Objective: To detect the expression of micro RNA(miRNA, miR)-30bin pancreatic cancer stem cells (PCSCs) and to observe the regulatory effect of miR-30b on epithelial-mesenchymal transformation (EMT) process, migration and invasion of PCSCs. Methods: CD24, CD44 and EpCAM triple-positive PCSCs in pancreatic ductal adenocarcinoma(PDAC) cell line PANC-1 were sorted out by flow cytometry and the expression of Nanog and Oct4 were evaluated. The expression profile of miR-30 family in PCSCs and common cancer cells was analyzed, and the memberwith the most obvious differential expression was selected.miR-30b mimic was transfected into PCSCs and then RT-qPCR or Western Blot were performed to investigate the expression of EMT markers. The effect of miR-30b on the migration and invasion ability of PCSCs was detected by Transwell assay. Then, miR-30b agomir was transfected into PCSCs and inoculated in nude mice to study the effect of mir-30b on the tumorigenic ability. Results: PCSCs accounted for 4.52-8.09% of the total. The mRNA and protein levels of Oct4 and Nanog of PCSCswere significantly higher than those of PANC-1(P<0.001). The expression levels of miR-30b, c and d were significantly down-regulated, among which miR-30b was the most significant. After miR-30b overexpression in PCSCs, E-cadherin was significantly up-regulated (P<0.001), N-cadherin (P<0.001) and transcription factor Snail (P<0.001) were significantly down-regulated, while vimentin expression was not significantly changed. Transwell assay showed that both migration and invasiveness of PCSCs were significantly decreased after transfection (P<0.001). In vivo experiments, the tumor volume and weight of the nude mice injected with PCSCs overexpressing miR-30b were also significantly lower than those of the control group (P<0.01). Conclusions: CD24, CD44 and EpCAMtriple positive PCSCs in pancreatic cancer cells showed obvious stemness characteristics. miR-30b can reverse the EMT process of PCSCs, reduce their migration and invasion, and inhibit the tumorigenicity of PCSCs.
目的： 检测微小RNA（miRNA，miR）-30b在胰腺癌干细胞（PCSC）中的表达并观察miR-30b对PCSC上皮-间质转化（EMT）及迁移侵袭能力的调控作用。 方法： 采用流式细胞仪分选胰腺导管腺癌（PDAC）细胞系PANC-1中CD24、CD44和EpCAM三阳性的PCSC，并检测胚胎干性基因情况；分析miR-30家族在PCSC和普通癌细胞的表达谱，筛选出差异表达最明显的成员；miR-30b mimic转染PCSC，检测EMT特征基因表达变化，Transwell检测miR-30b对PCSC迁移和侵袭能力的影响；再将miR-30b agomir转染PCSC后接种于裸鼠，研究miR-30b对PCSC成瘤能力的影响。 结果： 分选出的PCSC占PANC-1总数的4.52%~8.09%，Oct4和Nanog在mRNA及蛋白水平均显著高于PANC-1（P<0.001），miR-30b、c、d的表达水平显著下调，其中miR-30b最为显著。PCSC中miR-30b过表达后，E-钙黏蛋白显著上调（P<0.001），N-钙黏蛋白（P<0.001）、转录因子Snail（P<0.001）显著下调，而波形蛋白（vimentin）的表达无明显变化，Transwell实验显示受染后PCSC迁移和侵袭性均明显下降（P<0.001）。体内实验中过表达miR-30b的PCSC注射的裸鼠成瘤体积、质量也显著低于对照组（P<0.01）。 结论： 胰腺癌细胞中CD24、CD44和EpCAM三阳性PCSC存在明显的干性特征。miR-30b能够逆转PCSC的EMT过程，降低其迁移和侵袭作用，同时抑制PCSC的成瘤性。.
Keywords: Epithelial-mesenchymal transformation; MicroRNA; Pancreatic cancer stem cells; Pancreatic neoplasms.