Effect of miR-192-5p on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Idiopathic Scoliosis by Regulating Wnt/β-catenin Signaling Pathway via RSPO1


 BackgroundIdiopathic scoliosis (IS) is the most common structural scoliosis, which seriously affects not only patient’s physical and mental health but also quality of patient’s life. Abnormal osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is one of the causes of IS. However, the regulation mechanism of osteogenic differentiation of BMSCs in patients with IS remains to be further studied.MethodsSerum samples of 135 patients with IS were collected, and the expression of miRNA were detected by RT-qPCR. BMSCs from patients with IS were collected and the expression of miR-192-5p in BMSCs from IS patients and normal BMSCs was detected by RT-qPCR. Double luciferase reporter genes assay was used to verify the targeting relationship between miR-192-5p and RSPO1. The levels of RSPO1, osteogenic related proteins (OC, OPN and RUNX2) and Wnt/β-catenin signaling pathway related proteins (WNT3A and β-catenin) were detected by Western blotting. Alkaline phosphatase staining and alizarin red staining were used to evaluate the osteogenesis of BMSCs.ResultsmiR-192-5p was significantly up-regulated in serum and BMSCs of patients with IS. Alkaline phosphatase staining and alizarin red staining showed that miR-192-5p inhibitor promoted the osteogenic differentiation of BMSCs from IS patients. miR-192-5p targeted down-regulated the expression of RSPO1 in BMSCs from IS patients. In addition, overexpression of RSPO1 activated Wnt/β-catenin signaling pathway in BMSCs from IS patients. Furthermore, miR-192-5p/RSPO1 axis regulated levels of osteogenic related proteins (OC, OPN and RUNX2) in BMSCs from IS patients through Wnt/β-catenin signaling pathway, and affected the osteogenic differentiation of BMSCs.Conclusion﻿miR-192-5p, which was highly expressed in patients with IS, inhibited Wnt/β-catenin signaling pathway by down-regulating RSPO1 protein and then reduced the osteogenic differentiation ability of BMSCs.

Idiopathic scoliosis (IS) is the most common structural scoliosis, which seriously affects not only patient's physical and mental health but also quality of patient's life. Abnormal osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is one of the causes of IS. However, the regulation mechanism of osteogenic differentiation of BMSCs in patients with IS remains to be further studied.

Methods
Serum samples of 135 patients with IS were collected, and the expression of miRNA were detected by RT Background Scoliosis refers to the deviation in the normal vertical spine, which is the most common deformity of the spine. [1] As one pattern of scoliosis, idiopathic scoliosis (IS) seriously affects not only patient's physical and mental health but also quality of patient's life. [2] According to the research on IS, the available causes included genetic factors, hormonal factors, bone and connective tissue anomalies. [3] Focus on the role of bone loss plays in cause of IS, there has been reported that the mechanism and causes of bone loss in adolescent IS might be decrease osteogenic differentiation of mesenchymal stem cells. [4] Emerging evidence showed that microRNAs (miRNAs) are deregulated in scoliosis. [5] Importantly, there was also a study found that the differential expression of miRNAs in Bone marrow mesenchymal stem cells (BMSCs) from adolescent IS patients might provide a deep insight into the cause of adolescent IS.
[6] Nevertheless, the mechanism that miRNAs regulate the osteogenic differentiation of BMSCs involve in the pathogenesis of IS is still not clear.
R-spondin (RSPO) family belong to a superfamily of thrombospondin type 1 repeat-containing proteins, and contain four secreted proteins, RSPO1-4. RSPO secreted cysteine-rich glycoproteins and play an important role in maintenance of stem cells. [7] Moreover, RSPO together with WNT proteins enhance the activity of canonical Wnt/β-catenin signaling pathway. [8] Interestingly, activation of Wnt/β-catenin signaling pathway in BMSCs promoted osteogenic differentiation and reversed bone loss. [9] However, there was no study on the relationship between RSPO and IS.

Western blotting
Total proteins were extracted by using RIPA lysis buffer (#20101ES60, Yeasen, Shanghai, China) and the concentration of protein was detected by using BCA protein quanti cation kit (#20201ES76, Yeasen). Proteins were separated and transferred using 10% SDS-PAGE and then transferred to PVDF membranes.
The PVDF membranes were blocked in 5% skimmed milk at room temperature for 2h. And then incubated with primary antibodies at 4 °C overnight. Primary antibodies were CD44 (1: Double luciferase reporter genes assay The sequence of the RSPO1 wild type (RSPO1-WT) or RSPO1 mutant type (RSPO1-MUT) and miR-192-5p mimics were co-transfected into 293T cells (#HZ-H321, ATCC, USA) by using Lipofectamine2000. After transfection for 48 h, the luciferase activity was measured by dual luciferase reporter assay system.

Statistical analysis
All the data in this study were expressed as means ± standard deviation and calculated by SPSS 19.0. The difference between two groups was assessed by Student's t test and the difference in multiple groups was analyzed by one-way ANOVA. The differences of p<0.05 was considered statistically signi cant.

Results
Expression of miR-192-5p in IS patients' serum and BMSCs It has been reported that miRNAs participate in the process of IS. According to the heat map results (Fig.   1a), this study focused on the role of miR-192-5p played in IS. RT-qPCR results showed that the expression of miR-192-5p was signi cant higher in the serum samples of IS patients than normal people (Fig. 1b). In order to explored the function of miR-192-5p in BMSCs of IS, the BMSCs were obtained from IS patients, and western blotting showed that the BMSCs marker CD44 and CD90 were positive, CD34 and CD45 were negative (Fig. 1c). In addition, compared with the normal BMSCs, there was increased miR-192-5p expression in BMSCs from IS patients (Fig. 1d). Hence, the abnormal high expression of miR-192-5p in serum and BMSCs from IS patients might contribute to occurrence and deterioration of IS.

Function of miR-192-5p in BMSCs from IS patient
Based on the above results, miR-192-5p expressed abnormality greatly in BMSCs from IS patient, which might play an important role in osteogenic differentiation of BMSCs. In order to explored the function of miR-192-5p in BMSCs from IS patient, miR-192-5p inhibitor was transfected into the cells at rst (Fig. 2a).
Western blotting results showed that the expression of osteogenic related proteins (OC, OPN and RUNX2) increased in miR-192-5p inhibitor group compared with the NC group (Fig. 2b). Alkaline phosphatase staining (Fig. 2c) and alizarin red staining (Fig. 2d) also revealed that knockdown of miR-192-5p enhanced the osteogenic differentiation of BMSCs in IS. Therefore, miR-192-5p effected on the osteogenic differentiation of BMSCs in IS patient.
Relationship of miR-192-5p, RSPO1 and Wnt/β-catenin signaling pathway miRNAs play some roles in cells through interact with target gene. The binding sites between miR-192-5p and RSPO1 were showed as Fig. 3a. Moreover, double luciferase reporter gene results showed that compared with the NC group, the luciferase activity of RSPO1-WT signi cantly reduced in miR-192-5p mimics group, and the luciferase activity of RSPO1-MUT showed no signi cantly differences between NC and miR-192-5p mimics group (Fig. 3b). In addition, transfected miR-192-5p mimics in the BMSCs from IS patient, after testify the transfection e ciency (Fig. 3c), Western blotting was used to detect the expression of RSPO1 protein, the results showed that overexpression of miR-192-5p downregulated RSPO1 (Fig. 3d). Besides, RSPO1 is a regulator of Wnt/β-catenin signaling pathway. The RSPO1 was overexpressed or knock downed by pcDNA-RSPO1 or si-RSPO1 in the BMSCs from IS patient (Fig. 3e), Western blotting results showed that the expression of WNT3A and β-catenin increased with the increase of RSPO1, and the expression of WNT3A and β-catenin decreased with the decrease of RSPO1 (Fig. 3f). Thus, miR-192-5p inhibited Wnt/β-catenin signaling pathway through down-regulated the expression of RSPO1.

miR-192-5p effected on osteogenic differentiation of BMSCs in IS by regulating Wnt/β-catenin signaling pathway via RSPO1
Furthermore, the mechanism that miR-192-5p effected on osteogenic differentiation of BMSCs in IS by regulating Wnt/β-catenin signaling pathway via RSPO1 was studied. FH535 is inhibitor of Wnt/β-catenin signaling pathway. Western blotting results showed that compared with the NC group, the expression of Wnt/β-catenin signaling pathway related proteins (WNT3A and β-catenin) and osteogenic related proteins (OC, OPN and RUNX2) markedly raised in the OE-RSPO1 group, but there was no signi cant difference between the OE-RSPO1+miR-192-5p mimics group and the NC group; besides, the expression of WNT3A, β-catenin, OC, OPN and RUNX2 remarkable reduced in the OE-RSPO1+FH535 group compared with the OE-RSPO1 group (Fig. 4a). Alkaline phosphatase staining (Fig. 4b) and alizarin red staining (Fig. 4c) results showed that miR-192-5p mimics or FH535 reversed the promotion of osteogenic differentiation induced by OE-RSPO1 in BMSCs. Therefore, miR-192-5p restrained osteogenic differentiation of BMSCs in IS by down-regulated RSPO1 and inhibited Wnt/β-catenin signaling pathway.

Discussion
IS identify when scoliosis patients have no other underlying disease. For those IS patients who have not yet reached skeletal maturity, the treatment is often prophylactic. For adult IS patients, the treatment is most frequently because of symptoms, such as back pain, deformities, respiratory and cardiology problems.
[10] [11] Due to a currently recognizable cause of IS has not been found, it is urgent to explore the underlying mechanism of IS pathogeny.
Bone loss is one of the most important causes of IS. [12] With the research on miRNAs, there was a study reported that miRNAs can be potentially used as a clinical tool for molecular IS diagnosis, and this study also evidence for the deregulated miRNAs that participate in osteoblast or osteoclast differentiation mechanisms in IS. [13] For example, Bcl2-targeted miR-15a was downregulated in IS patients, which related to chondrocyte proliferation and participate in the development and progression of IS. [14] Besides, recent report con rmed that aberrant miR-145-5p impaired osteocyte function in adolescent IS. [15] Osteogenic differentiation of BMSCs is closely related to bone formation. [16] In this study, we found that miR-192-5p highly expressed in not only the serum but also BMSCs of IS patients. Moreover, knockdown of miR-192-5p BMSCs from IS patients inhibited osteogenic differentiation. The results elucidated that miR-192-5p might involve in IS through regulating osteogenic differentiation of BMSCs.
miRNAs contribute to differentiation of stem cells by regulating target genes and important cellular pathways. Based on the prediction of starbase, RSPO1 as a target gene of miR-192-5p participated in apoptosis of BMSCs. [17] Additionally, RSPO1 may interact with Wnt/β-catenin signaling pathway to induce differentiation in osteoblasts. [18] Our study results illuminated that overexpression of miR-192-5p or inhibition of Wnt/β-catenin signaling pathway in BMSCs reversed the promotion of osteogenic differentiation induced by overexpression of RSPO1.
About the osteogenic differentiation related proteins OC, OPN and RUNX2 in IS, a new study claimed that the level of OC in the severe adolescent IS patients was signi cantly lower. [19] Besides, OPN was at a low level in adolescent IS bone tissues. [15] And RNA and protein analyses revealed lower RUNX2 levels in adolescent IS.
[20] The results in this study showed that inhibition of miR-192-5p or overexpression of RSPO1 increased the level of OC, OPN and RUNX2 in BMSCs from IS patients, this results emphasized that miR-192-5p and RSPO1 might be exploited as a potential target for IS therapy.

Conclusion
In conclusion, miR-192-5p, which was highly expressed in patients with IS, inhibited Wnt/β-catenin signaling pathway by down-regulating RSPO1 protein and then reduced the osteogenic differentiation ability of BMSCs.   Relationship of miR-192-5p, RSPO1 and Wnt/β-catenin signaling pathway. a The binding sites between miR-192-5p and RSPO1. b Target relationship between miR-192-5p and RSPO1 was veri ed by Double luciferase reporter genes assay. c Transfection e ciency of miR-192-5p mimics in BMSCs from IS patients was examined by RT-qPCR. d Expression of RSPO1 in BMSCs from IS patients was measured by Western blotting. e Transfection e ciency of pcDNA-RSPO1 or si-RSPO1 in BMSCs from IS patients was examined by RT-qPCR. f Expression of RSPO1 and Wnt/β-catenin signaling pathway related proteins (WNT3A and β-catenin) in BMSCs from IS patients was measured by Western blotting.**P<0.01 vs NC group Figure 4 miR-192-5p effected on osteogenic differentiation of BMSCs in IS by regulating Wnt/β-catenin signaling pathway via RSPO1. a Expression of osteogenic related proteins (OC, OPN and RUNX2) and Wnt/βcatenin signaling pathway related proteins (WNT3A and β-catenin) in BMSCs from IS patients was measured by Western blotting. b Alkaline phosphatase (ALP) staining results of BMSCs from IS patients. c Alizarin red S (ARS) staining results of BMSCs from IS patients. **P<0.01 vs NC group, ##P<0.01 vs OE-RSPO1 group