lncRNA NEAT1靶向miR-497-5p对胰腺癌细胞增殖、凋亡及上皮-间质转化的影响

Effects of lncRNA NEAT1 targeting miR-497-5p on proliferation,apoptosis and epithelial-mesenchymal transition of pancreatic cancer cells

目的:探讨长链非编码RNA核富集转录体1(lncRNA NEAT1)靶向miR-497-5p对胰腺癌细胞增殖、凋亡及上皮-间质转化(EMT)的影响。方法:体外培养PANC-1细胞株,对其转染并分为空白对照组(NG组)、阴性转染组(siRNA-NC组)、沉默NEAT1组(NEAT1-siRNA组)、共转染组(NEAT1-siRNA+miR-497-5p-inhibitor组)。采用实时荧光定量PCR法检测NEAT1、miR-497-5p水平;CCK-8法、流式细胞术检测各组细胞增殖、凋亡情况;Western blot检测增殖细胞核抗原(Ki-67)、凋亡相关蛋白B淋巴细胞瘤基因-2(Bcl-2)、Bcl-2相关X蛋白(Bax)及EMT标志蛋白E-钙黏附蛋白(E-cadherin)、N-钙黏附蛋白(Ncadherin)、波形蛋白(Vimentin)表达情况;应用TargetScan数据库预测NEAT1与miR-497-5p靶向关系并用双荧光素酶报告基因实验验证。结果:与NG组、siRNA-NC组比较,NEAT1-siRNA组PANC-1细胞增殖抑制率、凋亡率、miR-497-5p及Bax、E-cadherin蛋白表达均显著升高(P<0.05),NEAT1水平、Ki-67、Bcl-2、N-cadherin、Vimentin蛋白表达显著降低(P<0.05)。与NEAT1-siRNA组比较,NEAT1-siRNA+miR-497-5p-inhibitor组PANC-1细胞增殖抑制率、凋亡率、miR-497-5p及Bax、E-cadherin蛋白表达均显著降低(P<0.05),Ki-67、Bcl-2、N-cadherin、Vimentin蛋白表达显著升高(P<0.05)。TargetScan数据库预测显示miR-497-5p是NEAT1的潜在靶基因,双荧光素酶报告基因实验结果显示miR-497-5p-inhibitor-NEAT1-WT组荧光素酶活性显著高于miR-497-5p-inhibitor-NC-NEAT1-WT组(P<0.05)。结论:沉默lncRNA NEAT1表达能够靶向上调miR-497-5p表达,抑制胰腺癌细胞增殖及EMT过程,并诱导细胞凋亡,有望成为胰腺癌新的潜在治疗靶点。

Objective:To investigate the effects of long non-coding RNA nuclear enriched abundant transcript 1(lncRNA NEAT1)targeting miR-497-5p on proliferation,apoptosis and epithelial-tmesenchymal transition(EMT)of pancreatic cancer cells.Methods:PANC-1 cell line was cultured in vitro,transfected and divided into blank control group(NG group),negative transfection group(siRNA-NC group),silencing NEAT1 group(NEAT1-siRNA group),cotransfection group(NEAT1-siRNA+miR-497-5p-inhibitor group). The levels of NEAT1 and miR-497-5p were detected by real-time quantitative PCR;the cell proliferation and apoptosis were detected by CCK-8 method and flow cytometry;the expression of MKI67 affinity purified polyclonal Ab(Ki-67),apoptosis associated protein B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),EMT marker protein E-cadherin,N-cadherin and Vimentin were detected by Western blot;the targeting relationship between NEAT1 and miR-497-5p were predicted and verified by TargetScan database and double luciferase reporter gene experiment.Results:Compared with NG group and siRNA-NC group,the proliferation inhibition rate,apoptosis rate,miR-497-5p,Bax and E-cadherin protein expression of PANC-1 cells in NEAT1-siRNA group were significantly increased(P<0.05),the NEAT1 level,Ki-67,Bcl-2,N-cadherin and Vimentin protein expressions were significantly decreased(P<0.05). Compared with NEAT1-siRNA group,the proliferation inhibition rate,apoptosis rate,miR-497-5p,Bax and E-cadherin protein expressions of PANC-1 cells in NEAT1-siRNA+miR-497-5p-inhibitor group were significantly decreased(P<0.05),the NEAT1 level,Ki-67,Bcl-2,N-cadherin and Vimentin protein expressions were significantly increased(P<0.05). TargetScan database predicted that miR-497-5p was a potential target gene for NEAT1. The double luciferase reporter gene experiment showed that the luciferase activity of miR-497-5p-inhibitor-NEAT1-WT group was significantly higher than that of miR-497-5p-inhibitor-NC-NEAT1-WT group(P<0.05).Conclusion:Silencing lncRNA NEAT1 can targetingly up-regulate the expression of miR-497-5p,inhibit the proliferation and EMT process of pancreatic cancer cells,and induce apoptosis,which may be a new potential therapeutic target for pancreatic cancer.