目的探讨Mex3C-1对卡巴胆碱诱导的Fos表达的影响。方法将具有可持续表达人Mex3C-1序列的质粒pLV-CMV-Mex3C(OE)在HEK293T细胞中包装成慢病毒载体,同时制备非特异性对照(NC)CmiR0001-MR03的慢病毒载体,分别感染人神经母细胞瘤细胞SH-SY5Y...目的探讨Mex3C-1对卡巴胆碱诱导的Fos表达的影响。方法将具有可持续表达人Mex3C-1序列的质粒pLV-CMV-Mex3C(OE)在HEK293T细胞中包装成慢病毒载体,同时制备非特异性对照(NC)CmiR0001-MR03的慢病毒载体,分别感染人神经母细胞瘤细胞SH-SY5Y,经嘌呤霉素筛选后得到稳定高表达Mex3C-1和阴性对照细胞系,用Real time PCR和Western blot方法检测Mex3C-1的基因和蛋白表达效果。之后用卡巴胆碱诱导Fos表达,在诱导0、30、60、90和120min后分别提取mRNA,采用Real time PCR方法检测Fos mRNA的相对表达量。结果Real time PCR检测结果显示,OE组的Mex3C-1 mRNA的相对表达量(21.11±0.60)高于NC组(1.03±0.13)(t=32.63,P=0.000)。Western blot结果显示,在82kDa处OE组的Mex3C-1蛋白表达量高于NC组(P<0.001)。Real time PCR检测不同干预时间两组Fos mRNA,除0min外各个时间点OE组均高于NC组,表明Mex3C-1的过表达可以明显上调Fos mRNA的表达;NC组于120min时已基本恢复至基础值,而OE组120min时Fos mRNA表达量仍然较高,Mex3C-1过表达可以延长Fos mRNA的半衰期,增强其稳定性,OE组与阴性对照NC组比较,Fos mRNA表达量差异有统计学意义(F=287.069,P=0.000)。结论持续过表达Mex3C-1的人神经母细胞瘤SH-SY5Y细胞系建立成功,且Mex3C-1过表达能够明显增强卡巴胆碱诱导的Fos表达程度并增强其稳定性。展开更多
Background:Although Mex3 RNA-binding family member A(Mex3a)has demonstrated an important role in multiple cancers,its role and regulatory mechanism in CRC is unclear.In this study,we aimed to investigate the role and ...Background:Although Mex3 RNA-binding family member A(Mex3a)has demonstrated an important role in multiple cancers,its role and regulatory mechanism in CRC is unclear.In this study,we aimed to investigate the role and clinical significance of Mex3a in CRC and to explore its underlying mechanism.Methods:Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)were performed to detect the expression levels of genes.5-Ethynyl-2’-deoxyuridine(EDU)and transwell assays were utilized to examine CRC cell proliferation and metastatic ability.The R software was used to do hierarchical clustering analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.Overexpression and rescue experiments which included U0126,a specific mitogen activated protein kinase kinase/extracellular regulated protein kinase(MEK/ERK)inhibitor,and PX-478,a hypoxia-inducible factor 1 subunit alpha(HIF-1α)inhibitor,were used to study the molecularmechanisms of Mex3a in CRC cells.Co-immunoprecipitation(Co-IP)assay was performed to detect the interaction between two proteins.Bioinformatics analysis including available public database and Starbase software(starbase.sysu.edu.cn)were used to evaluate the expression and prognostic significance of genes.TargetScan(www.targetscan.org)and the miRDB(mirdb.org)website were used to predict the combination site between microRNA and target mRNA.BALB/c nude micewere used to study the function of Mex3a and hsa-miR-6887-3p in vivo.Results:Clinicopathological and immunohistochemical(IHC)studies of 101 CRC tissues and 79 normal tissues demonstrated that Mex3a was a significant prognostic factor for overall survival(OS)in CRC patients.Mex3a knockdown substantially inhibited the migration,invasion,and proliferation of CRC cells.Transcriptome analysis and mechanism verification showed that Mex3a regulated the RAP1 GTPase activating protein(RAP1GAP)/MEK/ERK/HIF-1αpathway.Furthermore,RAP1GAP was identified to interact with Mex3a in Co-IP experiments.Bioinformatics and dual-luciferase reporter experiments revealed that hsa-miR-6887-3p could bind to the 3’-untranslated regions(3’-UTR)of the Mex3amRNA.hsa-miR-6887-3p downregulated Mex3a expression and inhibited the tumorigenesis of CRC both in vitro and in vivo.Conclusions:Our study demonstrated that the hsa-miR-6887-3p/Mex3a/RAP1GAP signaling axis was a key regulator of CRC and Mex3a has the potential to be a new diagnostic marker and treatment target for CRC.展开更多
文摘目的探讨Mex3C-1对卡巴胆碱诱导的Fos表达的影响。方法将具有可持续表达人Mex3C-1序列的质粒pLV-CMV-Mex3C(OE)在HEK293T细胞中包装成慢病毒载体,同时制备非特异性对照(NC)CmiR0001-MR03的慢病毒载体,分别感染人神经母细胞瘤细胞SH-SY5Y,经嘌呤霉素筛选后得到稳定高表达Mex3C-1和阴性对照细胞系,用Real time PCR和Western blot方法检测Mex3C-1的基因和蛋白表达效果。之后用卡巴胆碱诱导Fos表达,在诱导0、30、60、90和120min后分别提取mRNA,采用Real time PCR方法检测Fos mRNA的相对表达量。结果Real time PCR检测结果显示,OE组的Mex3C-1 mRNA的相对表达量(21.11±0.60)高于NC组(1.03±0.13)(t=32.63,P=0.000)。Western blot结果显示,在82kDa处OE组的Mex3C-1蛋白表达量高于NC组(P<0.001)。Real time PCR检测不同干预时间两组Fos mRNA,除0min外各个时间点OE组均高于NC组,表明Mex3C-1的过表达可以明显上调Fos mRNA的表达;NC组于120min时已基本恢复至基础值,而OE组120min时Fos mRNA表达量仍然较高,Mex3C-1过表达可以延长Fos mRNA的半衰期,增强其稳定性,OE组与阴性对照NC组比较,Fos mRNA表达量差异有统计学意义(F=287.069,P=0.000)。结论持续过表达Mex3C-1的人神经母细胞瘤SH-SY5Y细胞系建立成功,且Mex3C-1过表达能够明显增强卡巴胆碱诱导的Fos表达程度并增强其稳定性。
基金supported by the National Scientific Foundation of China(NSFC32071127 and 31871160)。
文摘Background:Although Mex3 RNA-binding family member A(Mex3a)has demonstrated an important role in multiple cancers,its role and regulatory mechanism in CRC is unclear.In this study,we aimed to investigate the role and clinical significance of Mex3a in CRC and to explore its underlying mechanism.Methods:Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)were performed to detect the expression levels of genes.5-Ethynyl-2’-deoxyuridine(EDU)and transwell assays were utilized to examine CRC cell proliferation and metastatic ability.The R software was used to do hierarchical clustering analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.Overexpression and rescue experiments which included U0126,a specific mitogen activated protein kinase kinase/extracellular regulated protein kinase(MEK/ERK)inhibitor,and PX-478,a hypoxia-inducible factor 1 subunit alpha(HIF-1α)inhibitor,were used to study the molecularmechanisms of Mex3a in CRC cells.Co-immunoprecipitation(Co-IP)assay was performed to detect the interaction between two proteins.Bioinformatics analysis including available public database and Starbase software(starbase.sysu.edu.cn)were used to evaluate the expression and prognostic significance of genes.TargetScan(www.targetscan.org)and the miRDB(mirdb.org)website were used to predict the combination site between microRNA and target mRNA.BALB/c nude micewere used to study the function of Mex3a and hsa-miR-6887-3p in vivo.Results:Clinicopathological and immunohistochemical(IHC)studies of 101 CRC tissues and 79 normal tissues demonstrated that Mex3a was a significant prognostic factor for overall survival(OS)in CRC patients.Mex3a knockdown substantially inhibited the migration,invasion,and proliferation of CRC cells.Transcriptome analysis and mechanism verification showed that Mex3a regulated the RAP1 GTPase activating protein(RAP1GAP)/MEK/ERK/HIF-1αpathway.Furthermore,RAP1GAP was identified to interact with Mex3a in Co-IP experiments.Bioinformatics and dual-luciferase reporter experiments revealed that hsa-miR-6887-3p could bind to the 3’-untranslated regions(3’-UTR)of the Mex3amRNA.hsa-miR-6887-3p downregulated Mex3a expression and inhibited the tumorigenesis of CRC both in vitro and in vivo.Conclusions:Our study demonstrated that the hsa-miR-6887-3p/Mex3a/RAP1GAP signaling axis was a key regulator of CRC and Mex3a has the potential to be a new diagnostic marker and treatment target for CRC.
