Dataset Information

Novel Alternatively Spliced Variants of Smad4 Expressed in TGF-?-Induced EMT Regulating Proliferation and Migration of A549 Cells.

ABSTRACT: Introduction:Non-small cell lung cancer (NSCLC) is a worldwide malignance threatening human life. TGF-?/Smad signaling is known to regulate cell proliferation, differentiation, migration and growth. As the only co-Smad playing crucial roles in TGF-? signaling, Smad4 is reported to be frequently mutated or to occur as alternatively spliced in tumor cells. Smad4 was reported to be involved in the TGF-?-induced EMT process. However, whether the alternative splicing occurs in the TGF-?-induced EMT process in NSCLC was not clear. Methods:In our current study, we explored the alternative splicing of Smad4 during the process of TGF-?-induced EMT in A549 cells. 10 ng/mL TGF-? was used to induce EMT. Then, nest-PCR and agarose electrophoresis were performed to detect the expression of Smad4 variants and sequencing to get the variant DNA sequences. For recombinant expression of variants of Smad4 in A549 cells, we used lentiviral variants to infect cells. In order to explore the effects of variants on the proliferation and migration of A549 cells, the MTT assay, colony formation assay and wound-healing assay were done. The effects of variants on E-cad and VIM protein expression were explored through Western blot. Results:There were several novel gene fragments expressed in TGF-?-induced A549 cells, and the sequencing results showed that they were indeed the Smad4 variants that were not reported. For recombinant expression of Smad4 variants in A549 cells, we found that they have significant effects on the proliferation and migration of cells, and also regulated the E-cad and VIM protein expression. Conclusion:Our results indicated that novel Smad4 variants were expressed in TGF-?-induced EMT process. The functional study showed that these novel variants regulate cell proliferation and migration and affect E-cad and VIM protein expression, showing the potential as targets for cancer therapy.

SUBMITTER: Wan R

PROVIDER: S-EPMC7073448 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Novel Alternatively Spliced Variants of Smad4 Expressed in TGF-β-Induced EMT Regulating Proliferation and Migration of A549 Cells.

Wan Rongxue R Xu Xichao X Ma Lunkun L Chen Ying Y Tang Liling L Feng Jianguo J

OncoTargets and therapy 20200311

<h4>Introduction</h4>Non-small cell lung cancer (NSCLC) is a worldwide malignance threatening human life. TGF-β/Smad signaling is known to regulate cell proliferation, differentiation, migration and growth. As the only co-Smad playing crucial roles in TGF-β signaling, Smad4 is reported to be frequently mutated or to occur as alternatively spliced in tumor cells. Smad4 was reported to be involved in the TGF-β-induced EMT process. However, whether the alternative splicing occurs in the TGF-β-induc ...[more]

PMID: 32210586

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Project description:Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples.We developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens.We found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively).Whole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM.

Project description:Tumors and the tumor microenvironment produce multiple growth factors that influence cancer cell behavior via various signal transduction pathways. Growth factors, like transforming growth factor β (TGFβ) and epidermal growth factor (EGF), have been shown to induce proliferation, migration, and invasion in different cell models. Both factors are frequently overexpressed in cancer and will often act in combination. Although both factors are being used as rational targets in clinical oncology, the similarities and differences of their contributions to cancer cell migration and invasion are not fully understood. Here we compared the impact of treating A549 lung adenocarcinoma cells with TGFβ, EGF, and both in combination by applying videomicroscopy, functional assays, immunoblotting, real-time PCR, and proteomics. Treatment with both factors stimulated A549 migration to a similar extent, but with different kinetics. The combination had an additive effect. EGF-induced migration depended on activation of the mitogen-activated protein kinase (MAPK) pathway. However, this pathway was dispensable for TGFβ-induced migration, despite a strong activation of this pathway by TGFβ. Proteome analysis (data are available via ProteomeXchange with identifier PXD023024) revealed an overlap in expression patterns of migration-related proteins and associated gene ontology (GO) terms by TGFβ and EGF. Further, only TGFβ induced the expression of epithelial to mesenchymal transition (EMT)-related proteins like matrix metalloproteinase 2 (MMP2). EGF, in contrast, made no major contribution to EMT marker expression on either the protein or the transcript level. In line with these expression patterns, TGFβ treatment significantly increased the invasive capacity of A549 cells, while EGF treatment did not. Moreover, the addition of EGF failed to enhance TGFβ-induced invasion. Overall, these data suggest that TGFβ and EGF can partly compensate for each other for stimulation of cell migration, but abrogation of TGFβ signaling may be more suitable to suppress cell invasion.

Dataset Information

Novel Alternatively Spliced Variants of Smad4 Expressed in TGF-?-Induced EMT Regulating Proliferation and Migration of A549 Cells.

Publications

Novel Alternatively Spliced Variants of Smad4 Expressed in TGF-β-Induced EMT Regulating Proliferation and Migration of A549 Cells.

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