| Background and objectionsColorectal cancer(CRC),a common malignant tumor of the digestive system,has high incidence and poor prognosis.Currently,metastastic colorectal cancer(mCRC)is regarded as the main reason for its therapeutic effect and death of CRC patients.And the majority of mCRC patients face the difficulties and challenges such as beyond cure and hard to benefit from treatments.Therefore,exploring the mechanism network of CRC metastasis may provide significant ideas and clues for early intervention and treatment of CRC.Moreover,studies on the mechanism network of CRC occurrence and development has begun to focus on post-transcriptional regulation in recent years.DDX39B,a type of RNA helicase,modulates almost all the RNA metabolic processes.Sudies on its functions are very rich and abundant,and it covers spliceosome and mRNA nuclear export complexes.While the role of DDX39B in disease,especially in tumors,and the undelying molecules mechanism are still unclear.In addition,DDX39B is mainly responsible for the splicing and the nuclear export of gene transcription products.Therefore,the abnormal expression or function of DDX39B,which is responsible for post-transcriptional regulation,could exert effects on the expression of corresponding genes(tumor promoting/suppressor factors),and then influences the occurrence and development of diseases.Firstly,bioinformatics analysis and molecular biology experiments were applied in our study to explore the expression of DDX39B in CRC.Then,the role of DDX39B in CRC invasion and metastasis were explored through a series of functional experiments(cell invasion and metastasis related).Secondly,the application of transcriptomics sequencing could provides ideas and cules for searching of the downstream targets of DDX39B.Finally,biological experiments were used to verify and in depth disccuss the post-transcriptionally regulatory mechanism and potential molecular interactions of DDX39B in the progression of CRC.The above mentioned may be conducive to add certain theoretical foundation for the diagnosis,therapy and prognosis of CRC and the potential molecular targets.Materials and Methods1.DDX39B expression in CRC tissues and cells1)The expression of DDX39B in CRC tissues(paired and unpaired)were analyzed by means of bioinformatics(TCGA?GEO database).And the correlation between DDX39B expression and the prognosis of CRC patients was also explored using public database.2)DDX39B expression in paired CRC tissues and CRC cell lines were evaluated using qRT-PCR,western blotting and IHC.2.The effect of DDX39B on the invasion and metastasis of CRC cells.1)Stable cell lines with DDX39B overexpression and transient silenced DDX39B celllines were constructed,western blotting and qRT-PCR were applied for validating the efficiency of transfection.Then the migrated and invasive capabilities in DDX39B overexpression and silenced group were detected using transwell and wound healing assay.2)And the interference sequence of DDX39B siRNA was used for the construction of stable silenced DDX39B cell lines,then the metastatic ability of CRC cells in vivo was explored using the ileocecal orthotopic implanted tumors in nude mice.3.The role of DDX39B in the epithelial-mesenchymal transition(EMT)program of CRC cells1)EMT markers expression(mRNA and protein)in both DDX39B overexpression and DDX39B knockdown groups were evaluated using qRT-PCR and western blotting.2)Cell tight junctions and morphological changes in DDX39B overexpression andknockdown group were analyzed using immunofluorescence of cytoskeleton staining.3)Immunofluorescence using tissue sections of the ileocecal orthotopic implanted tumors in nude mice was performed to detect the expression of EMT markers.4.Molecular mechanism on DDX39B regulating the downstream targeted genes in CRC.1)High-throughput sequencing such as RNA-seq and RNA-binding protein immunoprecipitation-sequencing(RIP-seq)was applied to provide clues for the searching of downstream target genes DDX39B may regulate in CRC.2)qRT-PCR assay was conducted to validate the RNA-seq results,and FUT3 whose expression changes were significant with the alterations of DDX39B expression was selected.Moreover,relationship of DDX39B expression with FUT3 expression was explored by means of IH C on serial sections of paired CRC tissues and qRT-PCR.3)Based on the RIP-seq results,splicing experiments in vitro using FUT3 minigene assay were conducted to validate that DDX39B modulates FUT3 splicing.And nuclear and cytoplasmic RNA separation assay was conducted to determine that DDX39B participates in the nuclear export of mature FUT3 mRNA(spliced).5.The DDX39B-FUT3-TGF?R-I regulates EMT,invasion and metastasis of CRC.1)Targeted gene expression of TGF?/SMADs signaling pathway in DDX39B overexpression and silenced group were evaluated using nuclear and cytoplasmic separation assay(protein).2)Aleuria Aurantia Lectin(AAL)preferentially recognizes Fuc-1,3GlcNac.Then lectin blotting was conducted to evaluate the fucosylated and total TGF?R-I expression.And immunofluorescence was also applied to validate AAL and TGF?R-I expression.3)The DDX39B overexpression group was treated with the TGF?R-I inhibitor SB431542,and we explored whether SB431542 could affect the ability of DDX39B to activate TGF?/SMADs signaling pathway and promote the invasion and metastasis of CRC cells.4)The DDX39B knockdown group was treated with TGF?1,and the response of the control group and the DDX39B silencing group to TGF?1 stimulation was explored.5)The DDX39B overexpression group was treated with the siRNA of FUT3.Then whether siFUT3 could affect the ability of DDX39B to activate TGF?/SMADs signaling pathway and promote the invasion and metastasis of CRC cells was investigated.Statistical analysesStatistical analyses were performed using Graph-Pad Prism software 6.0(GraphPad Software;San Diego,CA,USA)and Microsoft Excel 2016(Microsoft,Redmond,WA,USA).Student's t test or nonparametric ANOVA were applied for comparisons between groups.Correlation analysis was evaluated using the Spearman's rank correlation coefficient.P<0.05 indicated significance.Results1.Bioinformatics analysis data suggested that DDX39B was overexpressed in CRC samples compared with normal samples(paired and unpaired tissues).And the DDX39B expression was higher to varying degrees in cancers of different histological types and stages.Overall survival analysis revealed that patients with high DDX39B expression levels had a shorter overall survival time than patients with low DDX39B expression levels(GSE17536,GSE17538).2.The data of our research center shows that the expression level of DDX39B in tissues,protein and mRNA in CRC is higher than that in normal tissues.3.DDX39B promotes CRC migration and invasion,validated using transwell,wound healing assay and ileocecal orthotopic implanted tumors in nude mice.4.DDX39B promotes EMT of CRC cells,validated using western blotting,qRT-PCR,cytoskeleton stain and immunofluorescence of tissue sections.5.RNA-seq and RIP-seq results showed that DDX39 regulates FUT3 expression via directly binding with FUT3 pre-mRNA.6.IHC on serial sections of paired CRC tissues and qRT-PCR assay demonstrated that DDX39B expression is strongly correlated with FUT3 expression.7.Splicing experiments in vitro using Minigene assay showed that DDX39B enhances the splicing efficiency of FUT3 pre-mRNA.Nuclear and cytoplasmic RNA separation assay presented that DDX39B enhances the nuclear export of matured FUT3 mRNA,then the corresponding expression of FUT3 is upregulated.8.Nuclear and cytoplasmic protein assay suggested that DDX39B promotes the activation of TGF?/SMADs signaling pathway.9.Lectin blotting suggested that DDX39B promotes the fucosylation of TGF?R-I via FUT3.10.Complementary experiments(TGF?R-I inhibitor,TGF?1 stimulate,FUT3 knockdown)confirmed that the DDX39B-FUT3-TGF?R-I promotes EMT,invasion and metastasis of CRC.ConclusionsDDX39B expression is upregulated in CRC tissues and cells.And DDX39B promotes CRC metastasis and EMT via the DDX39B-FUT3-TGF?R-I. |
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