成骨细胞外泌体对前列腺癌细胞生物学特性的影响及潜在机制研究

Impact and underlying mechanisms of osteoblast-derived exosomes on biological characteristics of prostate cancer cells

目的 研究成骨细胞外泌体对前列腺癌细胞生物学特性的影响和潜在的机制。方法 将人骨髓间充质干细胞诱导分化为成骨细胞,超速离心提取细胞培养基上清中的外泌体并鉴定。克隆形成试验分析成骨细胞外泌体对前列腺癌细胞增殖的影响。PCR芯片分析外泌体处理后前列腺癌细胞中表达变化的miRNA。通过荧光标记实验证明成骨细胞和肿瘤细胞中存在miRNA传递。在细胞和动物水平研究关键miRNA对前列腺癌细胞生物学特性的影响。生信分析并实验验证关键miRNA的靶基因。结果 成功提取并鉴定了成骨细胞外泌体。相比于对照组,加入成骨细胞外泌体的前列腺癌细胞克隆形成能力明显增强(P<0.05)。PCR芯片筛选发现miR-223可能是其中的关键分子。膜交换实验显示成骨细胞与前列腺癌细胞存在膜交换,FAM标记miRNA显示miR-223能够在细胞间传递。功能实验表明,过表达miR-223能够增强前列腺癌细胞的增殖、抗凋亡和迁移侵袭能力(P<0.05)。生信分析提示WNT通路抑制蛋白APC可能是miR-223的靶基因,实验证实过表达miR-223能够降低前列腺癌细胞中APC的表达(P<0.05)。Rescue实验显示过表达APC能够逆转miR-223对前列腺癌细胞增殖、迁移、侵袭的促进作用。结论 成骨细胞外泌体是前列腺癌的促癌因子。外泌体中miR-223可能是其中的关键作用分子,其通过影响细胞中APC的表达发挥促癌作用。

Objective To investigate the effects and potential mechanisms of osteoblast-derived exosomes on the biological properties of prostate cancer(PC)cells.Methods Human bone marrow mesenchymal stem cells was induced to differentiate into osteoblasts,and then the exosomes were extracted from the supernatant of cell culture medium by ultracentrifugation and identified.Clone formation assay was employed to determine the effect of derived exosomes on the proliferation of PC cells,and PCR microarray was used to analyze miRNA changes after exosome treatment.Fluorescence labeling was applied to validate the transfer of miRNA between osteoblasts and tumor cells.The impact of key miRNA on the biological characteristics of PC cells was studied at the cellular and animal levels.Bioinformatics analysis was conducted to explore the target genes of key miRNA and then further validated.Results Osteoblast-derived exosomes were successfully extracted and then identified.These exosomes significantly promoted the clonogenic ability of PC cells when compared with the cells without treatment(P<0.05).PCR microarray showed that miR-223 might be a key molecule in this process.Membrane exchange experiments demonstrated the presence of membrane exchange between osteoblasts and PC cells,and miRNA FAM labeling displayed that miR-223 was transferred within above cells.Functional experiments indicated that overexpression of miR-223 enhanced the proliferation,anti-apoptosis,and migration/invasion ability of PC cells(P<0.05).Bioinformatics analysis indicated that APC,an inhibitor of WNT signaling pathway,may be a target gene of miR-223,which was further confirmed that overexpressing miR-223 down-regulated APC expression.Rescue experiment showed that overexpression of APC reversed the promoting effect of miR-223 on the proliferation,migration and invasion of PC cells.Conclusion Osteoblast-derived exosomes are pro-carcinogenic factors in PC,and miR-223 may be a key molecule in exosomes,which plays a oncogenic role by regulating APC in PC cells.