Dynamic modelling of microRNA regulation during mesenchymal stem cell differentiation
© Weber et al.; licensee BioMed Central Ltd. 2013
Received: 4 March 2013
Accepted: 30 October 2013
Published: 12 November 2013
Network inference from gene expression data is a typical approach to reconstruct gene regulatory networks. During chondrogenic differentiation of human mesenchymal stem cells (hMSCs), a complex transcriptional network is active and regulates the temporal differentiation progress. As modulators of transcriptional regulation, microRNAs (miRNAs) play a critical role in stem cell differentiation. Integrated network inference aimes at determining interrelations between miRNAs and mRNAs on the basis of expression data as well as miRNA target predictions. We applied the NetGenerator tool in order to infer an integrated gene regulatory network.
Time series experiments were performed to measure mRNA and miRNA abundances of TGF-beta1+BMP2 stimulated hMSCs. Network nodes were identified by analysing temporal expression changes, miRNA target gene predictions, time series correlation and literature knowledge. Network inference was performed using NetGenerator to reconstruct a dynamical regulatory model based on the measured data and prior knowledge. The resulting model is robust against noise and shows an optimal trade-off between fitting precision and inclusion of prior knowledge. It predicts the influence of miRNAs on the expression of chondrogenic marker genes and therefore proposes novel regulatory relations in differentiation control. By analysing the inferred network, we identified a previously unknown regulatory effect of miR-524-5p on the expression of the transcription factor SOX9 and the chondrogenic marker genes COL2A1, ACAN and COL10A1.
Genome-wide exploration of miRNA-mRNA regulatory relationships is a reasonable approach to identify miRNAs which have so far not been associated with the investigated differentiation process. The NetGenerator tool is able to identify valid gene regulatory networks on the basis of miRNA and mRNA time series data.
Modelling of gene regulatory networks (GRNs) has become a widely used computational approach in systems biology . This development has been greatly promoted by the availability of high-throughput data of adequate amount and quality, such as genome-wide expression data. A major task is the inference of regulatory dependencies between genes on the basis of such data. The inferred gene interactions constitute a network, which contains predictions about cellular regulation. They can motivate the design of new experiments, which might validate predicted dependencies and potentially elucidate unknown regulatory interactions. Successful applications of GRNs have been presented in studies of specific human diseases like cancer  and rheumatoid arthritis ), murine hepatocytes , E. coli[5, 6] and fungal infection .
In this study, we focus on the involvement of microRNAs (miRNAs) in the gene regulation of human mesenchymal stem cells (hMSCs) which differentiate towards chondrocytes. Therefore, we provide a biological background about hMSCs, characteristics and function of miRNAs and modelling approaches which integrate miRNA regulation. HMSCs are multi-potent adult stem cells, which have the capacity to differentiate into multiple cell types, such as chondrocytes, osteoblasts and adipocytes [8, 9]. Lineage commitment towards a certain type of cell depends on specific environmental factors. Those factors can activate intracellular signalling pathways which control developmental genes and other signalling pathways. Here, we focus on chondrogenic differentiation, which is characterised by a sequence of intermediate developmental stages, including cell condensation, proliferation, differentiation and hypertrophy . Each of the individual processes is associated with the activity and regulation of lineage-specific genes  encoding transcription factors (e.g. SOX9, MEF2C) or ligands of distinct signalling pathways (e.g. TGF-beta1, BMP2, IHH, WNT) . Stimulation of hMSCs by TGF-beta1 initiates the process of chondrogenic differentiation . Although key regulatory genes have been determined, the entire process of regulation in chondrogenesis is still not fully understood. In the recent years, it has become apparent that miRNAs are active regulators in the development of stem cells [14, 15].
MiRNAs are short (∼ 22 nucleotides), noncoding RNA molecules, which are able to bind to complementary sequences in target mRNAs, thereby repressing translation or inducing degradation of mRNA molecules . Silencing of gene expression by such post-transcriptional processes has been identified as a new level of gene regulation which is capable of modulating expression levels. In the human genome, more than two thousand mature miRNAs have been identified . Much effort has been put into the revelation of the complex functional network of miRNA and target gene regulation. According to sequence-based predictions, a single miRNA can target hundreds of genes, while a gene can be regulated by multiple miRNAs . Considering the biological function of the target genes, miRNAs were found to regulate various signalling pathways as well as the cell cycle. Interestingly, transcription factor genes are preferred targets of miRNAs [19, 20].
Network inference approaches have considered the emerging knowledge about miRNA-dependent regulation by taking account of interactions between miRNAs and mRNAs. Such approaches utilise miRNA target predictions as well as miRNA and mRNA expression data. Consideration of post-transcriptional gene regulation has been contributing to the extension and refinement of GRNs. This new feature has promoted the analysis of dependencies between miRNAs and target genes. For example, tools like MAGIA , MMIA  and mirConnX  perform integrated network analysis on the basis of miRNA target predictions and correlation between miRNA and mRNA expression profiles.
This study aims to present the inference of miRNA regulation as a novel application for the previously published NetGenerator V2.0 tool. The focus of this work is integrated network inference based on mRNA and miRNA time series data and prior knowledge [6, 24, 25]. The resulting network predicts the role of selected miRNAs in the chondrogenic regulatory network. In comparison to correlation-based approaches (e.g. MAGIA), the NetGenerator tool applies a dynamical model, which is based on linear ordinary differential equations (ODE). Previous network inference studies have successfully shown the ability of linear approaches to result in network models of high biological relevance [4, 26–29]. Experimental verification of predicted interactions underscored their validity, while it is often emphasised that biological processes can include more complex relationships .
Results and discussion
Chondrogenesis data and node selection
Predicted miRNA targets and associated time series correlation
miranda, mirtarget2, targetscan
As reported in the literature, there are prominent chondrogenesis marker genes such as COL2A1, ACAN (aggrecan) and COL10A1, whose expression level indicates the progress of differentiation [37, 38]. They encode for structural proteins of the extracellular matrix (ECM) and are differentially expressed in our time series data. Therefore, we added them to the selection of network nodes, because marker genes help to monitor the effects of regulation by miRNAs and transcription factors on chondrogenic differentiation.
In summary, the applied multi-step selection procedure resulted in a set of 11 network components, including 4 miRNAs (miR-524-5p, miR-494, miR-298 and miR-500), 4 transcription factor genes (SOX9, TRPS1, MEF2C and SATB2) and 3 chondrogenic marker genes coding for components of the extracellular matrix (COL2A1, ACAN and COL10A1).
The NetGenerator tool was applied to infer a system of linear ordinary differential equations, which describes a network of regulatory interactions between the components and the influence of the external stimulus (TGF-beta1+BMP2). The general model structure and the utilised optimisation approach is explained in the Methods section. Input data for the tool comprised time series data and prior knowledge about potential regulatory interactions between the components. Time series data were extracted from the available miRNA and mRNA microarray datasets, averaged across replicates at each time point, centered and scaled by their maximum absolute value (see Methods). The resulting time series matrix has 9 rows (time points) and 11 columns (nodes) (Additional file 1). Prior knowledge about regulatory interactions between the nodes was collected from diverse sources, which will be described below.
Extraction of prior knowledge
We considered knowledge about the general regulatory potential of each component as well as knowledge about regulatory interactions among the components for GRN inference. For the three component classes ((1) miRNA, (2) transcription factor gene, (3) marker/target gene), prior knowledge regarding the typical biological function was derived as follows: (1) miRNAs primarily function by degradation of their target mRNAs . Therefore, they are expected to downregulate the expression of their respective target genes. (2) Transcription factors positively or negatively regulate the expression of their target genes, which can be protein-coding genes as well as miRNA precursor genes. (3) Genes encoding structural components of the extracellular matrix are not known to have an effect on the expression of neither protein-coding genes nor miRNA genes. Therefore, they were considered to be pure target genes, whose expression is regulated by transcription factors and miRNAs. In addition to this annotation-based knowledge, a set of potential regulatory interactions was obtained from miRNA target predictions, as described in the previous section, and scientific literature. This included four predicted interactions between miRNAs and target genes, which have not been reported in previous studies (see Table 1). To extract regulatory interactions from published work, Pathway Studio V9 was applied, which provides a database of interactions automatically derived from PubMed . In total, four interactions from transcription factors on target genes were retrieved from the database. SOX9 was found to regulate the expression of COL2A1, ACAN and COL10A1 by specifically binding to regulatory elements in the promoter region of these genes . The chondrocyte hypertrophic marker COL10A1 is activated by MEF2C, which binds to conserved sequences in the promoter region of COL10A1 . Finally, the collected prior knowledge was stored in form of interaction matrices (see Methods), which can be processed by NetGenerator.
Model inference and interpretation
The biological interpretation of the network will be based on transcription factor nodes (SOX9, MEF2C, TRPS1, SATB2), by identifying regulator and target nodes for each of them. This promotes the understanding of the model, particularly of the mechanism that enables miRNAs to interfere with transcriptional regulation in order to control the differentiation process. As seen in the final model, the input stimulus (TGF-beta1+BMP2) inhibits the expression of 3 miRNAs (miR-494, miR-524-5p, miR-298) and activates miR-500, which is in turn suppressed by TRPS1. Consequently, the negative effect of downregulated miRNAs on their target genes is attenuated, which leads to the activation of the transcription factor genes SOX9, MEF2C, TRPS1 and SATB2. SOX9, the main regulatory factor in chondrogenesis , is inhibited by miR-524-5p, a finding which is supported by a predicted miRNA target site (Table 1). Since miR-524-5p expression is suppressed by the TGF-beta1+BMP2 stimulus, SOX9 expression increases and leads to activation of the differentiation markers COL2A1, ACAN and COL10A1. This transactivation is enabled by the presence of a consensus binding motif ((A/T)(A/T)CAA(A/T)G), which is shared by the SOX family members . In COL2A1, multiple copies of this motif could be identified in an enhancer located in intron 1. Activation of ACAN could be associated with the binding of SOX9 in its first intron  and the COL10A1 promoter contains a distal enhancer element 4.3 kb upstream of the transcription start site . Therefore, primary chondrogenesis might be under control of miR-524-5p through the modulation of the expression of SOX9 and its target genes. The MADS box transcription factor MEF2C, which controls chondrogenic hypertrophy, positively regulates expression of COL10A1 through binding to conserved sequences in the promoter region . Negative regulation of MEF2C by miR-298 might be a mechanism to prevent early activation of hypertrophic genes. The transcriptional repressor TRPS1 is known to be activated by a specific type of BMP-signalling and promotes chondrogenic differentiation by transcriptional repression of only a few known target genes . In our model, its expression is regulated by the stimulus as well as by miR-494 and miR-524-5p. The interaction with miR-494 is underpinned by prior knowledge (blue connection in Figure 4), but surprisingly there is also a predicted binding site for miR-524-5p within the TRPS1 mRNA. However, the positive sign of the connection suggests that the assumed inhibitory effect may be not reflected by the given data. In recent literature, extensive control of TRPS1 by at least 7 miRNAs has been described for the process of skeletal development . In the network, TRPS1 inhibits the expression of miR-500 and miR-298, which controls the chondrogenic transcription factor MEF2C. While knowledge about target genes of TRPS1 is rare, it is known that TRPS1 can upregulate the chondrogenic marker gene COL10A1 and thereby promote chondrogenic differentiation . SATB2, a transcription factor mainly associated with osteogenesis , is repressed by miR-500 and miR-494, as predicted by the model. A potential regulation of SATB2 by miR-500 is supported by the associated binding sequence (Table 1). However, since there is no influence of SATB2 on the expression of chondrogenic marker genes in the network, it is less relevant for chondrogenesis according to our model.
To summarise, the involvement of transcription factor genes is a central part of the model. The model integrates transcriptional regulation (by transcription factor genes) and post-transcriptional regulation (by miRNAs) and thereby displays the interrelationship between miRNAs and transcription factors. Since all four investigated miRNAs are ultimately downregulated, the model proposes the suppression of miRNA activity, which gives rise to the activation of the transcriptional regulators of chondrogenic differentiation such as SOX9. The model comprises miRNAs acting on different stages of the differentiation process including early proliferation and late hypertrophic stages. The downregulation of miR-524-5p provides an interesting explanation about how chondrogenic differentiation might be modulated on the level of post-transcriptional mRNA interference. Furthermore, we found expression of miR-524-5p to be differently regulated during osteogenic and adipogenic hMSC differentiation (Additional file 3). This indicates that the repression of miR-524-5p activity may be relevant for lineage specificity during hMSC differentiation. Previous studies have reported that miR-524-5p is active in glioma cells and interacts with two components of the Notch signalling pathway .
This study presented and analysed large-scale miRNA time series expression data, which captures the post-transcriptional level of chondrogenic differentiation of hMSCs. The combination of miRNA and mRNA microarray data enabled the identification of miRNAs which potentially act in this developmental process. To detect the most relevant miRNAs, a custom filtering based on diverse biological prior knowledge (literature knowledge, transcription factor annotation, miRNA target predictions) in conjunction with statistical criteria was applied. Four miRNAs (miR-524-5p, miR-494, miR-298 and miR-500) were found to be potentially involved in the regulation of chondrogenesis. To infer an integrated network of miRNA and gene regulation, we presented a novel application of the NetGenerator tool i.e. its capability to integrate miRNA and mRNA time series data into a single network inference. The good quality of the resulting network model with regard to complexity, data fit and robustness underlines the tool’s utility to infer the post-transcriptional level of gene regulation. Analysis of the network resulted in hypotheses and additional experiments which verified model predictions by showing that miR-524-5p can affect the expression of the central transcription factor gene SOX9 and differentiation marker genes. Therefore, this work demonstrated how dynamic modelling of miRNA regulation can enhance the understanding of a specific biological process and lead to the discovery of new regulatory interactions.
Culture and differentiation of human mesenchymal stem cells
Human mesenchymal stem cells (hMSCs), harvested from normal human bone marrow, were purchased from Lonza (Walkersville, MD) at passage 2. Cells were tested by the manufacturer and were found to be positive by flow cytometry for expression of CD105, CD166, CD29 and CD44 and negative for CD14, CD34 and CD45. We confirmed multipotency of all donor batches based on in vitro osteo-, chondro- and adipogenic differentiation capacity . The cells were expanded for no more than 5 passages in ‘mesenchymal stem cell growth medium’ (MSCGM, Lonza, Walkersville, MD) at 37°C in a humidified atmosphere containing 7.5% CO2. Studies were performed with hMSCs from multiple donors, including 5F0138, 5F0138 and 1F1061. For chondrogenic differentiation, hMSCs were trypsinised and 2.5x 105 cells pelleted in a 10 ml round bottom tube (Greiner Bio-One, Monroe, NC) for 10 min at 250xg. Cell pellets were subsequently cultured for 21 days in chondrogenic differentiation medium, consisting of proliferation medium supplemented with 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml sodium selenite, 5.35 µg/ml linoleic acid, 400 µg/ml proline, 1 mg/ml sodium pyruvate, 10-7 M dexamethasone, 50 µg/ml sodium L-ascorbate (all obtained from Sigma-Aldrich, St. Louis, MO), in the absence (incomplete or control) or presence of 10 ng/ml recombinant TGF-beta1 in combination with 50 ng/ml recombinant human BMP2 (TGF-beta1+BMP2). Growth factors were obtained from R&D Systems.
mRNA and microRNA profiling
Affymetrix Human Genome U133A (HG-U133A) microarrays were employed in triplicate experiments at 9 time points (0, 3, 6, 12, 24, 48, 72, 120 and 192 hours after onset of treatment with TGF-beta1+BMP2). Further experimental details can be found in . For miRNA profiling, 18 RNA samples were obtained from duplicate experiments, one biological condition and measured at 9 time points (0, 3, 6, 12, 24, 48, 72, 120 and 192 hours after onset of treatment with TGF-beta1+BMP2). RNA was extracted using TRIzol Ⓡ according to the protocol provided by the manufacturer (Invitrogen). For each sample, 5 µg of RNA was used for miRNA profiling. Hybridisation and profiling were performed using Exiqon (Vedbaek, Denmark) capture probe sets spotted on Schott Nexterion Hi-Sense E glass slides .
Determination of relative expression levels of chondrogenic marker genes using quantitative PCR (qPCR)
Total RNA was isolated from chondrogenic pellets using the Mirvana (Ambion) kit according to manufacturer’s instructions. The isolated total RNA (≈100 ng) was then used as a template in a 20 µl reverse transcriptase reaction using superscript reverse transcriptase from Invitrogen according to manufacturer’s instructions using random hexamers to prime the reaction. The following cycling conditions were used: 10 min at 20°C, 45 min at 42°C and 10 min at 94°C. The resulting cDNA solution was diluted 5x by adding 80µl water. qPCR of chondrogenic markers was performed using the following human primers: COL2A1 (forward: 5’-CTGCCAGTGGGCAACCA-3’, reverse: 5’-TTTGGGTCCTACAATATCCTTGATG-3’), COL10A1 (forward: 5’-AAAGCTGCCAAGGCACCAT3’ and reverse: 5’-AGGATACTAGCAGCAAAAAGGGTATT-3’), ACAN (forward: 5’-GACAGAGGGACACGTCATATGC-3’ and reverse: 5’-CGGGAAGTGGCGGTAACA-3’) and SOX9 (forward: 5’-GCAAGCTCTGGAGACTTCTGAAC-3’ and reverse: 5’-ACTTGTAATCCGGGTGGTCCTT-3’), expression values were normalised and corrected using RPS27a housekeeping gene (Forward: 5’-GTTAAGCTGGCTGTCCTGAAA-3’ and reverse: 5’-CATCAGAAGGGCACTCTCG-3’). Relative expression was calculated using the following formula: Relative expression: 2-C t·106 marker gene / 2-C t·106 RPS27a. Data are presented as a fraction of RPS27a expression and all qPCRs were performed in duplicates (Additional file 4).
Microarray data analysis
Microarray data pre-processing and network inference were entirely performed in the statistical programming environment R  using Bioconductor software tools . Pre-processing aims to remove non-biological noise from the data and to estimate gene expression levels.
Pre-processing of mRNA microarray data
Data from mRNA microarray experiments were pre-processed using the customised chip definition package “gahgu133a” and the robust multi-array average (RMA) procedures . The chip definition package provides custom probe-sets for the Affymetrix HG-U133A chip, which reduces the number of cross-hybridising probes . The remaining probes allow for a one-to-one correspondence between probe-set and gene. RMA procedures were applied for background correction, quantile normalisation and summarisation. The resulting signal matrix contains the logarithmised gene expression estimates for 12,175 genes.
Pre-processing of miRNA microarray data
First, mean signal values were extracted for each of the measured miRNAs. Secondly, quantile normalisation was applied, which is provided by the RMA package. This led to logarithmised miRNAs expression estimates for 1,023 miRNAs. In contrast to mRNA microarray data, there can be multiple probe-sets representing the same miRNA.
We applied the LIMMA package of the Bioconductor software suite  to the miRNA and the mRNA dataset, respectively. It provides routines using an empirical bayes approach for the identification of differentially expressed genes. Time series data can be analysed by contrast terms, which were defined by subtracting the control group from the stimulus group at each time point. Statistical significance was determined by applying a moderated F-statistics. Finally, LIMMA returned a ranked table, which contains columns for gene name, fold-change and adjusted p-values. While for mRNA selection a 2-fold-change criterion was combined with a p-value threshold (Benjamini-Hochberg adjusted p-value ≤10-10), miRNA selection was merely based on a 2-fold-change criterion, due to the low replicate number in the miRNA dataset (2 replicates per time point).
Time series standardisation
Time series standardisation is a pre-processing step required by the NetGenerator tool . It includes centering and scaling of each time series. Centering implies subtraction of the first value from all values such that the transformed time series starts from zero. Subsequent scaling divides the centered time series by its maximum absolute value, which leads to gene-wise scaled data varying within -1 and 1.
Dynamic change of expression x i of component i is described by the sum of weighted gene expressions of N genes and the weighted input u(t), which is a stepwise constant function representing the external stimulus (e.g. TGF-beta1+BMP2). The values of x i can be interpreted as standardised expression changes of component i between stimulated and non-stimulated (control) state, which serves as a reference point.
Regulatory interactions are modelled by the interaction parameters ai,j and the input parameters b i . A positive parameter value denotes an activating connection, a negative value denotes an inhibitory connection and the value zero denotes no connection. Consequently, the GRN structure is determined by the model’s interaction parameters, which have to be identified by the NetGenerator algorithm. The algorithm’s central part is a heuristic algorithm, which performs network structure and parameter optimisation. Structure optimisation applies the principle of sparseness. Iterative development of sparse sub-models explicitly restricts the number of identified connections. In each development step, parameter optimisation is applied to obtain interaction and input parameter values. The resulting model contains a minimal number of parameters that is necessary to obtain a good fit between simulated model and measured time series. A more detailed description of the algorithm can be found in [6, 24, 25].
NetGenerator also allows for integration of additional information about regulation between the components, referred to as prior knowledge. As this knowledge is independent of the time series data, it represents valuable additional input for the network inference. NetGenerator is capable of using prior knowledge during the structure optimisation process, while also dealing with contradictions between prior knowledge and time series data. Knowledge is provided in form of an interaction matrix which contains values assigned to particular connections, coded in the following way: no connection (0), activation (10), inhibition (-10), activation or inhibition (1) or not available (NA). NetGenerator provides a flexible integration mode which ignores prior knowledge in case the model fit is worsened.
Since NetGenerator contains a heuristic core, it depends on the setting of configuration parameters. The central parameter “allowedError” controls the permitted total deviation between simulated and measured data for each time series. To achieve an optimal result, we performed a series of network inference runs varying the value of this parameter (0.001, 0.01 (0.005) 0.05) resulting in ten models (see Figure 2). The resulting models were assessed on the basis of the actual model error J and the number of successfully integrated known connections. An optimal model reproduces the data with a low error (high accuracy), while attaining a relatively low model complexity (number of interactions). Considering the ten models, we found the second model (allowedError=0.01) to be optimal with respect to model error (J=0.0833), model complexity (21 interactions) and integrated prior knowledge connections (8). The network model is shown in Figure 4 and simulated time courses are shown in Figure 3.
For model validation, robustness of the inferred network against small distortions of the time series data was tested. Inaccuracy may occur in the data due to technical or biological variance. A robust inference result is expected to maintain a similar network structure when the input data is slightly perturbed. Therefore, we applied random perturbation of the time series data by sampling from a Gaussian noise distribution () and subsequent network inference. This procedure was repeated 100 times leading to a series of models, from which relative frequencies for each of the connections of the final model were derived. Connections which were inferred with a frequency of at least 50% were considered stable and therefore reliable.
Maintenance, lentiviral transfection and induced chondrogenesis of hMSCs
hMSCs were maintained in DMEM medium supplemented with 10% FBS, 1% pyruvate, 1% L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (referred to as proliferation medium, PM) and incubated at 37°C and an humidified atmosphere containing 7,5% CO2. The day before lentiviral transduction, about 5·105 cells were transferred to 25 cm2 flasks in PM and incubated for 18 hours, as before. Then, cells were transfected using lentivirus containing either the empty pMIRNA backbone vector (control) or pMIRNA vector with mir-524-5p premature DNA sequences (purchased from System Biosciences). Lentiviruses were added in various concentrations (20 ng, 40 ng and 80 ng virus/30.000 cells) in addition to 1 mg/L polybrene (Milipore). The transfected cells were incubated for 2-3 days to allow for lentiviral integration and expression of the introduced transgenes. Transfected hMSCs were grown as pellets (by centrifugation) in high-glucose DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1% L-glutamate 6,25 µg/ml insulin, 6,25 ng/ml sodium selenite, 6,25 µg/ml transferrin, 5,35 µg/ml linoleic acid, 400 µg/ml proline, 1% pyruvate, 100 nM dexamethasone, 50 µg/ml sodium ascorbate and 1,25 mg/ml bovine albumin (listed compound from Sigma). This medium will be further referred to as incomplete medium. Differentiation experiments were performed using incomplete medium in the presence or absence of 10 ng/ml TGF-beta1 and 50 ng/ml BMP2 (both purchased from R&D Systems). Differentiation of hMSCs chondrogenic pellets was allowed for 14 days.
We would like to thank all our LINCONET project partners of the ERASysBio+ initiative. Also, we kindly acknowledge the support of this work by the BMBF (German Federal Ministry of Education and Research) funding MW and PK within this initiative (Fkz. 0315719). We are grateful to Dr. Joris Pothof (ErasmusMC, Rotterdam) for his contribution to the microarray analysis of the miRNAs.
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