circ-Iqsec1 induces bone marrow-derived mesenchymal stem cell (BMSC) osteogenic differentiation through the miR-187-3p/Satb2 signaling pathway

Background Bone marrow-derived mesenchymal stem cells (BMSCs) are general progenitor cells of osteoblasts and adipocytes and they are characterized as a fundamental mediator for bone formation. The current research studied the molecular mechanisms underlying circRNA-regulated BMSC osteogenic differentiation. Methods Next-generation sequencing (NGS) was employed to study abnormal circRNA and mRNA expression in BMSCs before and after osteogenic differentiation induction. Bioinformatics analysis and luciferase reporting analysis were employed to confirm correlations among miRNA, circRNA, and mRNA. RT-qPCR, ALP staining, and alizarin red staining illustrated the osteogenic differentiation ability of BMSCs. Results Data showed that circ-Iqsec1 expression increased during BMSC osteogenic differentiation. circ-Iqsec1 downregulation reduced BMSC osteogenic differentiation ability. The present investigation discovered that Satb2 played a role during BMSC osteogenic differentiation. Satb2 downregulation decreased BMSC osteogenic differentiation ability. Bioinformatics and luciferase data showed that miR-187-3p linked circ-Iqsec1 and Satb2. miR-187-3p downregulation or Satb2 overexpression restored the osteogenic differentiation capability of BMSCs post silencing circ-Iqsec1 in in vivo and in vitro experiments. Satb2 upregulation restored osteogenic differentiation capability of BMSCs post miR-187-3p overexpression. Conclusion Taken together, our study found that circ-Iqsec1 induced BMSC osteogenic differentiation through the miR-187-3p/Satb2 signaling pathway.


Background
Osteoporosis (OP) is a widespread metabolic bone trait identified by reduced bone mineral density and bone quality, resulting in increased risk regarding bone fracture. It has a hazard ratio of 1.45 and 1.70 [1,2]. It is broadly recognized that a disproportionate balance between osteoblast-related bone formation and osteoclast-mediated bone absorption in the bone marrow microenvironment leads to OP pathogenesis [3][4][5][6]. Mesenchymal stem cells (MSCs) differentiate directly into osteoblasts and then deposit mineralized extracellular matrix. MSCs have been most broadly investigated and applied in transplantation and therapy, both in basic experiments and in clinical trials [7]. Among these cells, BMSCs belong to the class of adult stem cells with the *Correspondence: wuwenliang@sdu.edu.cn abilities of self-renewal and multi-directional division potential, which makes them able to transform into adipocytes, chondrocytes, osteoblasts, and nerve cells under different induction conditions [8,9]. Nevertheless, the regulatory mechanisms are not clear.
Circular RNAs (circRNAs) are newly discovered RNAs that can form closed continuous rings covalently [10]. circRNAs are more stable than linear RNAs because they lack a free end for RNA enzyme-mediated degradation [11]. Research revealed that circRNA can regulate the microenvironment during osteogenic differentiation [6,12,13]. Former investigations suggested that circ-0016624 might sponge miR-98 to regulate BMP2 expression during postmenopausal OP [14]. circ-CDR1as regulates osteoblastic differentiation of periodontal ligament stem cells through miR-7/SMAD/GDF5 and p38 MAPK signaling pathways [15]. circ-AFF4 modulates osteogenic differentiation of BMSCs through SMAD1/5 pathway activation via the miR-135a-5p/Irisin/FNDC5 axis [16]. However, circRNA roles in regulating osteoblastic differentiation is unclear. Therefore, more research is needed to investigate the basic molecular mechanisms of the circRNA regulatory network with respect to bone regeneration.
The current study determined that circ-Iqsec1 promoted the induction of osteogenic differentiation and that knockdown of circ-Iqsec1 significantly suppressed osteogenesis (OS) in BMSCs. miR-187-3p expression decreased as differentiation proceeded. In addition, circ-Iqsec1 induced BMSC osteogenic differentiation via the miR-187-3p/Satb2 signaling pathway. These data have proposed novel functions of circ-Iqsec1 during BMSC osteogenic differentiation and highlighted its potential application as a novel therapy target in bone formationrelated traits.

BMSC preparation, culture, and identification
Tibiae and femurs from BALB/c mice were extracted under sterile conditions to expose the bone marrow cavity, which was rinsed with saline. We collected and centrifuged the bone marrow filtrate at 225 × g for 5 min. The supernatant was discarded, and we resuspended the cells in HyClone low glucose (LG)-DMEM at 1×10 6 cells per 100 μL. We gradually added the cell suspension to mouse lymphocyte separation medium (Sigma-Aldrich) in a 1:1 (v:v) ratio and centrifuged it at 1000 × g for 20 min. A milky turbid mononuclear cell layer was obtained. The cells were resuspended in LG-DMEM without FBS at 1×10 6 cells per 100 μL before centrifuging at 225 × g for 5 min. The cells that were pelleted were resuspended in LG-DMEM complete medium containing 10% FBS and incubated in 5% CO 2 -saturated humidity at 37°C. The culture medium was changed every 3 days. The cells were sub-cultured in a 1:3 ratio when the cell confluence achieved 80~90%. BMSCs were passaged 3-4 times and utilized for the next steps. Fluorescein isothiocyanate (FITC-F) or phycoerythrin (PE) was applied for phenotypic analyses. CD44, CD54, CD31, CD29, CD90, integrin-β1, and vWF marker expressions were detected. IgG-matched isotype served as the internal control for all antibodies.

Multilineage bone marrow stem cell (BMSC) differentiation
To characterize BMSC abilities for multilineage differentiation, we cultivated 3rd-passage mouse BMSCs under various differentiation conditions. For adipocyte differentiation, we cultivated BMSCs in adipogenic differentiation medium. After 2 weeks, we determined adipocyte differentiation using Oil Red O staining. For osteoblast differentiation, we cultivated BMSCs in osteogenic differentiation medium and then stained them with alizarin red post 3 weeks to monitor overall survival (OS).

Strand-specific NGS RNA-seq library preparation
Total RNA from BMSCs induced for 0 and 21 days was obtained utilizing TRIzol reagent (Invitrogen, CA, USA). Our group utilized VAHTS with 3 μg RNA from each sample. RNA-seq (H/M/R) library prep kits from Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) were used to remove ribosomal RNA. RNA types, such as mRNAs and ncRNAs, were retained. RNA was treated applying 40 U RNase R (Epicenter) at 37°C for 3 h, followed by TRIzol purification. An RNA-seq library was prepared through KAPA stranded RNA-seq library prep kits (Roche, Basel, Switzerland), which we employed for NGS (Illumina HiSeq 4000 at Aksomics, Inc., Shanghai, China).

RNA isolation and real-time PCR
Total RNA was obtained using TRIzol reagent (Invitrogen), followed by cDNA synthesis applying TransScript All-in-One First-Strand cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China). Polymerase chain reaction (PCR) was conducted utilizing a Bio-Rad PCR instrument (Bio-Rad, CA, USA) and 2× Taq PCR master mix (Solarbio, Beijing, China) following all protocols. We calculated fold changes using the 2 −ΔΔCt approach. PCR primers were as follows: circ-Iqsec1:

Animals and cell transplantation
We infected BMSCs at the 4th passage utilizing lentivirus (si-circ-Iqsec1, miR-187-3p mimic, or Satb2 overexpression vector) and cultured in osteogenic differentiation medium for 7 days prior to in vivo research. After the BMSCs were trypsinized and directly resuspended in DMEM, we cultured the BMSCs with SynthoGraft (β-tricalcium phosphate; Bicon) for 1 h at 37°C. They were then centrifuged at 150 × g for 5 min and implanted into 2 symmetrical sites in the dorsal subcutaneous space in six 6-week-old BALB/c nude mice. The ethical review committee of Qilu Hospital of Shandong University approved the animal experiments.

Immunohistochemical analyses
We harvested specimens 8 weeks post-transplantation from mice euthanized by CO 2 asphyxiation. We decalcified specimens in 10% EDTA (pH 7.4), which we dehydrated and embedded in paraffin. We cultured sections overnight at 4°C with primary antibodies against OCN and RUNX2 and then for 1 h at 37°C with secondary antibodies (Abcam). We stained the sections using 3,3-diaminobenzidine. We counterstained with hematoxylin and examined under an Axiophot light microscope (Zeiss, Oberkochen, Germany).

Statistical analyses
The continuous variables are represented as the mean ± SD. For comparisons, one-way variance of analysis was conducted through GraphPad Prism (GraphPad, CA, USA). P ≤ 0.05 informed statistical significance.

BMSC characterization and differentiation
We made BMSCs from bone marrow obtained from BALB/c mice tibiae and femurs. The cultured BMSCs Results are denoted by the mean ± SD. ** P < 0.01, *** P < 0.001 vs. NC. E RT-qPCR data giving circ-Iqsec1 expression during osteogenic differentiation of BMSCs. Output is denoted as the mean ± SD. ** P < 0.01, *** P < 0.001 vs. 0 day. F Genomic loci of Iqsec1 and mmu_circ_0001493 had a typical morphology with a spindle structure (Fig. 1A). They were positive for known MSC markers CD44, CD90, CD54, CD29, and integrin-β1, yet did not express endothelial cell markers CD31 and vWF (Fig. 1B-I). The study also found that the isolated BMSCs had osteoblast and adipocyte differentiation ability, as demonstrated by Oil Red O (Fig. 1J) and alizarin red (Fig. 1K) staining.

circ-Iqsec1 plays a role during BMSC osteogenic differentiation
To identify the osteogenic differentiation ability, we induced BMSCs with an osteogenic differentiation induction medium for 0, 14, and 24 days. ALP staining ( Fig. 2A) and alizarin red staining (Fig. 2B) showed that the osteogenic differentiation ability was timedependent. High-throughput sequencing showed that osteogenic differentiation resulted in the abnormal expression of circRNA (Fig. 2C). RT-qPCR detection showed nine high-expression circRNAs based on the sequencing data. The data showed that only mmu_ circ_0001493 expression increased significantly in the osteogenic differentiation-induced group (Fig. 2D). Furthermore, the RT-qPCR results illustrated that increased mmu_circ_0001493 expression in BMSCs depended on the induction time (Fig. 2E). This suggested that mmu_circ_0001493 functions in the osteogenic differentiation of BMSCs. mmu_circ_0001493 originated by cyclizing two exons from Iqsec1, located at chr6:90639573-90644844. Iqsec1 was 5271 bp, and the spliced mature circRNA was 1539 bp (Fig. 2F). Therefore, mmu_circ_0001493 was also called circ-Iqsec1 by our laboratory.

Satb2 plays a role during osteogenic differentiation of BMSCs
High-throughput sequencing showed that osteogenic differentiation resulted in the abnormal expression of mRNA (Fig. 4A). RT-qPCR detection and sequencing data showed seven high-expression mRNAs: Satb2, Snx32, Egfl7, Bmp3, Susd5, Clec4a3, and Fmo4. However, only Satb2 expression increased significantly in the osteogenic differentiation-induced group (Fig. 4B). RT-qPCR data further showed that Satb2 expression increased in BMSCs in a time-dependent way following induction (Fig. 4C). This suggests that Satb2 functioned in BMSC osteogenic differentiation.
To further identify whether Satb2 participates in BMSC osteogenic differentiation, siRNA against Satb2 (si-Satb2) was constructed and transfected into BMSCs. The result showed that Satb2 expression decreased significantly after silencing of Satb2 (Fig. 4D). RT-qPCR detection showed that downregulation of Satb2 inhibited RUNX2 (Fig. 4E) and OCN (Fig. 4F) expression. Immunohistochemical staining for ALP and alizarin red staining for calcium showed that Satb2 downregulation reduced BMSC osteogenic differentiation ability (Fig. 4G). This suggested that Satb2 functioned during BMSC osteogenic differentiation.

Discussion
OP is a well-defined trait that leads to increased mortality and morbidity [18]. Because of the pluralistic differentiation potential, BMSCs function to regulate the microenvironment and bone mass and strength [19,20]. The present investigation discovered that BMSCs have osteogenic differentiation potential. In addition, circRNA was reported to function importantly in osteogenic differentiation [21]. High-throughput sequencing elucidated that circRNA had abnormal expression in BMSCs during osteogenic differentiation and that circ-Iqsec1 expression increased in BMSCs during osteogenic differentiation. circ-Iqsec1 downregulation inhibited RUNX2 and OCN expression and BMSC osteogenic differentiation. The data suggested that circ-Iqsec1 functioned in BMSC osteogenic differentiation.
High-throughput sequencing for mRNA expression detection showed abnormal mRNA expression in  OCN (B). Data are expressed as the mean ± SD. * P < 0.05, *** P < 0.001 vs NC ### P < 0.001 vs mimic. E, F Immunohistochemical staining for ALP and alizarin red staining show the osteogenic differentiation ability of BMSCs BMSCs regarding osteogenic differentiation. Special ATrich sequence-binding protein 2 (Satb2) expression was increased in BMSCs during osteogenic differentiation. Enhanced SATB2 has been reported to promote osteogenic differentiation of BMSCs from patients with osteonecrosis induced by ethanol [22,23]. Previous studies have found that Satb2 was a particular immunohistochemical biomarker of osteoblastic differentiation and has been helpful regarding bone and soft tissue tumors [24,25]. Satb2 downregulation inhibited RUNX2 and OCN expression and BMSC osteogenic differentiation. This is consistent with former investigations showing that Satb2 functions in BMSC osteogenic differentiation.
Previous studies have shown that circRNAs might regulate gene expression by influencing transcription, mRNA turnover, and translation via sponging RNA-binding proteins and microRNAs [18,26,27]. This study aimed to identify the connecting target miRNA for Satb2 and circ-Iqsec1. Bioinformatics analysis and luciferase reporter analysis confirmed that miR-187-3p functioned to link circ-Iqsec1 and Satb2. Former investigations showed that miR-187-3p expression decreased during osteogenic differentiation of human adipose-derived mesenchymal stem cells [28]. miR-187-3p expression upregulation inhibited the osteogenic differentiation of osteoblast precursor cells by inhibiting cannabinoid receptor type 2 [29]. The present investigation verified that miR-187-3p expression decreased in BMSCs during osteogenic differentiation.
The data discovered that miR-187-3p downregulation or Satb2 overexpression restored the osteogenic differentiation capability of BMSCs post-silencing circ-Iqsec1 in in vivo and in vitro investigations. Satb2 upregulation restored BMSC osteogenic differentiation capability post-miR-187-3p overexpression.

Conclusion
The present study revealed that circ-Iqsec1 functioned during osteogenic differentiation of BMSCs. circ-Iqsec1 induced BMSC osteogenic differentiation by regulating the miR-187-3p/Satb2/RUNX2/OCN signaling pathway. In addition, the effect of circ-Iqsec1 on osteogenic differentiation may be explored in the near future.