肿瘤相关成纤维细胞外泌体miR-34c-5p调控喉癌干细胞样生物学特性的体外实验研究

Cancer-associated fibroblast-derived exosomal miR-34c-5p regulates stem-like properties of laryngeal cancer cells in vitro

目的研究喉癌肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)外泌体miR-34c-5p对喉癌细胞体外干细胞样生物学特性的调控作用。方法喉癌CAFs外泌体中的miR-34c-5p为低表达,将其过表达后收集不同miR-34c-5p表达水平CAFs培养液上清中的外泌体,制备不同外泌体条件培养基(CM)母液,将不同培养基母液加入到喉癌细胞培养液中,观察其对喉癌细胞体外增殖、侵袭、无血清球体形成及平板克隆形成等干细胞样生物学特性的影响,并进一步检测肿瘤干细胞相关基因表达水平的变化。结果喉癌CAFs外泌体CM能够明显增强喉癌细胞的体外增殖、侵袭、无血清球体形成及平板克隆形成能力,同时上调肿瘤干细胞相关基因表达水平;而将CAFs的miR-34c-5p过表达后,其外泌体CM促进喉癌细胞以上干细胞样生物学特性的作用明显减弱。结论喉癌CAFs外泌体miR-34c-5p表达下调对于喉癌细胞体外干细胞样生物学特性的维持至关重要,可作为潜在的阻断治疗靶点。

Objective To investigate the influence of exosomal miR-34c-5p derived from cancer-associated fibroblasts(CAFs)on stem-like properties of laryngeal cancer cells in vitro.Methods Downregulation of exosomal miR-34c-5p in CAFs of laryngeal squamous cell carcinoma(LSCC)specimens was validated.Exosomes were collected from CAFs with downregulated or overexpressed exosomal miR-34c-5p as conditioned medium(CM).Laryngeal cancer cells were cultured in different exosomal CM.Cancer stem cell(CSC)-associated assays,including proliferation,invasion,sphere formation assay,plate colony formation assay and expression of self-renewal and CSC marker genes were evaluated.Results In vitro assays revealed that laryngeal cancer cells cultured with CAF-derived exosomes with downregulated miR-34c-5p had a higher capacity for proliferation,invasion,sphere formation and plate colony formation,and they also exhibited enhanced expression of CSC marker genes when compared with control cells not exposed to CAF-derived exosomes.However,when the CAFs miR-34c-5p was overexpressed,the tumor-promoting effects of CAFs exosomes were decreased significantly.Conclusions Downregulation of CAFs exosomal miR-34c-5p was critical for the maintenance of stem-like biological properties of LSCC cells,which may be a potential therapeutic target for LSCC therapy.