影响了ccRCC的预后。进一步的生物信息学分析表明，ccRCC中ARL4C mRNA表达增加，这与ccRCC患者的不良预后相关。通过对人ccRCC组织样本进行qRT-PCR和蛋白质印迹法进一步验证了ARL4C表达的增加。敲低ARL4C显著抑制了ccRCC细胞的增殖、迁移和侵袭，并且Wnt/β-连环蛋白通路的激活促进了ARL4C的表达。作为Wnt信号通路的重要下游效应分子，ARL4C增加了细胞周期蛋白D1和c-myc的表达，从而增强了细胞发生上皮-间质转化（EMT）和ccRCC进展的能力。
作为Wnt/β-连环蛋白通路中的关键因子，ARL4C调节ccRCC中的EMT和进展。
