Zhang Mingkun, Bi Baibin, Liu Guangcun, Fan Xiaoyong
Department of Neurosurgery, the First Affiliated Hospital of Shandong First Medical University, Shandong Medicine and Health Key Laboratory of Neurosurgery, Jinan, PR China.
Histol Histopathol. 2025 Jan;40(1):89-100. doi: 10.14670/HH-18-762. Epub 2024 May 15.
Cancer-associated fibroblasts (CAFs) play important roles in tumor microenvironments. Pyrroline-5-carboxylate reductase 1 (PYCR1) is a potential cancer therapy target. This study aimed to explore the expression of PYCR1 in glioma-associated CAFs and analyze the effects of PYCR1 expression in CAFs on the proliferation of C6 glioma.
A rat glioma model was induced by injecting C6 cells in the right caudate putamen via a microliter syringe. After 14 days, tumor tissues were collected, and the levels of COL1A1 and PYCR1 were measured by immunohistochemistry. The colocalization of fibroblast activation protein α (FAP) and PYCR1 in tissues was measured by double-immunofluorescence. The CAFs were labeled by FAP and isolated from the tumor tissues using a fluorescence-activated cell sorting (FACS) machine. The isolated CAFs were further separated into CAFs with different PYCR1 expressions using the FACS machine. CAFs with different PYCR1 expressions were respectively cocultured with C6 cells or MUVECs for 48h using a Transwell permeable support. The invasion and proliferation of C6 cells were measured using a Transwell assay and colony formation assay, and the angiogenesis of MUVECs was measured using a Tube formation assay. The expression of COL1A1 and PYCR1 proteins in C6 cells and VEGF-A and EGF proteins in MUVECs was measured by western blotting. PYCR1 silencing in C6 cells was induced by PYCR1 siRNA transfection, the effects of which on the proliferation of C6 cells were measured using a wound healing assay, a Transwell assay, and western blotting.
The PYCR1 and COL1A1 upregulation co-occurred in the rat glioma, and PYCR1 was expressed in CAFs. The CAF coculture enhanced the invasion and proliferation of C6 cells and the angiogenesis of MUVECs. Meanwhile, the levels of COL1A1 protein in C6 cells, and the levels of VEGF-A and EGF proteins in MUVECs were increased after CAF coculture. Moreover, the effects of CAF coculture were increased with PYCR1 expression in the CAF. Silencing PYCR1 suppressed the migration and invasion of C6 cells, and decreased the levels of COL1A1 and VEGF-A proteins in C6 cells.
PYCR1 is expressed in glioma-associated CAFs, and promotes the proliferation of C6 cells and angiogenesis of MUVECs, suggesting that targeting PYCR1 may be a therapeutic strategy for glioma.
癌症相关成纤维细胞（CAFs）在肿瘤微环境中发挥重要作用。吡咯啉 - 5 - 羧酸还原酶1（PYCR1）是一个潜在的癌症治疗靶点。本研究旨在探讨PYCR1在胶质瘤相关CAFs中的表达，并分析CAFs中PYCR1表达对C6胶质瘤增殖的影响。
通过微量注射器将C6细胞注射到右侧尾状壳核诱导建立大鼠胶质瘤模型。14天后，收集肿瘤组织，采用免疫组织化学法检测COL1A1和PYCR1的水平。通过双重免疫荧光检测组织中成纤维细胞活化蛋白α（FAP）和PYCR1的共定位。用FAP标记CAFs，并使用荧光激活细胞分选仪（FACS）从肿瘤组织中分离出来。使用FACS仪将分离出的CAFs进一步分为具有不同PYCR1表达水平的CAFs。使用Transwell可渗透支持物将具有不同PYCR1表达水平的CAFs分别与C6细胞或MUVECs共培养48小时。使用Transwell试验和集落形成试验检测C6细胞的侵袭和增殖，使用管形成试验检测MUVECs的血管生成。通过蛋白质印迹法检测C6细胞中COL1A1和PYCR1蛋白以及MUVECs中VEGF - A和EGF蛋白的表达。通过PYCR1 siRNA转染诱导C6细胞中PYCR1沉默，使用伤口愈合试验、Transwell试验和蛋白质印迹法检测其对C6细胞增殖的影响。
大鼠胶质瘤中PYCR1和COL1A1上调同时出现，且PYCR1在CAFs中表达。CAF共培养增强了C6细胞的侵袭和增殖以及MUVECs的血管生成。同时，CAF共培养后C6细胞中COL1A1蛋白水平以及MUVECs中VEGF - A和EGF蛋白水平升高。此外，CAF共培养的作用随着CAF中PYCR1表达的增加而增强。沉默PYCR1抑制了C6细胞的迁移和侵袭，并降低了C6细胞中COL1A1和VEGF - A蛋白的水平。
PYCR1在胶质瘤相关CAFs中表达，促进C6细胞增殖和MUVECs血管生成，表明靶向PYCR1可能是胶质瘤的一种治疗策略。
