LncRNA DLEU7-AS1通过调控膜突蛋白MSN的表达促进胃癌细胞增殖与迁移的作用机制

LncRNA DLEU7-AS1 contributes to proliferation and migration of gastric cancer by regulating MSN transcription

背景与目的:越来越多的研究表明长链非编码RNA(long non-coding RNA,lncRNA)在肿瘤的发生、发展过程中发挥着重要作用,然而大多数lncRNA在胃癌中的作用和机制尚不明确。LncRNA DLEU7-AS1在胃癌中的作用和机制的研究鲜见报道。本文旨在研究DLEU7-AS1对胃癌恶性表型的影响并初步探讨其分子机制。方法:采用癌症基因组图谱(the Cancer Genome Atlas,TCGA)数据库分析DLEU7-AS1在胃癌组织中的表达及其对胃癌患者生存期的影响。采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)验证DLEU7-AS1在胃癌组织及胃癌细胞系中的表达情况。采用5-氮杂-2’-脱氧胞苷(5-aza-2’-deoxycytidine,DAC)和曲古抑菌素A(trichostatin A,TSA)处理胃癌细胞株,分析表观遗传学调控对DLEU7-AS1表达的影响。采用小干扰RNA(small interfering RNA,siRNA)下调HGC-27、AGS细胞株中DLEU7-AS1的表达,采用重组质粒上调MGC-803和MKN-45中DLEU7-AS1的表达,采用RTFQPCR验证效果;采细胞计数试剂盒(cellcountingkit-8,CCK-8)细胞增殖毒性实验、transwell小室迁移实验、平板克隆形成实验以及流式细胞术研究DLEU7-AS1对胃癌细胞增殖、迁移及凋亡和细胞周期的影响;采用RNA测序技术(RNAsequencing,RNA-seq)分析沉默DLEU7-AS1后的下游信号转导通路的变化,并采用RTFQ-PCR和蛋白质印迹法(Western blot)进行验证;采用RNA免疫共沉淀实验(co-immunoprecipitation,RIP)探讨DLEU7-AS1对下游信号分子的调控机制。结果:TCGA数据库分析及RTFQ-PCR检测均证明DLEU7-AS1在胃癌中表达升高。DLEU7-AS1的表达与胃癌患者生存期呈负相关。DAC和TSA处理胃癌细胞株后,DLEU7-AS1表达上调,说明其表达受表观遗传学调控。沉默DLEU7-AS1抑制胃癌细胞增殖、迁移,促进细胞凋亡;过表达DLEU7-AS1促进胃癌细胞增殖、迁移,抑制细胞凋亡。RNA-seq结果表明,DLEU7-AS1表达下调后会导致膜突蛋白(moesin,MSN)表达量的显著降低,RTFQ-PCR及Western blot的结果验证了这一结论。Rescue实验结果进一步证实,过表达MSN可部分回复干扰DLEU7-AS1对胃癌细胞增殖和迁移的抑制作用,提示MSN可能作为DLEU7-AS1下游效应分子。DLEU7-AS1主要定位在细胞核中,DLEU7-AS1与P300结合以及MSN启动子附近的H3K27的高度富集。结论:LncRNA DLEU7-AS1在胃癌中高表达并且其表达与胃癌患者生存期呈负相关,DLEU7-AS1可能通过招募P300调控MSN的转录进而促进胃癌的增殖和迁移等恶性表型。

Background and purpose:An increasing number of studies have demonstrated that lncRNAplays a critical role in the occurrence and development of tumors.However,the function of lncRNA in human gastric cancer remains largely unknown.So far,the role and mechanism of lncRNA DLEU7-AS1 in gastric cancer have not been reported.This study aimed to investigate the effect of DLEU7-AS1on the tumorigenesis and progression of gastric cancer and its mechanism.Methods:The public database the Cancer Genome Atlas(TCGA)was used to analyze the expression of DLEU7-AS1 in gastric cancer tissues and the correlation between its expression and the survival of gastric cancer patients.Then real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)was performed to verify the expression of DLEU7-AS1 in gastric cancer tissues and gastric cancer cell lines.Gastric cancer cell lines were treated with 5-aza-2’-deoxycytidine(DAC)and trichostatin A(TSA)to explore whether epigenetic regulation participated in DLEU7-AS1 transcription.SiRNA was used to down-regulate the expression of DLEU7-AS1 in HGC-27 and AGS cells,and recombinant plasmid was used to up-regulate the expression of DLEU7-AS1 in MGC-803 and MKN-45.The effect was verified by RTFQ-PCR.Cell biological experiments,such as cell counting kit-8(CCK-8)cell proliferation toxicity test,transwell chamber assay,plate colony formation assay and flow cytometry were used to investigate the effect of DLEU7-AS1 on the proliferation,migration,apoptosis and cell cycle progression of gastric cancer cells.RNAsequencing(RNA-seq)was used to analyze downstream signal pathways after silencing DLEU7-AS1 and tested by RTFQ-PCR and Western blot.And RNA Co-immunoprecipitation(RIP)was used to explore the regulatory mechanism of DLEU7-AS1 on downstream signal molecules.Results:The results of public database analysis and RTFQ-PCR demonstrated that DLEU7-AS1 was up-regulated in gastric cancer tissues compared with normal tissues.DLEU7-AS1 expression was negatively correlated with the survival of gastric cancer patients.DLEU7-AS1 was up-regulated in gastric cancer cell lines treated with DAC and TSA,indicating that its expression was epigenetically regulated.DLEU7-AS1 downregulation inhibited gastric cancer cells proliferation and migration and promoted cell apoptosis,while overexpression of DLEU7-AS1 promoted cell proliferation and metastasis and inhibited cell apoptosis.The results of RNA-seq showed that the downregulation of DLEU7-AS1 expression led to a significant decrease in moesin(MSN)expression,which was confirmed by RTFQ-PCR and Western blot.Rescue experiment results further verified that MSN overexpression could partially restore the inhibition effect of knockdown of DLEU7-AS1 on the proliferation and migration of gastric cancer cells.Considering that DLEU7-AS1 mainly located in the nucleus,DLEU7-AS1 binding to P300 and H3K27 highly enriched near the MSN promoter,it was proposed that DLEU7-AS1 might regulate the expression of MSN by recruiting P300,thus contributing to the proliferation and migration of gastric cancer cells.Conclusion:LncRNA DLEU7-AS1 is abnormally up-regulated in gastric cancer and negatively correlated with survival of gastric cancer patients.DLEU7-AS1 may promote the proliferation and migration of gastric cancer by recruiting P300 to regulate the transcription of MSN,which provides a new idea for the diagnosis and treatment of gastric cancer..