Timecourse bioinformatic identification of miR-199 as a key regulator of chondrogenic gene expression.

A) Overrepresentation analysis (ORA) of the differentially expressed miRNAs and mRNAs at each time point contrasted to D0. For each significantly enriched pathway identified, the number of associated genes found from the pathway is shown on the x-axis. B) Line plots displaying scaled log2fc values over the 14-day time course for the indicated pathways. Acronyms defined in results text, MRCC = Metabolic reprogramming in colon cancer. Here, individual genes found in the filtered miRNA-mRNA interactions for each pathway are plotted along a time course. Only genes (miRNAs or mRNAs) that have a scaled log2FC value of at least 1 at any point of the line plot are highlighted and labelled.

Modulation of miR-199 affects chondrogenesis gene expression and ECM production.

MSCs were transfected for 24 hours with miR-199a-5p, miR-199b-5p or non-targetting miRNA control mimics or inhibitors prior to the induction of chondrogenesis. A) Overexpression of miR-199a-5p. B-D) Inhibition of (B) miR-199a-5p (C), miR-199b-5p, or (D) miR-199a-5p and -199b-5p. A-D) At days: 0, 1, 3 and 7 after initiation of chondrogenesis, RNA was extracted and measurements of ACAN, COL2A1 and SOX9 gene expression were taken. qPCR results for day 0 were undetectable for COL2A1. Gene expression was normalised to 18S. Values are the mean ± SEM of data pooled from 3-4 separate MSC donors, with 4-6 biological replicates per donor. Presented as % of non-targeting control levels. The p-values calculated by paired two-tailed student’s t test, NS = not significant, * = <0.05, ** = < 0.01, *** = < 0.001.

Identification of miR-199 targets during early chondrogenesis.

Results from RNAseq analysis of control miRNA, miR-199a-5p and miR-199b-5p inhibition experiments. A) GO analysis for the significantly differentially expressed genes found from miR-199a-5p or miR-199b-5p inhibition at day 0 and day 1 of chondrogenesis. Up to five activated and five suppressed pathways are displayed for each contrast. All GO terms shown have an adjusted P value of <0.05. Count size represents the genes found in a pathway and this determined the size of the circles. B) Volcano plots to display gene expression changes following inhibition of miR199a or miR199b at day 0 (D0) or day 1(D1). The miRNAtap-selected 21 miR-199a/b-5p targets are identified (and labelled, space permitting) in red or blue if up- or down-regulated The cut-off for significance was an adjusted (BH) P value of <0.05.

Effect of miR-199a/b-5p inhibition on putative miR-199 targets.

A1-2) MSCs were transfected for 24 hours with miR-199a-5p, miR-199a/b-5p, miR-199b-5p or non-targetting miRNA control inhibitors prior to the induction of chondrogenesis. At days: 0, 1, 3 and 7 after initiation of chondrogenesis, RNA was extracted and A1) FZD6, ITGA3 and CAV1 expression was measured after miR-199a-5p and miR-199a/b-5p inhibition, or A2) CAV1 levels were also measured after miR-199b-5p inhibition. Gene expression was normalised to 18S. Presented as % of non-targetting control levels. B1-2) Luciferase expression in SW1353 cells following co-transfection of miR-199a-5p or non-targetting control mimic and miR-199a-5p target 3’UTR reporter constructs for 24 hours. B1) FZD6 and ITGA3 3’UTR-regulated expression normalised to renilla luciferase. B2) Wildtype and mutant FZD6 and ITGA3 3’UTR-regulated expression normalised to renilla and presented as percentage of non-targetting control levels. Values shown are the mean +/- SEM of data pooled from A) three separate MSC donors, with 4-6 biological replicates per donor, or B) three independent experiments. p-values were calculated using paired two-tailed student’s t test, NS = not significant, * = <0.05, ** = < 0.01, *** = < 0.001.

Initial kinetic modelling of miR-199a/b-5p regulation of chondrogenesis.

A) Schematic of how miR-199a/b-5p modulation effects the predicted miR-199a/b-5p FZD6, ITGA3 and CAV1, and the chondrogenic biomarkers SOX9, COL2A1, ACAN and GAG. Made using BioRender. B) Simulations (blue lines) from the kinetic modelling were contrasted against the experimental data – if available (red line) and a MSE score is provided in these cases. Alternatively, if no experimental data was available, a dashed blue line displays the predicted behaviour of the gene. If multiple measurements were available, they have been displayed using red crosses. C) A more detailed model displaying how miR-199a/b-5p regulates chondrogenesis via FZD6, ITGA3 and CAV1 mRNAs, in GRN form. Here information from literature was added and miR-140-5p was added to the model. The GRN shown here is a minimalistic version of Supplementary Figure 1. This was used to inform the topology of a kinetic model which aimed to explain how miR-199a-5p and miR-199b-5p act as pro-chondrogenic regulators by downregulating activity of FZD6, ITGA3 and CAV1 mRNAs. This GRN contained 18 species including: two proteins (TGFB3, SOX9), one phospho-protein (phospho-SOX9) three mRNAs (SOX9, ACAN, COL2A1), three miRNAs (miR-140-5p, miR-199a-5p, miR-199b-5p), two drugs (hpmiR-199a-5p, hpmiR-199b-5p), six Gene Activity (SRC, CAV1, FZD6, ITGA3, OtherTargets, OtherTargetsRegulator), and one phenotype (GAG). Each species has a sink and a source. Species are also shaped based on their properties: Proteins are rectangles, RNAs are rhombus, Phenotypes are hexagons, Drugs are oval, Gene Activity are rectangles with dotted lines. Species are also highlighted with a white box if they are found in the ECM or pink if they are found within a chondrocyte. Edges between species are solid if there is literature/ data supporting an interaction or dotted if there the interaction is hypothetical. Species are also colour coded: green if there is associated data, blue if there is some data and the rest has been inferred based on literature, or grey if there is no data associated with the species. D) Simulations from modelling the more detailed miR-199a/b-5p chondrogenesis model were shown here. Notations follow Figure 5B.

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