Global transcriptome study of Dip2B-deficient mouse embryonic lung fibroblast reveals its important roles in cell proliferation and development

Abstract

Disco-interacting protein 2 homolog B (Dip2B) is a member of Dip2 family encoded by Dip2b gene. Dip2B has been reported to regulate murine epithelial KIT⁺ progenitor cell expansion and differentiation epigenetically via exosomal miRNA targeting during salivary gland organogenesis. However, its molecular functions, cellular activities and biological process remain unstudied. Here, we investigated the transcriptome of Dip2B-deficient mouse embryonic lung fibroblasts (MELFs) isolated from E14.5 embryos by RNA-Seq. Expression profiling identified 1369 and 1104 differentially expressed genes (DEGs) from Dip2b^-/- and Dip2b^+/- MELFs in comparisons to wild-type (Dip2b^+/+ ). Functional clustering of DEGs revealed that many gene ontology terms belong to membrane activities such as 'integral component of plasma membrane', and 'ion channel activity', suggesting possible roles of Dip2B in membrane integrity and membrane function. KEGG pathway analysis revealed that multiple metabolic pathways are affected in Dip2b^{- / -} and Dip2b ^{+/ -} when compared to Dip2b ^+/+ MELFs. These include 'protein digestion and absorption', 'pancreatic secretion' and 'steroid hormone synthesis pathway'. These results suggest that Dip2B may play important roles in metabolism. Molecular function analysis shows transcription factors including Hox-genes, bHLH-genes, and Forkhead-genes are significantly down-regulated in Dip2b^{- / -} MELFs. These genes are critical in embryo development and cell differentiation. In addition, Dip2B-deficient MELFs demonstrated a reduction in cell proliferation and migration, and an increase in apoptosis. All results indicate that Dip2B plays multiple roles in cell proliferation, migration and apoptosis during embryogenesis and may participate in control of metabolism. This study provides valuable information for further understanding of the function and regulatory mechanisms of Dip2B.

Keywords: DEG; Dip2B knockout; Gene ontology; KEGG; MELFs; Transcriptome.

Graphical abstract

Fig. 1

MELF cell isolation and Dip2b mRNA expression analysis by qPCR (A) Cell images of MELF cultures at low (Top penal) and high (Bottom panel) density. (B) Relative expression levels of Dip2b mRNA in MELFs by qPCR. (C) Gel electrophoresis image showing PCR products.

Fig. 2

Overview of gene expression profiling. (A) Venn diagram showing unique and overlapping DEGs between heterozygous and homozygous MELFs (FC ≥ 1 and FDR ≤ 0.001). (B) Number of up- and down-regulated DEGs in Dip2b^−/− and Dip2b^+/− vs to Dip2b^+/+ (C) Volcano plots highlighting significant DEG among three comparative samples. Each dot in plot corresponds to one differentially expressed gene, the y-axis represents −log₁₀ (FDR) and the x-axis displays the differences of FC values in samples. Blue and red dots represent up- and down-regulated differentially expressed genes, whereas gray dots indicate genes with no change. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

GO analysis. Top seven GO terms of up- and down-regulated DEGs of Dip2b^+/+ vs Dip2b^−/− (FC ≥ 2, FDR < 0.01).

Fig. 4

KEGG pathway analysis of DEGs from Dip2b^+/+ vs Dip2b^−/− MELFs. Adjusted p-values (Q-values) depicting significant enrichment (q-value < 0.05).

Fig. 5

Effect of Dip2B on cell proliferation and growth. Heatmap for DEGs associated with GO biological process terms of (A) ‘negative regulation of cell proliferation’ and (B) ‘negative regulation of cell growth’. (C) MTT viability assay of MELFs derived from Dip2b^−/−, Dip2b^+/− and Dip2b^+/+.

Fig. 6

Apoptosis and cell cycle analysis. (A) Heatmap for DEGs associated with GO terms ‘positive regulation of apoptotic process’. (B) Apoptotic cells measured using a Becton-Dickinson FACScan cytofluorometer. (C) Histogram of flow cytometric analysis of cell cycle showing the distribution of cell phase.

Fig. 7

Scratching assay for cell migration. Images were taken at 0, 24, 48hrs post-scratching.

Fig. 8

Gene expression validation of DEGs from RNA-Seq by qPCR.