Guo Q S, Wang P, Huang Y, Guo Y B, Zhu M Y, Xiong Y C
Zhonghua Yi Xue Za Zhi. 2019 Oct 15;99(38):3019-3023. doi: 10.3760/cma.j.issn.0376-2491.2019.38.011.
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. 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. 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(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 (0.001), N-cadherin (0.001) and transcription factor Snail (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 (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 (0.01). 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.
检测微小RNA（miRNA，miR）-30b在胰腺癌干细胞（PCSCs）中的表达，并观察miR-30b对上皮-间质转化（EMT）过程、PCSCs迁移和侵袭的调控作用。通过流式细胞术分选胰腺导管腺癌（PDAC）细胞系PANC-1中CD24、CD44和EpCAM三阳性的PCSCs，并评估Nanog和Oct4的表达。分析PCSCs和普通癌细胞中miR-30家族的表达谱，选择差异表达最明显的成员。将miR-30b模拟物转染到PCSCs中，然后进行RT-qPCR或蛋白质免疫印迹法检测EMT标志物的表达。通过Transwell实验检测miR-30b对PCSCs迁移和侵袭能力的影响。随后，将miR-30b激动剂转染到PCSCs中并接种于裸鼠，研究miR-30b对致瘤能力的影响。PCSCs占总数的4.52 - 8.09%。PCSCs中Oct4和Nanog的mRNA和蛋白水平显著高于PANC-1（P < 0.001）。miR-30b、c和d的表达水平显著下调，其中miR-30b最为明显。PCSCs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）和转录因子Snail（P < 0.001）显著下调，而波形蛋白表达无明显变化。Transwell实验表明，转染后PCSCs的迁移和侵袭能力均显著降低（P < 0.001）。体内实验中，注射过表达miR-30b的PCSCs的裸鼠的肿瘤体积和重量也显著低于对照组（P < 0.01）。胰腺癌细胞中CD24、CD44和EpCAM三阳性的PCSCs表现出明显的干性特征。miR-30b可逆转PCSCs的EMT过程，降低其迁移和侵袭能力，并抑制PCSCs的致瘤性。
