沉默Oct4和Nanog基因抑制胰腺癌干细胞的生物学特征

Silencing Oct4and Nanog inhibit stem-related biological characteristics of pancreatic cancer stem cells

目的探讨特异短发夹RNA(shRNA)沉默胰腺癌PANC-1肿瘤干细胞(PCSCs)生物学特征的变化及其机制。方法从PANC-1细胞株中分离出CD44+CD24+上皮特异性抗原(ESA)+PCSCs,检测Oct4和Nanog蛋白表达。用慢病毒载体介导的shRNA沉默PCSCs中Oct4和Nanog表达,用荧光定量RT-PCR和Western blot检测干扰效率。通过单克隆形成实验、Transwell小室模型、细胞计数试剂盒8(CCK8)检测Oct4和Nanog对PCSCs增殖、迁移、侵袭和药物敏感性的影响。NOD/SCID小鼠致瘤实验观察Oct4和Nanog对PCSCs致瘤性的影响。结果 PANC-1细胞株中PCSCs占0.1%-0.8%,Oct4和Nanog在PCSCs中呈高表达(P<0.05);慢病毒载体介导的shRNA降低PCSCs中Oct4和Nanog的表达(P<0.05)。沉默Oct4、Nanog后,PCSCs的单克隆形成、增殖、迁移、侵袭能力下降,药物敏感性增强并诱导凋亡(P<0.05)。致瘤实验表明,沉默Oct4和Nanog后,PCSCs致瘤能力下降(P<0.05)。结论 Oct4和Nanog在PCSCs中高表达,慢病毒介导的shRNA可沉默PCSCs中Oct4、Nanog的表达。沉默PCSCs中Oct4和Nanog的表达可抑制PCSCs相关生物学特征,增强其药物敏感性并诱导细胞凋亡。

Objective To study the changes and underlying mechanism of stem-related biological characteristics of pancreatic cancer stem cells （PCSCs） inhibited by silencing Oct4 and Nanog. Methods The PCSCs of CD44^＋ CD24^＋ ESA^＋ phenotypes in human pancreatic cancer cell line PANC-1 were isolated and the expressions of Oct4 and Nanog in PCSCs were detected on both transcriptional and translational levels. The interference efficiency of Oct4 and Nanog in PCSCs at the transcriptional and translational levels was determined by the reported gene GFP （lentiviral vector-GFP, LV/GFP）-mediated shRNA. The proliferation, migration, invasion ability and the drug sensitivity of PCSCs influenced by Oct4 and Nanog were observed by monoclonal formation as well as the CCK8 assay, transwell chamber model. The mechanisms were investigated by FQRT-PCR and Western blot. NOD/SCID mice tumorigenicity experiment was used to observe the tumorigenic potential of PCSCs influenced by Oct4 and Nanog. Results The PCSCs accounted for 0. 1%-0.8% in PANC-1 cells. Oct4 and Nanog were highly expressed in PCSCs（P〈0. 05）, which were strongly inhibited by lentiviral vector （ LV）-mediated shRNA （P%0. 05 ）. The proliferation, monoclonal formation, migration and invasion ability of PCSCs were decreased and drug sensitivity and apoptosis were enhanced significantly after Oct4 and Nanog were silenced （P〈0. 05）. Tumorigenicity experiment showed that silencing of Oct4 and Nanog significantly decreased the tumorigenic capacity of PCSCs （P〈0. 05）. Conclusion Oct4 and Nanog are highly expressed in PCSCs, which can be silenced by LV-mediated shRNA with inhibited biological characteristics and enhanced drug sensitivity and apoptosis.