鼻咽癌CNE2干样细胞具有干细胞特性及较高水平的自噬表达

CNE2 stem-like cells of nasopharyngeal carcinoma have stem cell characteristics and high level of autophagy

背景:鼻咽癌是具有高侵袭转移潜能的头颈部恶性肿瘤,对鼻咽癌晚期患者尚未取得满意治疗效果。目前,已有一些自噬抑制剂与抗肿瘤药物同时应用于临床研究,但可能机制尚未明确。目的:探索鼻咽癌干样细胞是否存在较高水平自噬表达。方法:通过无血清悬浮培养法从人源化鼻咽癌CNE2细胞株中富集得到肿瘤干样细胞球。通过细胞分化实验、流式细胞术、透射电镜、Western blot及RT-PCR检测鼻咽癌CNE2亲本细胞和CNE2干样细胞的干细胞特性及细胞自噬水平。结果与结论:①通过无血清悬浮培养法能富集得到鼻咽癌CNE2干样细胞,在含血清培养基中培养能贴壁分化成鼻咽癌CNE2亲本细胞;②鼻咽癌CNE2干样细胞CD133阳性标记的细胞比例(9.6%)高于鼻咽癌CNE2亲本细胞(0.3%),差异有显著性意义(P <0.01);③与鼻咽癌CNE2亲本细胞相比,鼻咽癌CNE2干样细胞高表达干性相关基因(Bmi-1、Twist1)和自噬相关基因(Beclin1、LC3B),差异有显著性意义(P <0.01);④透射电镜观察鼻咽癌CNE2干样细胞自噬小体较鼻咽癌CNE2亲本细胞形成明显增多;⑤结果表明,通过无血清悬浮培养法可有效富集鼻咽癌CNE2干样细胞,鼻咽癌干样细胞具有干细胞生物学特性,并存在较高水平自噬表达。

BACKGROUND: Nasopharyngeal carcinoma is a head and neck malignant tumor with high invasion and metastatic potential. It has not yet achieved satisfactory effects in the treatment of advanced nasopharyngeal carcinoma. Some autophagy inhibitors and anti-tumor drugs have been used in clinical research, but the possible mechanism has not been clarified.OBJECTIVE: To explore whether high-level autophagy occurs in stem-like cells of nasopharyngeal carcinoma.METHODS: Tumor stem-like cell spheres were enriched from humanized nasopharyngeal carcinoma CNE2 cell line by serum-free suspension culture. The stem cell characteristics and expression levels of autophagy-related genes and proteins in CNE2 parental cells and CNE2 stem-like cells were detected using cell differentiation assay, flow cytometry, transmission electron microscope, western blot and RT-PCR.RESULTS AND CONCLUSION: The nasopharyngeal carcinoma CNE2 stem-like cells were enriched by serum-free suspension culture. The suspension spheres were cultured in serum-containing medium to differentiate into CNE2 parental cells. Proportion of CD133^+ cells in CNE2 stem-like cells(9.6%) was significantly higher than that in CNE2 parental cells(0.3%;P < 0.01). Compared with CNE2 parental cells, CNE2 stem-like cells highly expressed stemness-related genes Bmi-1, Twist1 and autophagy-related genes Beclin1 and LC3 B(P < 0.01). Under the transmission electron microscope, the autophagic bodies of CNE2 stem-like cells in nasopharyngeal carcinoma were significantly increased compared with CNE2 parental cells. Therefore, nasopharyngeal carcinoma CNE2 stem-like cells can be effectively enriched using the serum-free suspension culture method, and be verified to have the biological characteristics of stem cells and exhibit high-level autophagy.