Effect of FOS and JUN expression in inmortalized human mesenchymal progenitor cells

Mesenchymal progenitor cells (MPCs) have been proposed as cells of origin for sarcomas, while c-Fos and c-Jun transcription factors have been proposed has a oncogenes in sarcoma tumors. Here we study the effects of the induction of c-fos or c-Jun expression in immortalized hMPCs. C-Fos expression in these cells not only generated modifications in cell cycle, but also morphological changes, reduced mobility capacity and impaired adipogenic and osteogenic differentiation potentials. Even more interesting, c-Fos expressing immortalized hMPCs generated tumors with chondrogenic phenotype when implanted in immunodeficient mice. In accordance with these results, c-Fos is expressed at protein level in many human bone tumors, specifically in chondrosarcomas (76% c-fos positive samples) and osteosarcomas (49% c-fos positive samples). c-Jun expression in immortalized hMPCs induces increased proliferation and cell transformation and, once implanted in immunodeficient mice, these cells generate fibroblastic osteosarcomas. Doble c-jun and c-fos expression in immortalized hMPCs also induces cell transformation and yields high grade pleomorphic osteosarcoma tumors when implanted in immunodeficient mice. c-DNA was cloned into pOTB7 plasmid, which carries Tomato gene as reporter fluorescent protein. Lentiviral particles were produced by transient calcium phosphate transfection of HEK-293 cells. hMPCs were transduced with those lentiviral particles. Tomato expression was assessed by flow cytometry and c-fos expression was confirmed via qPCR, western blot and immunofluorescence. Cells transduced with lentivirus coding only for fluorescent protein were used as controls. Three independent replicas are provided from each treatment: 3H 1, 3H 2, 3H 3 correspond to hMPCs transduced with control lentiviral particles. 3HF 1, 3HF 2 and 3HF 3 correspond to hMPCs tranduced with lentiviral particles designed for c-fos expression. 3HJ 1, 3HJ 2 and 3HJ 3 correspond to hMPCs tranduced with lentiviral particles designed for c-jun expression. 3HJF 1, 3HJF 2 and 3HJF 3 correspond to hMPCs tranduced with lentiviral particles designed for c-jun and c-fos expression.

间充质祖细胞（Mesenchymal progenitor cells, MPCs）已被提出为肉瘤的起源细胞，而c-Fos与c-Jun转录因子也被认定为肉瘤中的致癌基因。本研究聚焦于在永生化人源间充质祖细胞（human MPCs, hMPCs）中诱导c-fos或c-Jun表达所产生的生物学效应。在该类细胞中诱导c-Fos表达，不仅会引发细胞周期紊乱，还会诱导形态学改变、迁移能力下降，同时损害成脂与成骨分化潜能。更值得关注的是，将过表达c-Fos的永生化hMPCs接种至免疫缺陷小鼠体内时，可形成具有软骨分化表型的肿瘤。与上述实验结果相符，c-Fos蛋白在多种人类骨肿瘤中均有表达，其中软骨肉瘤（阳性样本占比76%）与骨肉瘤（阳性样本占比49%）的表达尤为显著。在永生化hMPCs中诱导c-Jun表达，则可促进细胞增殖与细胞转化；将此类细胞接种至免疫缺陷小鼠体内后，可形成纤维母细胞型骨肉瘤。在永生化hMPCs中同时诱导c-jun与c-fos表达，同样可诱导细胞转化；将其接种至免疫缺陷小鼠体内后，可形成高级别多形性骨肉瘤。将c-DNA克隆至携带Tomato报告荧光蛋白基因的pOTB7质粒中。通过磷酸钙瞬时转染HEK-293细胞，制备重组慢病毒颗粒。利用上述重组慢病毒颗粒转导hMPCs。通过流式细胞术检测Tomato的表达情况，并通过qPCR、蛋白质印迹（western blot）与免疫荧光实验验证c-fos的表达水平。仅转导了编码荧光蛋白的慢病毒的细胞作为空白对照。本研究提供了各处理组的3次独立生物学重复：3H 1、3H 2与3H 3为转导对照慢病毒颗粒的hMPCs；3HF 1、3HF 2与3HF 3为转导靶向c-fos表达的慢病毒颗粒的hMPCs；3HJ 1、3HJ 2与3HJ 3为转导靶向c-jun表达的慢病毒颗粒的hMPCs；3HJF 1、3HJF 2与3HJF 3为转导靶向c-jun与c-fos共表达的慢病毒颗粒的hMPCs。