Fig 1.
Acute restraint stress induces long-lasting changes in BLC miRNA expression.
(A) Overview of the experimental timecourse. (B) Heatmap of the 18 miRNAs differentially expressed in the BLC of mice that went through acute restraint stress 30 days earlier. Red colors indicate upregulation and blue indicate downregulation of a given miRNA. (C) qPCR validation of the smRNA-seq was performed for 3 miRNAs. t indicates a trend in the same direction as the smRNA-seq. * indicates p<0.05. Error +/- s.e.m. (D) The top 10 significant pathways of predicted target genes for the list of stress induced miRNAs obtained using DIANA’s miRPATH software. To the right of each pathway are listed the number of target genes that belong to the pathway and a yes (Y) or no (N) to indicate if the pathway is targeted by mir-29a-5p. (E) qPCR experiments were run to examine mir-29a-5p at 60 minutes and 24 hours post-stress, no significant differences were observed between groups.
Fig 2.
Acute restraint stress induces long last proteomic changes in the BLC.
(A) A summary of the global proteomics profile of the BLC in mice that underwent a single acute restraint session 30 days prior. (B) The top 25 most differentially regulated proteins in the BLC of stressed mice and their log2 fold change (Log2FC) values. (C+D) Ingenuity Pathway Analysis of the global proteomics profile identified the top 10 most significant canonical pathways (C) and functional annotations (D) of the gene list. Upregulated pathways are shown in green and downregulated in orange, with the number of molecules belonging to each pathway indicated to the right. (E) Representative images of western blot validation of Ube2d3 protein. On the left are the total protein stain for normalization and the image of Ube2d3. On the right is the quantification of the western blot data.
Fig 3.
Acute restraint stress induces long lasting RNA changes in the BLC.
(A) A summary of the global transcriptome profile of the BLC in mice that underwent a single acute restraint session 30 days prior. (B) A volcano plot of the RNA-seq data with significantly changed genes colored in red and unchanged genes colored in gray. (C) qPCR validation of a subset of genes significantly upregulated in the RNA-seq. *p<0.05. Error +/- s.e.m. (D+E) Ingenuity Pathway Analysis of the global transcriptional profile identified the top 10 most significant canonical pathways (D) and functional annotations (E) of the gene list. Upregulated pathways are shown in green and downregulated in orange, with the number of molecules belonging to each pathway indicated to the right.
Fig 4.
Integration of RNA-seq, smRNA seq and proteomics datasets.
(A) Venn diagram displays the overlap of differentially expressed genes at the RNA and protein levels. 16 proteins were regulated at both levels and their log2 fold change (Log2FC) values for each measure are depicted in (B). Upregulated changed are highlighted in green and downregulated changes are highlighted in orange. (C) Integration of the datasets identified putative miRNA targets that are regulated in the opposite direction from significantly changed miRNAs in both the proteomics and RNA-seq datasets. (D-E) Matrices depict the differentially expressed miRNAs that also target the top 15 most regulated genes at the RNA (D) or protein level (E) in an opposing fashion. Upregulated miRNAs are highlighted in green and downregulated in orange.
Fig 5.
mir-29a-5p predicted targets are regulated in the transcriptome and proteome after acute restraint stress.
Tables of the predicted mir-29a-5p targets that are downregulated at the RNA level (A) and protein level (B) in the BLC 30 days after an acute restraint session, based on RNA-seq and quantitative mass spectrometry. The database that identified each target is listed to the right of the log2 fold change values.