Mechanisms Of IGF2BP3 Regulating A549 Cells By Post-transcriptional Alternative Splicing Effect Revealed By Transcriptome Analysis

Objective:1.Identify important target genes of Insulin-like growth factor 2 m RNA binding protein 3(IGF2BP3),and explore possible signal transduction pathways through bioinformatics analysis;2.Through the analysis of transcriptome data,we explored the molecular mechanism of IGF2BP3 regulating alternative splicing.Methods:1.Search and identification of target genes:(1)In order to understand the relationship between IGF2BP3 and cancer,firstly obtain the expression data of IGF2BP3 in various malignant tumors through TCGA and Oncomine databases;(2)In order to further explore the regulatory mechanism of IGF2BP3 on target genes in A549,A549 cells were divided into IGF2BP3 plasmid transfection group and control group.IGF2BP3 in A549 cells was over-expressed by plasmid transfection technology.Detection of IGF2BP3 expression in the two groups by q-PCR and Western blot;(3)Then use the modified RNA immunoprecipitation sequencing(i RIP-seq)technology to analyze the transcripts of IGF2BP3 transfection group and control group interacting with IGF2BP3;(4)GO and KEGG enrichment analysis of the binding peak data to reveal bioinformatics methods and reveal the signal pathways enriched in;(5)Next,identify the binding target of IGF2BP3 by quantitative RIP-PCR;2.Transcriptome data analysis:(1)In order to investigate the effect of IGF2BP3 on gene regulation in A549 cells,the transcriptome sequencing(RNA-seq)technology was used to analyze gene expression differences in the transfected and control groups,respectively;(2)Based on GO and KEGG enrichment analysis of differentially expressed genes to investigate the signal regulation pathways involved in these genes;(3)In order to further investigate whether IGF2BP3 plays an important role in alternative splicing,we performed alternative splicing analysis of transcriptome data to analyze the differential alternative splicing of target genes in A549 cells;(4)GO and KEGG enrichment analysis of genes with alternative splicing events to understand the signaling pathways involved;3.Integrated analysis of RNA-seq and i RIP-seq data:By analyzing the distribution of reads of genes with variable splicing events and q-PCR experiments,the changes of the alternative splicing events of key regulatory genes in A549 cells after overexpression of IGF2BP3 were further investigated.Results:1.Search and identification of target genes:(1)IGF2BP3 is highly expressed in various cancers,especially in lung adenocarcinoma and lung squamous cell carcinoma;(2)In A549 cells,compared with the control group,the expression level of the IGF2BP3 transfection group was significantly increased,and overexpression,indicating that IGF2BP3 was successfully transfected;(3)In A549 cells,IGF2BP3 binds tightly to m RNA,and the target genes that interact with IGF2BP3 are enriched in the CDS region,3’UTR region,and 5’UTR region,suggesting that they encode multiple protein products and are widely involved in the regulation processes on RNA levels and translation level;(4)IGF2BP3 binds to the target to participate in multiple signal pathways such as RNA splicing and spliceosome,suggesting that the target gene has an important role in the signal pathway;(5)IGF2BP3 can regulate BTF3 and PKM and other genes,suggesting that IGF2BP3 may participate in the proliferation and apoptosis process of A549 cells;2.Transcriptome data analysis:(1)In A549 cells,IGF2BP3 overexpression caused 1216 gene expression changes,suggesting that IGF2BP3 widely regulates gene expression in A549;(2)Differentially expressed genes are mainly enriched in multiple signaling pathways,such as the Notch signaling pathway and Ras signaling pathway,suggesting that differentially expressed genes may have regulatory effects on the proliferation and apoptosis-related signaling pathways;(3)After overexpression of IGF2BP3,121 alternative splicing events were detected to significantly change,suggesting that IGF2BP3 widely regulates alternative splicing events in A549 cells;(4)Genes that have alternative splicing events are mostly enriched in cell regulation and signaling pathways(such as the NF-κB signaling pathway,negative transcription regulation of the RNA polymerase II promoter,post-translational protein modification,etc.),suggesting alternative splicing genes exist extensively in multiple regulatory processes of A549 cells;3.Integrated analysis of RNA-seq and i RIP-seq data:The reads distribution and q-PCR results showed that the number of BTF3 alternative splicing events increased and the PKM alternative splicing events decreased after IGF2BP3 overexpression,suggesting that IGF2BP3 can regulate BTF3 and PKM’s alternative splicing events in A549 cells.Conclusion:This study explored and identified multiple important target genes of IGF2BP3,and confirmed that IGF2BP3 can regulate gene expression in multiple signaling pathways through post-transcriptional alternative splicing,thereby regulating the evolution of A549 cells.Our results not only offer new mechanisms for deeplyunderstanding malignancy,but also provide possible therapeutic targets.