目的研究肌肉过量RNA结合家族成员A(muscle excess RNA-binding family member A,MEX3A)表达在肺鳞癌组织的临床意义,并探讨MEX3A对肺鳞癌细胞增殖和转移能力的作用及分子机制。方法采用GEPIA在线软件分析MEX3A在肺鳞癌组织中的表达水...目的研究肌肉过量RNA结合家族成员A(muscle excess RNA-binding family member A,MEX3A)表达在肺鳞癌组织的临床意义,并探讨MEX3A对肺鳞癌细胞增殖和转移能力的作用及分子机制。方法采用GEPIA在线软件分析MEX3A在肺鳞癌组织中的表达水平。收集住院的肺鳞癌和癌旁组织标本各58例,采用免疫组化检测MEX3A蛋白在肺鳞癌组织和癌旁组织中的表达水平,统计分析MEX3A表达与患者临床病理参数及预后的关系。构建MEX3A干扰及MEX3A过表达质粒,常规培养肺鳞癌细胞株H266,分为NC组(5μl Lip 2000和5μg对照质粒)、sh-MEX3A组(5μl Lip 2000和5μg MEX3A敲减质粒)和oe-MEX3A组(5μl Lip 2000和5μg MEX3A过表达质粒)。CCK-8法检测各组细胞增殖能力,Transwell实验检测各组细胞转移能力,Western blot检测各组细胞PI3K/AKT信号通路PI3K、p-PI3K、AKT和p-AKT蛋白的表达。结果GEPIA分析和免疫组化结果显示与癌旁组织相比,MEX3A在肺鳞癌组织中的表达显著上调(P<0.05)。MEX3A高表达与患者淋巴结转移和TNM分期相关(P<0.05);与MEX3A低表达肺鳞癌患者相比,MEX3A高表达肺鳞癌患者预后较差(P<0.05)。与NC组相比,sh-MEX3A组肺鳞癌细胞增殖和转移能力显著下降(P<0.05),oe-MEX3A组肺鳞癌细胞增殖和转移能力显著增加(P<0.05);与NC组相比,sh-MEX3A组肺鳞癌细胞PI3K/AKT信号通路关键蛋白p-PI3K和p-AKT的表达降低(P<0.05),oe-MEX3A组肺鳞癌细胞PI3K/AKT信号通路关键蛋白p-PI3K和p-AKT的表达增加(P<0.01)。结论MEX3A促进肺鳞癌恶性进展,可望作为肺鳞癌的新型生物标志物和治疗靶点。展开更多
Insulin-like growth factor-1 receptor(IGF-1R) has been made an attractive anticancer target due to its overexpression in cancers.However,targeting it has often produced the disappointing results as the role played by ...Insulin-like growth factor-1 receptor(IGF-1R) has been made an attractive anticancer target due to its overexpression in cancers.However,targeting it has often produced the disappointing results as the role played by cross talk with numerous downstream signalings.Here,we report a disobliging IGF-1R signaling which promotes growth of cancer through triggering the E3 ubiquitin ligase MEX3A-mediated degradation of RIG-I.The active β-arrestin-2 scaffolds this disobliging signaling to talk with MEX3A.In response to ligands,IGF-1Rβ activated the basal βarr2 into its active state by phosphorylating the interdomain domain on Tyr64 and Tyr250,opening the middle loop(Leu130-Cys141) to the RING domain of MEX3A through the conformational changes of βarr2.The models of βarr2/IGF-1Rβ and βarr2/MEX3A could interpret the mechanism of the activated-IGF-1R in triggering degradation of RIG-I.The assay of the mutants βarr2Y64Aand βarr2Y250Afurther confirmed the role of these two Tyr residues of the interlobe in mediating the talk between IGF-1Rβ and the RING domain of MEX3A.The truncated-βarr2 and the peptide ATQAIRIF,which mimicked the RING domain of MEX3A could prevent the formation of βarr2/IGF-1Rβ and βarr2/MEX3A complexes,thus blocking the IGF-1R-triggered RIG-I degradation.Degradation of RIG-I resulted in the suppression of the IFN-I-associated immune cells in the TME due to the blockade of the RIG-I-MAVS-IFN-I pathway.Poly(I:C) could reverse anti-PD-L1 insensitivity by recovery of RIG-I.In summary,we revealed a disobliging IGF-1R signaling by which IGF-1Rβ promoted cancer growth through triggering the MEX3A-mediated degradation of RIG-I.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
基金supported by Beijing Natural Science Foundation(7222253,China)National Natural Science Foundation of China(81973350/82173841)supported by Beijing Natural Science Foundation(7212149,China)。
文摘Insulin-like growth factor-1 receptor(IGF-1R) has been made an attractive anticancer target due to its overexpression in cancers.However,targeting it has often produced the disappointing results as the role played by cross talk with numerous downstream signalings.Here,we report a disobliging IGF-1R signaling which promotes growth of cancer through triggering the E3 ubiquitin ligase MEX3A-mediated degradation of RIG-I.The active β-arrestin-2 scaffolds this disobliging signaling to talk with MEX3A.In response to ligands,IGF-1Rβ activated the basal βarr2 into its active state by phosphorylating the interdomain domain on Tyr64 and Tyr250,opening the middle loop(Leu130-Cys141) to the RING domain of MEX3A through the conformational changes of βarr2.The models of βarr2/IGF-1Rβ and βarr2/MEX3A could interpret the mechanism of the activated-IGF-1R in triggering degradation of RIG-I.The assay of the mutants βarr2Y64Aand βarr2Y250Afurther confirmed the role of these two Tyr residues of the interlobe in mediating the talk between IGF-1Rβ and the RING domain of MEX3A.The truncated-βarr2 and the peptide ATQAIRIF,which mimicked the RING domain of MEX3A could prevent the formation of βarr2/IGF-1Rβ and βarr2/MEX3A complexes,thus blocking the IGF-1R-triggered RIG-I degradation.Degradation of RIG-I resulted in the suppression of the IFN-I-associated immune cells in the TME due to the blockade of the RIG-I-MAVS-IFN-I pathway.Poly(I:C) could reverse anti-PD-L1 insensitivity by recovery of RIG-I.In summary,we revealed a disobliging IGF-1R signaling by which IGF-1Rβ promoted cancer growth through triggering the MEX3A-mediated degradation of RIG-I.
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.
