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. 2016 Oct 6;17(1):781.
doi: 10.1186/s12864-016-3122-3.

Multi-omics analysis reveals regulators of the response to PDGF-BB treatment in pulmonary artery smooth muscle cells

Jidong Chen  1   2 , Xiaolei Cui  1 , Zhengjiang Qian  1   2 , Yanjiao Li  1   2 , Kang Kang  3 , Junle Qu  2 , Li Li  4 , Deming Gou  5
Affiliations

Multi-omics analysis reveals regulators of the response to PDGF-BB treatment in pulmonary artery smooth muscle cells

Jidong Chen et al. BMC Genomics. .

Abstract

Background: Pulmonary arterial hypertension (PAH) is a lethal disease with pronounced narrowing of pulmonary vessels due to abnormal cell proliferation. The platelet-derived growth factor BB (PDGF-BB) is well known as a potent mitogen for smooth muscle cell proliferation. To better understand how this growth factor regulates pulmonary arterial smooth muscle cells (PASMCs) proliferation, we sought to characterize the response to PDGF-BB stimulation at system-wide levels, including the transcriptome and proteome.

Results: In this study, we identified 1611 mRNAs (transcriptome), 207 proteins (proteome) differentially expressed in response to PDGF-BB stimulation in PASMCs based on RNA-sequencing and isobaric tags for relative and absolute quantification (iTRAQ) assay. Transcription factor (TF)-target network analysis revealed that PDGF-BB regulated gene expression potentially via TFs including HIF1A, JUN, EST1, ETS1, SMAD1, FOS, SP1, STAT1, LEF1 and CEBPB. Among them, SMAD1-involved BMPR2/SMADs axis plays a significant role in PAH development. Interestingly, we observed that the expression of BMPR2 was decreased in both mRNA and protein level in response to PDGF-BB. Further study revealed that BMPR2 is the direct target of miR-376b that is up-regulated upon PDGF-BB treatment. Finally, EdU incorporation assay showed that miR-376b promoted proliferation of PASMCs.

Conclusion: This integrated analysis of PDGF-BB-regulated transcriptome and proteome was performed for the first time in normal PASMCs, which revealed a crosstalk between PDGF signaling and BMPR2/SMADs axis. Further study demonstrated that PDGF-BB-induced miR-376b upregulation mediated the downregulation of BMPR2, which led to expression change of its downstream targets and promoted proliferation of PASMCs.

Keywords: BMPR2; Platelet-derived growth factor-BB (PDGF-BB); Pulmonary arterial hypertension (PAH); RNA sequencing; iTRAQ; miR-376b.

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Figures

Fig. 1
Fig. 1
Transcriptome analysis of RPASMCs treated with PDGF-BB. a, RNA sequencing heatmap showing a subset of genes differentially expressed upon RPASMCs treated with PDGF-BB for 12 h (FC > 2, FKPM > 0.5 and Qvalue < 0.05 was shown); b, Gene ontology (GO) analysis was carried out on genes differentially expressed, heatmap showing total expression of genes in the most enriched GO terms; c, the most enriched pathways of differentially expressed genes, analyzing via KEGG pathway; d, The network was constructed with genes differentially expressed and their potential corresponded transcription factors (TFs), showing the 15 most enriched TFs (yellow), Color of line represented the enrichment level. As to a GO or KEGG term, Rich factor = (number of differentially expressed gene) / (total gene number), Qvalue is p-value adjusted by method “Benjamini and Hochberg”
Fig. 2
Fig. 2
Protenome analysis of RPASMCs treated with PDGF-BB. a, heatmap displaying a subset of proteins differentially expressed upon RPASMCs treated 12 or 24 h with PDGF-BB (FC > 1.6, p <0.05), measured via iTRAQ; b, GO analysis showing the most enriched GO term on differentially expressed proteins in response to PDGF-BB; c, the most enriched pathway of proteins differentially expressed, analyzing via KEGG pathway; d, The network was constructed with differentially expressed proteins and their potential corresponded transcription factors (TFs), displaying the 14 significantly enriched TFs (yellow), Color of line represented the enrichment level. As to a GO or KEGG term, Rich factor = (number of differentially expressed gene) / (total gene number), Qvalue is p-value adjusted by method “Benjamini and Hochberg”
Fig. 3
Fig. 3
Comparison and integration of differential transcriptome and proteome. a, Venn diagram revealing 56 genes expressed differentially in both mRNA and protein levels; b, Differential expression of these 56 genes was significantly correlated (r = 0.87032, p < 0.001, with Pearson test); c, Integration of TF-target network at proteome and transcriptome levels, Color of line represented the enrichment level; d, the most enriched pathway of differentially expressed genes corresponding to SMAD family, analyzing via KEGG pathway. As to a KEGG term, Rich factor = (number of differentially expressed gene) / (total gene number), Q value is p-value adjusted by method “Benjamini and Hochberg”
Fig. 4
Fig. 4
Differential expression of SMAD family TFs in response to PDGF-BB was due to downregulation of BMPR2. a, differentially expressed genes corresponding to SMAD involved in 3 major cellular procedures; b, q-PCR validated the expression of part of SMAD family targets, which were identified to express differentially in response to PDGF-BB via RNA-sequencing; BMPR2 was detected in RAPSMCs treated with PDGF-BB (time course and dose course) via Western blot at protein level c and qRT-PCR at mRNA level d, e, n = 3, *p < 0.05, **p < 0.01 vs 0 h or 0 ng/ml
Fig. 5
Fig. 5
BMPR2 is a potential direct target of miR-376b. a, Heatmap showed relative level of miRNAs following PDGF-BB treatment for different time; b, BMPR2 protein level was detected in RPASMCs transfected with miRNA mimics via Western Blot; c, The two conserved miR-376b binding sites in the 3′-UTR of BMPR2 along with the mutation sites, respectively; d, 3′-UTR luciferase reporter assay with target sites and their mutant along with miR-376b/miR-Con mimics. Bar charts of luciferase reporter analysis represent means ± SD (n = 3), *p < 0.05, **p < 0.01 vs miR-Con/3′UTR of targets
Fig. 6
Fig. 6
miR-376b promoted RPASMCs proliferation. a, RPASMCs transfected with miR-376b or control mimic were incubated with EDU solution for 4 h, and then were fixed and stained with Apollo dye (red) and DAPI (blue); b, Bar charts showing relative EdU incorporation rate in miR-376b transfected RPASMCs, n = 3, data are shown as mean ± SD. *p < 0.05 vs control mimic; c, Bar charts showing quantification of miR-376b in transfected RPASMCs. n = 3, data are shown as means ± SD, ***p < 0.001 vs control; d, Model depicting PDGF-BB promoted proliferation via regulating miR-376b-induced BMPR2 decrease
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