长链非编码RNA n336928对胃肠胰神经内分泌肿瘤细胞增殖及迁移的影响

Effects of Long Non-coding RNA N336928 on Proliferation and Migration of Gastrointestinal Pancreatic Neuroendocrine Tumor Cells

目的:从基础实验层面研究长链非编码RAN n336928(LncRNA n336928,即n336928)在胃肠胰神经内分泌肿瘤(Gastro-entero-pancreatic neuroendocrine tumors,GEP-NETs)进展中的作用与影响。方法:首先采用实时定量荧光聚合酶链反应(qRT-PCR)检测n336928在人正常胰腺导管上皮细胞系(hTERT-HPNE)、GEP-NETs细胞株(QGP-1、STC-1)中表达情况。采用靶向n336928的小干扰(si-RNA),以转染试剂lipo3000为载体进行敲降。通过CCK8法、克隆实验和EdU实验观察细胞增殖活力,通过Transwell实验观察细胞迁移能力变化,通过流式分析实验检测n336928敲低是否与周期阻滞和细胞凋亡相关,并用Western blotting检测周期和凋亡相关标志性蛋白表达。结果:QGP-1和STC-1中n336928的m RNA水平的表达相较于hTERT-HPNE明显升高(P<0.05),抑制QGP-1及STC-1中n336928的表达后,细胞的增殖及迁移能力明显减弱(P<0.05)。QGP-1细胞转染n336928小干扰后,细胞周期主要阻滞在G1/S期,且细胞凋亡明显增加。与对照组相比,敲降组的周期相关蛋白(CyclinD1)明显减少,相反凋亡相关蛋白(Caspase3)明显升高(P<0.05)。为了进一步研究基因作用机制,我们用RNA免疫沉淀反应(RIP)验证n336928可与EZH2结合,并共同调控下游基因P21。结论:LncRNA n336928可通过结合EZH2并表观调控下游基因P21,而参与胃肠胰神经内分泌肿瘤的发生发展过程。

Objective:To investigate the function and influence of long non-coding RAN n336928(LncRNA n336928,n336928)on Gastro-Entero-pancreatic neuroendocrine tumors(GEP-NETs)from the basic-experimental level.Methods:The expression of n336928 in human normal pancreatic ductal epithelial cell lines(hTERT-HPNE)and GEP-Nets cell lines(QGP-1,STC-1)was detected by real-time quantitative fluorescence polymerase chain reaction(qRT-PCR).LncRNA n336928 was knocked down by small interference using transfection reagent Lipo3000 as a carrier.Cell proliferation activity was observed by CCK8 assay,colony assay and EdU assay,cell migration ability was observed by transwell assay,flow cytometry assay was used to detect whether lessen expression of n336928was related to cell cycle arrest and cell apoptosis,and the expression of related proteins were detected by western blotting.Results:The m RNA expression of n336928 in QGP-1 and STC-1 was significantly higher than that of hTERT-HPNE(P<0.05).After inhibiting the expression of n336928 in QGP-1 and STC-1,the proliferation and migration of the cells were significantly decreased(P<0.05).After transfection of QGP-1 cells with si-n336928,the cell cycle was mainly arrested at G1/S phase,and cell apoptosis was significantly increased.Compared with the control group,cycle-related protein cyclinD1 was significantly decreased in knockout group,while apoptosis-related protein caspase3 was significantly increased(P<0.05).To furtherly investigate the relevant mechanism,RNA immunoprecipitation reaction(RIP)was used to verify that n336928 could bind to EZH2 and jointly regulate the downstream gene P21.Conclusion:LncRNA n336928 can be involved in the development of GEP-NETs by binding to EZH2 and epigeneticly regulation of downstream gene P21.