长链非编码RNA PCAT-1对胰腺癌细胞增殖和凋亡的影响及作用机制研究

Effect and mechanism of long non-coding RNA PCAT-1 on proliferation and apoptosis of pancreatic cancer cells

目的研究长链非编码RNA前列腺癌相关转录本1(lncRNA PCAT-1)对胰腺癌细胞增殖和凋亡的影响,并初步探讨其作用机制。方法qRT-PCR检测lncRNA PCAT-1在胰腺癌细胞系Capan-1、Capan-2、PANC-1、SW1990和正常胰腺细胞hTERT-HPNE中的表达水平。lncRNA PCAT-1表达最高的胰腺癌细胞系分为sh-NC组和sh-PCAT-1组,进行NC干扰慢病毒(shRNA)和PCAT-1 shRNA感染,qRT-PCR检测各组细胞中lncRNA PCAT-1的表达水平。3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑鎓(MTS)实验检测沉默lncRNA PCAT-1的表达对细胞增殖能力的影响;流式细胞仪检测沉默lncRNA PCAT-1的表达对细胞周期和凋亡的影响;皮下移植瘤实验检测沉默lncRNA PCAT-1的表达对胰腺癌细胞体内生长能力的影响;Western blot检测lncRNA PCAT-1对细胞周期相关蛋白、凋亡相关蛋白和PI3K/AKT信号通路关键蛋白的表达影响。结果qRT-PCR检测结果显示lncRNA PCAT-1在胰腺癌细胞系的表达水平高于在正常胰腺细胞hTERT-HPNE中的表达(P<0.05),与Capan-1、Capan-2和SW1990细胞相比,PANC-1细胞中lncRNA PCAT-1的表达最高。PANC-1细胞感染PCAT-1 shRNA,qRT-PCR结果显示,与sh-NC组相比,sh-PCAT-1组细胞中lncRNA PCAT-1的表达降低(P<0.05);沉默lncRNA PCAT-1的表达后,PANC-1细胞的增殖能力降低,细胞周期阻滞在G0/G1期,细胞凋亡增加,细胞体内裸鼠成瘤能力降低,细胞周期蛋白cyclinD1和CDK6蛋白的表达减少,抗凋亡蛋白Bcl-2蛋白减少,促凋亡蛋白Bax蛋白表达增加,PI3K/AKT信号通路关键蛋白pPI3K和pPAKT蛋白表达减少。结论沉默lncRNA PCAT-1的表达可能通过调控PI3K/AKT信号通路抑制胰腺癌细胞增殖和促进细胞凋亡。lncRNA PCAT-1可能是治疗胰腺癌的新型靶标。

Objective To study the effect of long non-coding RNA prostate cancer-associated transcript 1(lncRNA PCAT-1)on the proliferation and apoptosis of pancreatic cancer cells,and to explore its mechanism.Methods The expression level of lncRNA PCAT-1 in pancreatic cancer cell lines Capan-1,Capan-2,PANC-1,SW1990 and normal pancreatic cell hTERT-HPNE was detected by qRT-PCR.The highest lncRNA PCAT-1 expressing pancreatic cancer cell line was divided into sh-NC group and sh-PCAT-1 group.NC interfering with lentivirus(shRNA)and PCAT-1 shRNA infection were performed,qRT-PCR was used to detect the expression level of lncRNA PCAT-1 in each group of cells.[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,MTS]test was used to detect the effect of silenced lncRNA PCAT-1 expression on cell proliferation,flow cytometry was used to detect the effect of silenced lncRNA PCAT-1 on cell cycle and apoptosis,subcutaneous transplantation tumor test was used to detect the effects of silenced lncRNA PCAT-1 expression on pancreatic cell growth in vivo.Western blot was used to detect the effects of lncRNA PCAT-1 on the expression of cell cycle-related proteins,apoptosis-related proteins and key proteins of the PI3K/AKT signaling pathway.Results qRT-PCR results showed that the expression level of lncRNA PCAT-1 in pancreatic cancer cell lines was higher than that in normal pancreatic cells hTERT-HPNE(P<0.05).Compared with Capan-1,Capan-2 and SW1990 cells,the expression of lncRNA PCAT-1 was the highest in PANC-1 cells,and the highest expression of lncRNA PCAT-1 was in PANC-1 cells.PANC-1 cells were infected with PCAT-1 shRNA,and qRT-PCR results showed that compared with the sh-NC group,the expression of lncRNA PCAT-1 in the cells of the sh-PCAT-1 group reduced(P<0.05);after silenced the lncRNA PCAT-1expression in PANC-1 cells,cell proliferation ability reduced,cell cycle was arrested at G0/G1 phase,the apoptosis increased,tumorigenicity reduced in nude mice,cyclinD1 and CDK6 proteins expression decreased,and anti-apoptotic protein Bcl-2 expression decreased,the pro-apoptotic protein Bax expression increased,and the key proteins of PI3K/AKT signaling pathway pPI3K and pPAKT expression decreased.Conclusion Silencing the expression of lncRNA PCAT-1 may inhibit the proliferation and promote apoptosis of pancreatic cancer cells by regulating the PI3K/AKT signaling pathway.lncRNA PCAT-1 may be a new target for the treatment of pancreatic cancer.