Lu Xiaoming, Wang Jinfeng, Dong Binbin, Wang Liping, Liu Yadong
Department of Urology, The Yancheng School of Clinical Medicine of Nanjing Medical University.
Department of Urology, The Third People's Hospital of Yancheng, Yancheng, China.
Anticancer Drugs. 2023 Mar 1;34(3):439-450. doi: 10.1097/CAD.0000000000001453. Epub 2022 Nov 21.
Dysregulation of cancer-associated fibroblasts (CAFs) still greatly challenges the treatments for bladder cancer (BC), where exosomal miRNAs derived from CAFs are one of the essential effectors for tumor progression. miR-93-5p is reported to be upregulated in BC, however, it is barely investigated in BC-derived CAFs.
The CAF markers were immunofluorescent-labeled and examined by western blotting assay in CAFs and normal fibroblasts (NFs). CAFs- and NFs-derived exosomes (CAFs-exo/NFs-exo) were authenticated by transmission electron microscope and nanoparticle tracking analysis. Cell viability was determined by cell counting kit-8 assay, and cell mobility was evaluated by wound healing and transwell assays. Real-time quantitative PCR was used to quantify the RNA expressions, and a western blotting assay was used for protein expression. Interaction between miR-93-5p and Platelet-Activating Factor Acetylhydrolase IB Subunit Beta (PAFAH1B1) was verified by luciferase reporter assay. HE staining assay was applied to assess the histological changes of xenografts.
CAFs-exo notably enhanced cell mobility and the expression levels of miR-93-5p of BC cells compared to NFs-exo. However, inhibition of miR-93-5p in CAFs-exo exhibited attenuated pro-metastatic ability on BC cells. PAFAH1B1 was one of the predicted targets of miR-93-5p, whose mRNA level was most significantly downregulated after miR-93-5p transfection. The interaction between PAFAH1B1 and miR-93-5p was verified, and miR-93-5p negatively regulated the protein level of PAFAH1B1. Overexpression of PAFAH1B1 could efficiently reverse the effects of miR-93-5p mimic on BC cell mobility. Finally, inhibition of miR-93-5p was proved to impair the carcinogenic function of CAFs-exo in vivo .
Exosomal miR-93-5p derived from CAFs confers oncogenicity on BC cells via sponging PAFAH1B1, suggesting a novel therapeutic strategy for BC.
癌症相关成纤维细胞（CAFs）的失调仍然是膀胱癌（BC）治疗的巨大挑战，其中源自CAFs的外泌体miRNA是肿瘤进展的重要效应因子之一。据报道，miR-93-5p在膀胱癌中上调，然而，在源自膀胱癌的CAFs中对其研究甚少。
通过免疫荧光标记CAF标志物，并通过蛋白质免疫印迹法在CAFs和正常成纤维细胞（NFs）中进行检测。通过透射电子显微镜和纳米颗粒跟踪分析对CAFs和NFs来源的外泌体（CAFs-exo/NFs-exo）进行鉴定。采用细胞计数试剂盒-8法测定细胞活力，通过伤口愈合实验和Transwell实验评估细胞迁移能力。使用实时定量PCR定量RNA表达，采用蛋白质免疫印迹法检测蛋白质表达。通过荧光素酶报告基因实验验证miR-93-5p与血小板活化因子乙酰水解酶IB亚基β（PAFAH1B1）之间的相互作用。应用苏木精-伊红（HE）染色实验评估异种移植瘤的组织学变化。
与NFs-exo相比，CAFs-exo显著增强了膀胱癌细胞的迁移能力和miR-93-5p的表达水平。然而，抑制CAFs-exo中的miR-93-5p对膀胱癌细胞的促转移能力减弱。PAFAH1B1是miR-93-5p的预测靶标之一，在转染miR-93-5p后其mRNA水平下调最为显著。验证了PAFAH1B1与miR-93-5p之间的相互作用，且miR-93-5p负向调节PAFAH1B1的蛋白质水平。PAFAH1B1的过表达能够有效逆转miR-93-5p模拟物对膀胱癌细胞迁移的影响。最后，证明抑制miR-93-5p会损害CAFs-exo在体内的致癌功能。
源自CAFs的外泌体miR-93-5p通过靶向PAFAH1B1赋予膀胱癌细胞致癌性，为膀胱癌提供了一种新的治疗策略。
