CD133+CD44+喉鳞状细胞癌干细胞的致癌性研究及相关微小RNA的鉴定

摘要

Objective To compare the carcinogenic abilities of CD133+CD44+laryngeal cancer stem cells and general laryngeal cancer stem cells and to identify the mechanism underlying the action of miRNAs. Methods Solid tumor-derived laryngeal carcinoma stem cells and Hep-2-derived laryngeal carcinoma stem cells were cultured, and CD133+CD44+ laryngeal cancer stem cells were sorted by flow cytometry. Boden chamber invasion assay, cell migration assay and tumor formation assay were then performed to compare the invasion, migration and tumorigenic abilities of CD133+CD44+ laryngeal cancer stem cells and general laryngeal cancer stem cells. And then, miRNAs isolated from two laryngeal cancer stem cells were detected and analysed with miRNA chip. Results (1)In Boyden chamber invasion assay, the cell invasion rate of CD133+CD44+ laryngeal cancer stem cells was obviously higher (80.2％± 2.3％ vs. 63.9％± 3.2％, t=5.011,P=0.027);(2)CD133+CD44+laryngeal cancer stem cells also had higher mobility in cell migration assay (82.9％± 1.1％ vs. 70.9％± 0.6％, t=4.514,P=0.031); (3)In tumor formation assay, the tumor formation rate of CD133+CD44+ laryngeal cancer stem cells was also higher (80％ vs. 50％). What's more, we identified 15 miRNAs that were significantly upregulated in CD133+CD44+laryngeal cancer stem cells and 3 miRNAs that were significantly downregulated in CD133+CD44+ laryngeal cancer stem cells, compared with normal laryngeal cancer stem cells. Conclusions CD133+CD44+laryngeal cancer stem cells have stronger invasion, migration and tumorigenic abilities compared with normal laryngeal cancer stem cells, and the difference of miRNAs' expression is one of the possible causes.%目的 比较CD133+CD44+喉鳞状细胞癌(简称喉鳞癌)干细胞与一般喉鳞癌干细胞的致癌能力,并探讨微小RNA(miRNA)在其中发挥的作用.方法 培养实体瘤来源的喉鳞癌干细胞和Hep-2来源的喉鳞癌干细胞,通过流式细胞仪分选出CD133+CD44+喉鳞癌干细胞,通过Boyden小室侵袭实验、细胞迁移实验和裸鼠成瘤实验比较CD133+CD44+喉鳞癌干细胞与未筛选的喉鳞癌干细胞的侵袭、迁移和成瘤能力,然后提取两种喉鳞癌干细胞的总miRNA,通过丹麦Exiqon公司的miRNA芯片进行检测和分析比较.结果 (1)在Boyden小室侵袭实验中,CD133+CD44+喉鳞癌干细胞与未筛选的喉鳞癌干细胞比较,侵袭细胞更多,细胞侵袭率也更高(80.2％±2.3％比63.9％±3.2％,t=5.011,P=0.027);(2)在细胞迁移实验中,CD133+CD44+喉鳞癌干细胞比未筛选的喉鳞癌干细胞具有更高的迁移率(82.9％±1.1％比70.9％±0.6％,t=4.514,P=0.031);(3)在动物成瘤实验中,CD133+CD44+喉鳞癌干细胞比未筛选的喉鳞癌干细胞成瘤需要注射的细胞数少(1×103个比1×105个),成瘤率更高(80％比50％).另外,用miRNA芯片检测CD133+CD44+喉鳞癌干细胞和未筛选的喉鳞癌干细胞的miRNA水平后发现,在CD133+CD44+喉鳞癌干细胞中,有15条miRNA水平明显较高,有3条miRNA水平明显较低.结论 CD133+CD44+喉鳞癌干细胞具有更强的侵袭、迁移和成瘤能力,miRNAs的差异表达是其中的一个可能原因.