摘要
目的:探讨长链非编码RNA(Long non-coding RNA,LncRNA)人浆细胞瘤转化迁移基因1(Plasmacytoma Variant Translocation 1,PVT1)对真核翻译起始因子4A1(Eukaryotic Translation Initiation Factor 4A1,EIF4A1)可能的表达调控作用及机制。方法:通过对MGC-803细胞PVT1基因的表达进行干扰,对其干扰效果进行了鉴定,然后利用real time-PCR和Western Blot技术对EIF4A1基因mRNA、蛋白水平进行了测定;利用LncBase Predicted v.2预测PVT1作为分子海绵在分子上吸附的miRNA;然后用TargetScan和miRTarBase对调控EIF4A1表达的miRNA进行预测,结合两者结果得出中介miRNA;进一步通过LncBase Predicted v.2寻找PVT1与候选miRNA结合位点,并通过GEO数据库说明候选miRNA在胃癌中的表达情况;最后从干扰PVT1表达的细胞中通过real time-PCR技术测定候选miRNA的水平。结果:在胃癌细胞MGC-803中成功抑制了PVT1表达。在PVT1表达下调的胃癌细胞中EIF4A1的mRNA和蛋白质的表达水平均下调(P<0.05);通过LncBase Predicted v.2预测出277个PVT1可能结合吸附的miRNA;TargetScan和miRTarBase数据库预测出12个可能调控EIF4A1的miRNA;两者结果取交集,发现miR-493-3p可能为PVT1吸附降低的miRNA,且miR-493-3p可能下调EIF4A1的水平;通过LncBase Predicted v.2预测出PVT1与miR-493-3p的结合位点位于染色体的8:128048475-128048492;通过GEO数据库发现miR-493-3p在胃癌组织中处于表达下调状态。Real-time PCR结果显示PVT1水平降低后miR-493-3p水平有所增加(P<0.05)。结论:LncRNA PVT1可通过吸附降低miR-493-3p水平而正向调控EIF4A1的表达,miR-493-3p是PVT1调节EIF4A1表达的关键中间分子,为PVT1在胃癌中的作用机制提供线索。
Objective:To explore the role and mechanism of Long non-coding RNA(LncRNA)plasmacytoma variant translocation 1(PVT1)to regulate the expression of eukaryotic translation initiation factor 4A1(EIF4A1).Methods:By interfering with the expression of PVT1 gene in MGC-803 cells,the interference effect was identified,and then the mRNA and protein levels of EIF4A1 gene were determined by realtime-PCR and Western Blot techniques.LncBase Predicted v.2 was used to predict miRNAs adsorbed with lncRNA PVT1 as molecular sponge.Subsequently,TargetScan and miRTarBase databases were used to predict the miRNAs that might regulate EIF4A1,and the mediating miRNAs were obtained by combining the two prediction results.Furthermore,LncBase Predicted v.2 was used to find the specific binding sites of PVT1 and candidate miRNAs,and the expression of candidate miRNAs in gastric cancer was explained through GEO database.Finally,the levels of candidate miRNAs were detected by real time-PCR in cells that interfered with PVT1 expression.Results:The expression of PVT1 was successfully interfered in gastric cancer cell MGC-803.The expression levels of EIF4A1 mRNA and protein in gastric cancer cells with PVT1 knockdown were decreased(P<0.05).A total of 277 miRNAs that might bind to PVT1 adsorption were predicted by LncBase Predicted v.2.TargetScan and miRTarBase database predicted 12 miRNAs that might regulate EIF4A1.The intersection of these two results showed that miR-493-3p may be a miRNA with reduced PVT1 adsorption,and miR-493-3p may down-regulate the level of EIF4A1.LncBase Predicted v.2 predicted that the binding site of PVT1 and miR-493-3p was located at 8:128048475-128048492 on chromosome.The GEO database showed that miR-493-3p was down-regulated in gastric cancer tissues.The results of real-time PCR showed that the level of miR-493-3p was increased after the reduction of PVT1 level(P<0.05).Conclusions:LncRNA PVT1 can positively regulate the expression of EIF4A1 by reducing the level of miR-493-3p.miR-493-3p is a key intermediate molecule of PVT1 in regulating EIF4A1 expression and may be an important target for the treatment of gastric cancer.
作者
金迅
郭婷婷
孙建伟
卢雨林
王丹君
肖冰琪
杜诗尧
李冬妹
JIN Xun;GUO Ting-ting;SUN Jian-wei;LU Yu-lin;WANG Dan-jun;XIAO Bing-qi;DU Shi-yao;LI Dong-mei(Key Laboratory of Xinjiang Endemic and Ethnic Disease,Shihezi University School of Medicine,Xinjiang Shihezi,832002)
出处
《农垦医学》
2023年第2期106-111,共6页
Journal of Nongken Medicine
基金
国家自然科学基金项目(82160573)
新疆生产建设兵团指导性科技计划项目(2022ZD084)
国家级大学生创新创业训练计划项目(202210759023)。
