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Fascin actin-bundling protein 1 regulates non-small cell lung cancer progression by influencing transcription and splicing of tumorigenesis-related genes [RNA-seq]

ABSTRACT: Background. High mortality rates are prevalent among patients with non-small cell lung cancer (NSCLC), and effective therapeutic targets are key prognostic factors. Fascin actin-bundling protein 1 (FSCN1) promotes NSCLC; however, its role as an RNA-binding protein in NSCLC remains unexplored. Therefore, we aimed to explore FSCN1 expression and function in A549 cells. Method. We screened for alternative-splicing events and differentially expressed genes (DEGs) after FSCN1 silence via RNA-sequencing (RNA-seq). FSCN1 immunoprecipitation followed by RNA-seq were used to identify target genes whose mRNA expression and pre-mRNA alternative-splicing levels might be influenced by FSCN1. Results. Silencing FSCN1 in A549 cells affected malignant phenotypes; it inhibited proliferation, migration, and invasion, and promoted apoptosis. RNA-seq analysis revealed 2851 DEGs and 3057 alternatively spliced genes. Gene ontology-based functional enrichment analysis showed that downregulated DEGs and alternatively splicing genes were enriched for the cell-cycle. FSCN1 promoted the alternative splicing of cell-cycle-related mRNAs involved in tumorigenesis (i.e., BCCIP, DLGAP5, PRC1, RECQL5, WTAP, and SGO1). Combined analysis of FSCN1 RNA-binding targets and RNA-seq data suggested that FSCN1 might affect ACTG1, KRT7, and PDE3A expression by affecting the pre-mRNA alternative-splicing levels of NME4, NCOR2, and EEF1D FSCN1 to long non-coding RNA transcripts (RNASNHG20, NEAT1, NSD2, and FTH1), which were highly abundant. Overall, extensive transcriptome analysis of gene alternative splicing and expression levels was performed in cells transfected with FSCN1 short-interfering RNA. Our data provide global insights into the regulatory mechanisms mediated by FSCN1 and its target genes in lung cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE234859 | GEO | 2023/07/01

REPOSITORIES: GEO

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Project description:Accumulating evidence indicates that RNA-binding proteins (RBPs) are involved in tumorigenesis. As a member of DEAD-box RBPs, DDX17 has a significant role in various contexts, such as mRNA translation, pre-mRNA splicing, ribosome biogenesis, and DNA repair. However, whether DDX17 is involved in the regulation of gene expression and alternative splicing in lung adenocarcinoma (LUAD) remains unclear. Methods: CCK-8 assay was used to evaluate cells proliferation. Cell apoptosis was evaluated by an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit. We used high-throughput RNA sequencing (RNA-seq) to determine differentially expressed genes (DEGs) and alternative splicing (AS) events in DDX17 knockdown LUAD cells. Functional and pathway enrichment analysis was also performed. Results: Knockdown of DDX17 by siRNA significantly inhibited the proliferation and induced apoptosis in A549 cells. 5243 DEGs were identified. Functional enrichment analyses indicated that these genes were mainly enriched in biological processes including regulation of transcription, regulation of cell growth, DNA repair. DDX17 knockdown increased the level of pro-apoptotic gene expression and decreased the expression of pro-proliferation genes. We also showed that DDX17-regulated AS events revealed by computational analysis using ABLas software in A549 cells were highly validated by RT-qPCR, and also validated by TCGA-LUAD dataset. Functional enrichment analyses showed regulated alternatively spliced genes (RASGs) were mainly enriched in biological processes related to apoptotic process, DNA repair, positive regulation of translation, positive regulation of apoptotic process. Conclusion: The present findings suggest that DDX17 may function as an oncogene by regulating both the expression and the alternative splicing of genes associated with proliferation, invasion and apoptosis in LUAD cells. Our findings may bring new insights into understanding the molecular mechanisms of LUAD and provide a new direction for LUAD treatment.

Project description:Alternative splicing (AS) is particularly relevant to cancer progression and apoptosis. Although previous studies have shown that the apurinic-apyrimidinic endonuclease-1 (APEX1) is involved in tumor progression, it is unclear whether APEX1 can regulate AS on cell proliferation and apoptosis of Non-small-cell lung cancer (NSCLC). We performed a comprehensive analysis of the APEX1 expression in 517 lung NSCLC samples from the TCGA (Cancer Genome Atlas) database, and selected two sets of cancer samples with differentially expression APEX1 to analyze potential APEX1-regulated alternative splicing events (ASEs). Functional analysis of the APEX1 in A549 cells were performed in vitro. The APEX1 was overexpression in A549 cell by gene transfection. We identified AS targets regulated by APEX1, analyzed the GO biological process and KEGG functional pathways, and validated APEX1-regulated ASEs detected by RNA-seq and RT-PCR in A549 cells, then in clinical NSCLC samples these results were verified. The expression of the APEX1 was up-regulated in NSCLC samples, and overexpression of the APEX1 resulted in cell proliferation reduction and apoptosis induction. AS of many genes regulated by the APEX1 were enrich in cancer-related functional pathways. Results from A549 cell model and clinical samples showed that the MAPK signaling pathway, the Wnt signaling pathway were shared among the top ten enriched GO processes and KEGG functional pathways. According to our research, the validated AS events regulated by APEX1 mostly located in genes encoding transcription regulation factor in various signaling pathways, including the AXIN1 (axis inhibition protein 1), GCNT2 (N-acetyl glucosaminyl transferase 2), SMAD3 (SMAD Family Member 3), CTBP2 (C-Terminal Binding Protein 2). In this study, we successfully applied RNA-seq technology to demonstrate APEX1 regulation of AS. Our results underline that APEX1 was efficiently up-regulated in NSCLC samples, while overexpression of the APEX1 in A549 cells resulted in proliferation reduction and apoptosis induction. We confirm that the APEX1 regulates the AS of many genes which involved in cancer proliferation and apoptosis functional pathways, such as the MAPK signaling pathway and the Wnt signaling pathway, leading to mediate lung cancer progression. We found that high expression of the APEX1 in NSCLC is an independent prognostic factor related to tumor progression. Therefore, the APEX1 can serve as a molecular marker or therapeutic target for NSCLC treatment.

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Fascin actin-bundling protein 1 regulates non-small cell lung cancer progression by influencing transcription and splicing of tumorigenesis-related genes [RNA-seq]

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