Characterization of Differentially Expressed miRNAs by CXCL12/SDF-1 in Human Bone Marrow Stromal Cells

: Stromal cell-derived factor 1 (SDF-1) is known to influence bone marrow stromal cell (BMSC) migration, osteogenic differentiation, and fracture healing. We hypothesize that SDF-1 mediates some of its effects on BMSCs through epigenetic regulation, specifically via microRNAs (miRNAs). MiRNAs are small non-coding RNAs that target specific mRNA and prevent their translation. We performed global miRNA analysis and determined several miRNAs were differentially expressed in response to SDF-1 treatment. Gene Expression Omnibus (GEO) dataset analysis showed that these miRNAs play an important role in osteogenic differentiation and fracture healing. KEGG and GO analysis indicated that SDF-1 dependent miRNAs changes affect multiple cellular pathways, including fatty acid biosynthesis, thyroid hormone signaling, and mucin-type O-glycan biosynthesis pathways. Furthermore, bioinformatics analysis showed several miRNAs target genes related to stem cell migration and differentiation. This study’s findings indicated that SDF-1 induces some of its effects on BMSCs function through miRNA regulation.


Introduction
Recent developments in regenerative medicine seek to utilize adult stem cells as therapeutics to treat agerelated musculoskeletal disease. Gene reprogramming via manipulating the cellular microenvironment is a promising and unique opportunity to direct adult stem cell fate. Bone marrow stromal cells (BMSCs) are mesenchymal lineage progenitors that give rise to multiple cell types, including osteoblasts, chondrocytes, adipocytes, and other connective tissue cells [1,2]. BMSCs are thought to play an important role in the repair and maintenance of various musculoskeletal tissues. The migration and differentiation of adult stem cells, including BMSCs, is a highly regulated by interaction with the cellular milieu. Molecular factors within the milieu, such as growth factors and chemokines, play a vital role in the extracellular regulation of BMSCs [3].
Given the ability of SDF-1 to influence various signaling pathways within BMSCs, we postulate that it likely mediates its control via epigenetic regulation [16]. Epigenetic regulation is a mechanism by which gene expression changes occur without changes in genetic makeup [17]. Epigenetic factors, including DNA methylation and miRNAs, are known to be implicit in the differentiation of BMSCs and musculoskeletal development [18][19][20][21][22]. MiRNAs are small non-coding RNAs that bind mRNA at the 3'-untranslated regions (3'-UTR). Upon binding, the miRNA prevents translation or promotes degradation of the mRNA, thus negatively regulating gene expression at the post-transcriptional level [23,24]. Several reports suggest that miRNAs regulate almost all cellular events including cell proliferation, differentiation, and development [25][26][27][28]. We hypothesize that SDF-1 induces some of its effects via the regulation of miRNAs. In this study we investigate the SDF-1 dependent regulation of miRNAs within human BMSCs and their correlation with respect to survival, proliferation, migration, and differentiation.

Isolation of human BMSCs and SDF-1 treatment:
Human bone marrow (BM) from young (18-40 years of age) subjects were obtained under sterile conditions from orthopedic surgery patients as per the Institutional Review Board (IRB) of Augusta University. CD271+ MSCs were extracted from the bone marrow using an isolation kit (Miltenyi Biotec Inc., 130-092-283, Sunnyvale, CA, USA) and washed with a standard culture medium composed of DMEM medium (Corning, 10-013-CM, Corning, NY, USA), 1% antibiotics-antimycotics (AA; Invitrogen, 15240-062, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS). Cells were transferred to 100 mm culture dish and incubated at 37°C and 5% carbon dioxide (CO2) in a humidified environment. After 24 h, the media with nonadherent cells was removed. The adherent cells were washed in phosphate buffer saline (PBS) and further expanded by incubation in the fresh standard culture medium. Culture-expanded CD271+ BMSCs of passage 1 were used for treating with the SDF-1. Human BMSCs were cultured on 24 well plates treated with or without SDF-1 (50 ng/mL concentration) for 72 hrs. Media was changed every day with or without SDF-1. At the end of 72 hrs, miRNAs were isolated using a miRNA isolation kit (SABiosciences Corporation, Frederick, MD, USA).
Informed consent: Informed consent has been obtained from all individuals included in this study.
Ethical approval: The research related to human use has been complied with all the relevant national. regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the Institutional Review Board (IRB) of Augusta University.
MicroRNA Array and Bioinformatics Analysis; Total miRNA isolation was performed using a miRNA isolation kit (SABiosciences Corporation, Frederick, MD, USA) that captures small RNAs with lengths less than 200 nucleotides. RNA concentrations were determined using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The quality of RNA samples was characterized by an Agilent BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) using an RNA6000 Nano Chip (Agilent). miRNA microarrays were performed using an Affymetrix GeneChip® miRNA 4.0 array at the Integrated Genomics Core, Augusta University, GA, USA. The miRNA profile was analyzed for the hierarchical clustering of miRNA to generate heat maps and principal component analysis (PCA). T-tests were conducted to calculate the p-value and determine whether miRNA levels were significantly changed in SDF-1 treatment versus control groups. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation were performed on differentially expressed miRNAs using DIANA-miRPath v.3. Bioinformatics software (http://www.targetscan. org/vert_72/ and http://www.mirdb.org/) were used to predict gene targets of differentially expressed miRNAs considered to be novel.
GEO Database Analysis of Differentially Expressed MiRNA; To correlate our microarray results, we performed an analysis of existing microarray data in the Gene Expression Omnibus (GEO) public repository. GEO allows researchers to archive and distribute high throughput gene sequencing and microarray data sets [29]. Users can analyze genomic expression profiles of interest from previously performed pre-clinical studies. We searched the GEO database for microarray datasets of miRNA differentially expressed under three unique criteria. 1) GEO was queried for datasets of fracture healing models. This search yielded the study by Hadjiargyrou et al. (GEO accession GSE76197), which profiled miRNA in murine femur fracture across 14 days post-fracture versus intact control. 2) GEO was queried for datasets from osteogenic differentiation of mesenchymal stem cells or bone marrow stromal cells. Only datasets with significant results were included for analysis. The search yielded the following datasets: GSE159508, GSE134946 , GSE72429, GSE115197. 3) GEO was queried for datasets derived from BMP2 osteogenic growth factor treatment of cell lines derived from mesenchymal origin. The search yielded a dataset by Bae et al. (GEO accession GSE37036) showcasing miRNA expression in C2C12 myoblasts treated with BMP2 for 72 hours.
GEO2R interactive tool was used to determine differential expression between treatment and control groups by calculating log base 2 of fold change (log2(FC)). Significance in fold change was determined from the adjusted p-value parameter calculated by the GEO2R interactive tool. The Benjamini & Hochberg false discovery rate method for p-value adjustment was selected as it is the most commonly used adjustment for microarray data. It provides a balance between the discovery of statistically significant genes and the limitation of false positives.

Results
MiRNA Differentially Expressed Following SDF-1 Treatment: miRNA microarray analysis was conducted to compare miRNA profiles of human BMSCs with and without SDF-1 treatment. We found 104 miRNAs to be differentially regulated (p < 0.05) with an absolute fold change of 1.5 or greater following SDF-1 treatment. Out of these 104 miRNAs, 49 miRNAs were downregulated, and 55 miRNAs were upregulated. Table 1 presents the top 50 most differentially expressed miRNAs. A heat map with hierarchical clustering visually depicts the unique profiles of miRNA from SDF-1 treated and control groups (Figure 1).
Principal Component Analysis; Principle component analysis (PCA) was performed to distinguish the miRNA profile in SDF-1 treated versus control BMSCs. The PCA depicts unique clusters for treated samples versus control samples highlighting the clear distinction between these two groups (Figure 2).

Novel miRNAs Target Key Genes in Migration and Differentiation;
In silico analysis was performed on novel miRNAs with lesser-known functions scarcely reported in the existing literature. To elucidate their roles, miRNAs underwent bioinformatic analysis utilizing targetscan.org and mirdb.org. Criteria for target genes include complementary binding between miRNA seed sequence and the mRNA transcript for the gene of interest. Several target genes were identified that are known to modulate stem cell migration and differentiation into musculoskeletal tissues. Table 2 presents these novel miRNAs and their respective gene targets.

KEGG Pathway Annotation and GO Enrichment Analysis of Differentially Expressed MiRNAs;
The collective effect of differentially expressed miRNA was characterized with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and Gene Ontology (GO) enrichment analysis. KEGG annotation identified several cellular pathways that are affected by these miRNAs ( Table 3). Notable pathways include fatty acid biosynthesis, thyroid hormone signaling, and mucin-type O-glycan biosynthesis pathways. GO analysis determined 102 biological processes associated with the differentially expressed miRNAs ( Table 4). Common processes affected by both upregulated and downregulated miRNAs include gene expression, nucleic acid binding transcription factor activity, cellular protein modification process, enzyme binding, and small molecule metabolic process.    Table 4: GO analysis of biological processes (top 50) potentially affected by downregulated miRNAs (a) and upregulated miRNAs (b) due to SDF-1 treatment in human BMSCs.

SDF-1 Versus Fracture Healing and Osteogenic Induced MiRNA Expression Profile; GEO dataset archived by Hadjiargyrou et al. (GEO accession GSE76197
) was analyzed to determine the differential expression of miRNA in murine femur fracture model across 14 days, and results were compared to that of SDF-1. The analysis determined 12 common miRNAs were significantly upregulated in our study and fracture healing [Hadjiargyrou et al., 2016)] at one or more time points across the 14-day study. Conversely, two common miRNAs were significantly downregulated across both studies. These specific miRNAs are presented in Figure 3. Subsequent GEO search yielded several more datasets depicting differently regulated miRNAs during osteogenic differentiation of human mesenchymal stem/stromal cells (GSE159508, GSE134946 , GSE72429, GSE115197). The analysis determined that several common miRNAs were differentially expressed across these studies and with SDF-1 treatment. These results are presented in Table 5.

SDF-1 Versus BMP2 Induced MiRNA Expression Profile; GEO dataset archived by Bae et al. (GEO accession
GSE37036) was analyzed to determine the miRNA expression profile with BMP2 treatment, and results were compared to that of SDF-1. The analysis revealed several common miRNAs were differentially expressed in both studies. Specifically, let-7f-5p, miR-140-5p, miR-196a-5p, and miR-24-2-5p were upregulated in both SDF-1 and BMP2 treatment, as presented in Figure 4. miR-330-3p was downregulated with the treatment of either growth factor. This suggests that both BMP2 and SDF-1 induce osteogenic differentiation by regulating the same downstream osteogenic miRNAs. Furthermore, SDF-1 is known to have potential BMP2 induced bone formation, a phenomenon that could be explained in part by shared downstream osteogenic miRNA.

Discussion
Adult bone marrow stromal cells are the primary source of stem cells in the field of regenerative medicine. As mentioned previously, the extracellular milieu plays a paramount role in controlling the migration and differentiation of BMSCs. Manipulating this environment offers a unique opportunity to control cell fate and achieve enhanced therapeutic results. Based on previous findings from our group and others, extracellular SDF-1 is beneficial for osteogenesis and fracture healing [8][9][10][13][14][15]. We hypothesized that extracellular SDF-1 mediates its effects on BMSCs via miRNA-dependent gene regulation.
While various studies have shown the pervasive role of miRNAs regulating cellular events, no study has previously identified SDF-1 regulation of miRNAs in human BMSCs. Our identification of these miRNAs provides an important link in understanding the mechanism by which SDF-1 exerts its osteogenic effect on BMSCs. Herein we identified a list of miRNAs that were differentially expressed in BMSCs following SDF-1 treatment. Several of these miRNAs are also regulated in osteogenic differentiation and fracture healing. Comparing our findings with the GEO dataset by Hadjiargyrou et al. (in which they analyzed miRNA expression in murine femur fracture) 14 common miRNAs were expressed similarly across both studies (Figure 3). Furthermore, we identified several common miRNAs regulated in the presence of SDF-1 and during osteogenic differentiation of mesenchymal stem/stromal cells ( Table 5). We speculate that SDF-1 partially enhances fracture healing and osteogenic differentiation through the regulation of these miRNAs.
Several groups have demonstrated SDF-1 potentiates BMP2 induced bone formation [10,30,31]. BMP2 is known to enhance the proliferation and osteogenic differentiation of BMSCs [32][33][34]. Comparing our findings with the GEO dataset by Bae et al. (in which miRNA expression was analyzed in cells treated with BMP2) several common miRNAs were differentially expressed across both studies (Figure 4). These findings suggest SDF-1 and BMP2 could act synergistically via shared downstream osteogenic miRNA. Additionally, our results show SDF-1 downregulates miR-654-5p, a miRNA known to directly target BMP2 within BMSCs (verified by reporter assay) [35]. MiR-654-5p is also observed to be persistently decreased in patients during osteoblast differentiation [36]. Taken together, this is consistent with our prior finding that SDF-1 increases BMP2 mRNA levels in vitro [10]. BMP2 is approved by the FDA for lumbar fusion surgery and is used off-label in many other applications, despite concerns of pro-oncogenic effects [37]. Our findings support the use of lower BMP2 dosage in conjunction with synergistic molecules to achieve clinical outcomes with decreased oncogenic risk. Existing literature indicates that several differentially expressed miRNAs by SDF1 play an important role in osteogenic differentiation of BMSCs. MiR-339 has been found to inhibit the osteogenic differentiation of BMSC via the direct targeting of DLX5, a known factor in osteogenic differentiation [38]. We speculate that SDF-1 likely enhances BMSC osteogenesis via the downregulation of miR-339-3p. MiR-503-5p has been found to promote bone formation by targeting the negative osteogenic regulator SMURF1 within BMSCs [39]. Our results suggest SDF-1 decreases SMURF1 by upregulating miR-503-3p. Let-7f-5p has been found to promote cell survival in various cell lines by targeting caspase-3 and caspase-9 while increasing levels of anti-apoptotic factors Bcl-2 and Bcl-xL [40,41]. Taken with our results, this indicates an increase in cell survival of BMSCs mediated by SDF-1, consistent with previous reports [9]. We also identified several novel miRNAs with no prior known functions. Bioinformatic analysis revealed a considerable number of these miRNAs are involved in stem cell homing and commitment to musculoskeletal lineage ( Table 2).
To better understand the collective effect of differentially expressed miRNA, KEGG pathway annotation and GO enrichment analysis was conducted. KEGG annotation identified several cellular pathways potentially affected, with fatty acid biosynthesis, thyroid hormone signaling, and mucin-type O-glycan biosynthesis pathways most relevant. Fatty acids and their metabolites have been well linked to stem cell proliferation and differentiation [42]. Both saturated and unsaturated fatty acids have differential effects on BMSC survival [43]. Furthermore, polyunsaturated fatty acids have been found to modulate proliferation, migration, and differentiation of various tissue-specific sources of MSCs [44][45][46]. GO analysis identified several cellular processes (TLR signaling, mitotic cell cycle, post-translation protein modification, extracellular matrix disassembly, apoptotic signaling pathway) that are vital in BMSC survival, proliferation, migration, and differentiation.
SDF-1 signaling has been recognized for its chemotactic and osteogenic function in BMSCs. Our findings further solidify its expanded role in BMSC biology. Table 5: List of common miRNAs differently expressed after SDF-1 treatment and after various osteogenic differentiation protocols (retrieved via GEO database analysis). GSE159508 presents human periodontal ligament stem cells (hPDLSCs) cultured in osteogenic medium for 14 days compared to hPDLSCs cultured in normal medium (10% FBS in DMEM) for 14 days. GSE134946 and GSE115197 presents human BMSCs cultured in osteogenic medium for 7 days compared to non-induced BMSCs from day 0. GSE72429 presents human synovial membrane MSCs and adipose derived stem cells (ADSCs) osteogenically differentiated compared to undifferentiated controls. Differential expression is reported as miRNA fold change after treatment compared to respective control. All reported fold changes are statistically significant based upon GEO2R adjusted p-value.  We have elucidated several miRNAs that are upregulated and downregulated in response to SDF-1. Among these miRNAs, we correlated some of their functions with roles identified in existing literature, and we characterized novel miRNAs concerning their potential targets in BMSCs ( Table 2). We also determined key signaling pathways that are collectively influenced by the differentially expressed miRNAs. Limitations to our study do exist. Our study only utilized BMSCs in tissue culture and thus needs to be validated in vivo. Moreover, our study only utilized one concentration and one time period for SDF-1 treatment. It is valuable to understand the dose and time-dependent effects of SDF-1 in vitro and in vivo models. Overall, our study indicated that SDF-1 induces some of its osteogenic and chemotactic effects by regulating miRNAs in human BMSCs.