Identification of differentially expressed circRNAs, miRNAs and mRNAs
To investigate the early molecular events underlying osteogenic differentiation, we obtained three microarray datasets which were listed in Table S3. These datasets profiled the expression patterns at 7 days post chemical induction of osteogenic differentiation in hBMSCs. We included mRNA, miRNA, and circRNA expression profiles of BMSCs undergoing osteogenesis and normal cells in our study.
A total of 80 differentially expressed circRNAs (DE-circRNAs) were identified in the osteogenic induction group compared to the uninduced group in BMSCs at 7 days, with a significance threshold of P < 0.05 and |log2 fold change|> 0.5 (Fig. 1a). Among these, 41 circRNAs were upregulated, while 39 were downregulated. Additionally, 98 differentially expressed miRNAs (DE-miRNAs) were detected during osteogenic differentiation (P < 0.05, |log2 fold change|> 0.5) (Fig. 1b), with 41 upregulated and 57 downregulated DE-miRNAs. Furthermore, a total of 814 differentially expressed mRNAs (DE-mRNAs) were identified using a significance threshold of P < 0.05 and |log2 fold change|> 1.2, comprising 478 upregulated and 336 downregulated DE-mRNAs (Fig. 1c).
The volcano plot shows the differential expression of circRNAs, miRNAs, and mRNAs between the normal culture group and the osteogenic induction group hBMSCs. (a) DE-circRNAs (P < 0.05, |log2 fold change|> 0.5); (b) DE-miRNAs (P < 0.05, |log2 fold change|> 0.5); (c) DE-mRNAs(P < 0.05, |log2 fold change|> 1.2); Red dots represent upregulated gene expression, blue dots represent downregulated gene expression.
Subsequently, volcano plots were generated using the pheatmap package, and 80 DE-circRNAs, 98 DE-miRNAs, and 814 DE-mRNAs were selected for further analysis.
Function enrichment analysis
To explore the role of circRNAs as miRNA sponges in modulating the osteogenic differentiation of BMSCs, we utilized an online database to predict circRNAs corresponding to the identified miRNAs. Our analysis revealed that 61 DE-circRNAs interacted with 3184 miRNAs from circbank. Furthermore, by intersecting the circRNAs-targeted miRNAs with the DE-miRNAs from GEO, we identified 56 overlapping miRNAs (Fig. 2a). Subsequently, we predicted that 7,648 mRNAs contained binding sites for the overlapped miRNAs. Through further analysis, we detected 215 overlapping mRNAs by intersecting the miRNAs-targeted mRNAs with the DE-mRNAs (Fig. 2b).
Construction of the circRNA–miRNA–mRNA Interaction Network. (a) Venn diagram of the intersection of molecules predicted to be combined with circRNAs and DE-miRNAs by circBank database; (b) Venn diagram of the intersection of molecules predicted to be combined with miRNAs and DE-mRNAs by mirTarBase database. (c) GO function annotation bar graph(GO enrichment analysis includes Cellular Component (CC), Molecular Function (MF), and Biological Process (BP); P < 0.05.). (d) ceRNA interaction network diagram of circRNA-miRNA-mRNA (Green ellipses represent circRNA, blue rectangles represent miRNA, and orange rhombuses represent mRNA).
To elucidate the functional roles of these 215 mRNAs, we conducted GO enrichment analysis using the DAVID online database. Our analysis revealed that these mRNAs were significantly enriched in biological processes related to transcriptional regulation, proliferation, and differentiation (P < 0.05) (Fig. 2c). Notably, 35 of these mRNAs were specifically enriched in biological processes associated with osteogenesis and cell proliferation, including GO: 0,008,284 ~ positive regulation of cell proliferation, GO: 0,001,503 ~ ossification, GO: 0,008,285 ~ negative regulation of proliferation, and GO: 0,002,053 ~ positive regulation of mesenchymal cell proliferation. These processes are closely associated with osteogenesis, calcification, and cell differentiation. Consequently, we selected the 35 mRNAs enriched in these processes for further investigation.
Construction of the circRNA–miRNA–mRNA interaction network related to osteogenic differentiation
Based on the analysis of differential expression in microarray data and the ceRNA mechanism, we identified a set of interconnected molecules to construct a circRNA-miRNA-mRNA network relevant to osteogenic differentiation. Utilizing published literature, we selected 15 mRNAs closely associated with stem cell differentiation (TableS1). Among the 56 overlapping miRNAs, 17 were found to interact with the previously mentioned 15 mRNAs. Furthermore, out of the 61 differentially expressed circRNAs, 22 were found to interact with the 17 miRNAs. Consequently, we posit that these 22 circRNAs, 17 miRNAs, and 15 mRNAs mutually interact and are closely linked to hBMSCs osteogenic differentiation. Employing Cytoscape software, we constructed a ceRNA network diagram containing:
circRNAs: hsa_circ_0001063, hsa_circ_0001600, hsa_circ_0002415, hsa_circ_0002474, hsa_circ_0003552, hsa_circ_0003563, hsa_circ_0003611, hsa_circ_0004418, hsa_circ_0005991, hsa_circ_0006006, hsa_circ_0007933, hsa_circ_0008621, hsa_circ_0016956, hsa_circ_0022502, hsa_circ_0034293, hsa_circ_0057104, hsa_circ_0057105, hsa_circ_0063756, hsa_circ_0068465, hsa_circ_0072387, hsa_circ_0072678, hsa_circ_0088062
miRNAs: hsa-miR-199b-5p, hsa-miR-15a-5p, hsa-miR-424-5p, hsa-miR-504-5p, hsa-miR-383-5p, hsa-miR-335-5p, hsa-miR-20b-5p, hsa-miR-200b-3p, hsa-miR-30c-2-3p, hsa-miR-30a-5p, hsa-miR-203a-3p, hsa-let-7i-5p, hsa-miR-942-5p, hsa-miR-9-3p, hsa-miR-335-3p, hsa-miR-3200-3p, hsa-miR-542-3p
mRNAs: LIF, ZBTB16, VEGFA, SOST, SOD2, SMAD1, SFRP4, LRRC17, IL6, IGF2, FOXP1, FGF9, CLEC3B, BMP7, AREG (Fig. 2d).
Validation of key circRNAs and miRNAs in the ceRNA network
In the control group, hBMSCs were cultured in complete medium without osteogenic induction. In contrast, the experimental group hBMSCs were treated with osteogenic induction medium for 7 days, corresponding to the matrix maturation stage. This time point is critical for capturing early differentiation events17. RT-qPCR was performed to assess the expression levels of 11 molecules corresponding to differentially expressed circRNAs identified through bioinformatics analysis. Compared to the control group, the expression levels of hsa_circ_0001600, hsa_circ_0002415, hsa_circ_0063756, hsa_circ_0072678, and hsa_circ_0005991 were significantly upregulated in the 7-day osteogenic induction group, while the expression of hsa_circ_0008621 was downregulated (P < 0.05) (Fig. 3a). Similarly, compared to the control group, the expression levels of hsa-miR-20b-5p, hsa-miR-335-3p, hsa-miR-942-5p, hsa-miR-424-5p, and hsa-miR-542-3p were significantly decreased in the osteogenic induction group, whereas the expression of hsa-miR-203a-3p, hsa-miR-30a-5p, hsa-miR-30c-2-3p, and hsa-miR-199b-5p was increased (P < 0.05) (Fig. 3b).
Certain circRNA molecules involved in the regulation of BMSC osteogenic differentiation. (a) qPCR detection of the expression level changes of circRNAs screened by bioinformatics analysis after osteogenic differentiation induction. (b) qPCR detects the expression level changes of miRNAs screened by bioinformatics analysis after osteogenic differentiation induction. (c) qPCR detects the expression level changes of hsa_circ_0001600 and hsa-miR-542-3p during osteogenic differentiation at 0 days, 3 days, and 7 days. (d) qPCR detects the expression level changes of hsa_circ_0005991 and hsa-miR-424-5p during osteogenic differentiation at 0 days, 3 days, and 7 days. (e) Transfection of hsa_circ_0001600 expression in hBMSCs at a siRNA concentration of 50 nM for 48 h. (f) qPCR detects the changes in hsa-miR-542-3p levels in the si-hsa_circ_0001600 2 days after transfection. Compared with the si-NC, the expression of hsa-miR-542-3p increased in the si-hsa_circ_0001600. (si-hsa_circ_0001600 is the hsa_circ_0001600 siRNA transfected knockdown group, si-NC is the negative control group; * represents P < 0.05; ** represents p < 0.01; *** represents p < 0.001, **** represents p < 0.0001).
The interaction between circRNAs and miRNAs within the ceRNA network
During the osteogenic differentiation of hBMSCs, the expression of hsa_circ_0005991 and hsa_circ_0001600 shows an increasing trend, indicating that the two molecules may be involved in the regulation of cell osteogenic differentiation. hsa_circ_0001600 and hsa_circ_0005991 were selected as candidate circRNAs. According to the circBank database prediction, hsa_circ_0001600 (circBank ID: hsa_circFKBP5_002) has one binding site (position: 739) with hsa-miR-542-3p in the miRanda algorithm, and four binding sites (positions: 3624, 752, 3629, 758) in the Targetscan algorithm. hsa_circ_0005991 (circBank ID: hsa_circAPBB2_013) has one binding site (position: 584) with hsa-miR-424-5p in the miRanda algorithm prediction, and two binding sites (positions: 598, 604) according to the Targetscan algorithm. hBMSCs were osteogenically induced for 0, 3, and 7 days, and qPCR was employed to assess the expression levels of hsa_circ_0001600, hsa-miR-542-3p, hsa_circ_0005991, and hsa-miR-424-5p. The findings revealed a progressive increase in the expression of hsa_circ_0001600 throughout osteogenic induction (Fig. 3c), while the expression of hsa-miR-542-3p decreased gradually with prolonged osteogenic induction. Additionally, the expression of hsa_circ_0005991 exhibited a gradual increase over the osteogenic induction period (Fig. 3d), whereas the expression of hsa-miR-424-5p decreased progressively with the extension of osteogenic induction time.
The experimental design included the use of siRNA to suppress the expression of hsa_circ_0001600. Initial investigation was conducted to determine the optimal siRNA, transfection concentration, and duration. The determined concentration of the target gene siRNA was 50 nM, and the transfection duration was 48 h. qPCR analysis indicated a significant reduction in hsa_circ_0001600 expression with si-hsa_circ_0001600 2 in hsa_circ_0001600 siRNA, achieving a knockdown efficiency of 70% (Fig. 3e). The hsa_circ_0001600 siRNA transfection knockdown group exhibited an increase in hsa-miR-542-3p expression compared to the negative control group (Fig. 3f).
Downregulation of hsa_circ_0001600 expression reduces the osteogenic differentiation capacity of hBMSCs
The circular RNA hsa_circ_0001600 is generated through the reverse splicing of the FKBP5 gene, representing a newly discovered circular molecule with unknown biological function and molecular mechanism (Fig. 4a). Compared to linear RNAs, circRNAs are more stable and longer-lasting because they lack a free end for RNA enzyme-mediated degradation. Circular RNAs are generated by the back-splicing process, and they are covalently closed loops, keeping them highly stable to RNase R digestion. Using qPCR, the expression changes of hsa_circ_0001600 and its parental gene FKBP5 in RNase R-digested total RNA (RNase R +) were analyzed. The non-digested group (RNase R-) served as the control group, with the internal reference in the RNase R- group used as the calculation standard. The qPCR results demonstrated no significant alteration in the expression level of hsa_circ_0001600 in the experimental group compared to the control group. However, there was a notable reduction in the expression of FKBP5, indicating that hsa_circ_0001600 may be resistant to RNase R, whereas its parental gene FKBP5 is predominantly digested by RNase R (Fig. 4b).
(a) hsa_circ_0001600 generation model diagram; (b) qPCR detects the expression of hsa_circ_0001600 and FKBP5 before and after RNase R treatment. RNase R + represents the group treated with RNase R enzyme, RNase R- represents the group without RNase R enzyme treatment; (c) The left side shows alizarin red staining after 14 days of osteogenic induction, and the right side shows ALP staining after 3 days of osteogenic induction. (i) and (iii) are the control group, and (ii) and (iv) are the hsa_circ_0001600 siRNA transduction knockdown group; the results showed that compared with the control group, the degree of alizarin red and ALP staining was significantly weakened in the hsa_circ_0001600 siRNA transduction knockout group. (d) qPCR detection of changes in osteogenesis-related gene expression levels after 3 days of osteogenic induction. In comparison to the si-NC, the expression of COL1A1 and RUNX2 was downregulated in the si-hsa_circ_0001600. (e) Western Blot detection of protein changes after 3 days of osteogenic induction; the band diagram shows that the bands of COL1A1, OCN, and RUNX2 in the si-hsa_circ_0001600 are noticeably lighter compared to the si-NC. (f) The protein quantification diagram shows reduced protein expression levels of COL1A1, OCN, and RUNX2 in the si-hsa_circ_0001600 compared to the si-NC. (si-hsa_circ_0001600 is the hsa_circ_0001600 siRNA transfected knockdown group, si-NC is the negative control group; * represents P < 0.05; ** represents p < 0.01; *** represents p < 0.001, **** represents p < 0.0001).
hBMSCs were seeded into 6-well plates and induced with osteogenic induction medium for 14 days. Throughout this period, siRNA (si-hsa_circ_0001600 and si-NC) was refreshed every 3 days. Alizarin Red staining was performed for both the control and experimental groups. Microscopic observation revealed a significant reduction in red staining in the hsa_circ_0001600 siRNA knockdown group compared to the control group. Furthermore, after transfecting hBMSCs with hsa_circ_0001600 siRNA for 2 days, the medium was switched to osteogenic induction medium for 3 days. The control group comprised hBMSCs transfected with negative control siRNA for 2 days, followed by a 3-day osteogenic induction. ALP staining was conducted for both the control and experimental groups. Microscopic observation revealed a significant reduction in red staining in the hsa_circ_0001600 siRNA knockdown group compared to the control group, indicating a clear decrease in ALP expression (Fig. 4c). qPCR analysis was carried out for both the control and experimental groups, demonstrating reduced expression of osteogenic-related genes COL1A1 and RUNX2 in the knockdown group with hsa_circ_0001600 siRNA transfection, as compared to the negative control group (Fig. 4d). Furthermore, Western Blot analysis showed decreased expression of osteogenic-related proteins COL1A1, OCN, and RUNX2 in the knockdown group with hsa_circ_0001600 siRNA transfection, as compared to the negative control (Fig. 4e, 4f). The raw bands are shown in Figure S1.
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- Source: https://www.nature.com/articles/s41598-024-76136-z