Bone Marrow Mesenchymal Stem Cells Involved In The Pathogenesis Of Adolescent Idiopathic Scoliosis

BackgroundAdolescent idiopathic scoliosis(AIS)is an unexplained,three-dimensional deformity of the spine,which primarily occurs in girls aged 10 to 16 years.The general incidence of AIS is between 2%and 4%,with high disability rate.If AIS is not treated in time,it will not only affect the body shape,appearance and psychology of the patients,but may also develop into severe spinal deformity,which may lead to cardiopulmonary impairment,limb paralysis and even death.At present,the treatments of AIS include surgical treatments and non-surgical treatments.Most of them involve biomechanical intervention with great risks,rather than the causal-based treatments.Therefore,elucidating the pathogenesis and mechanism of AIS has important clinical significance for effective prediction and prevention of AIS.At the same time,it also provides a basis for developing more targeted treatments.Increasing reports demonstrate that AIS patients have persistent lower bone mineral density(BMD).Bone marrow mesenchymal stem cells(BMSCs)have multi-directional differentiation potential and can differentiate into osteoblasts and chondrocytes under specific induction conditions.BMSCs are essential in two types of bone formation modes:intramembranous and endochondral ossification.In the previous studies,our team has found that genes,proteins and miRNAs are abnormally expressed and there are dysregulated proliferation and developmental signaling pathways in BMSCs of AIS patients.Therefore,we hypothesized that the abnormal function of BMSCs is the cause of BMD in AIS patients.AimsIn order to determine the influence of BMSCs abnormalities affect the osteogenic differentiation ability in AIS patients,we isolated BMSCs from bone marrow specimens of AIS patients and age-matched healthy donors,and compared their osteogenic differentiation abilities in vitro in the first part.In the second part,combining the differentially expressed genes(DEG)profile with dysregulated MAPK signaling pathway in AIS patients,we focused on SPRY4 gene,the one displaying the most significant change and associated with the MAPK signaling pathway.Then,the effects of SPRY4 on osteogenic differentiation of BMSCs in vitro and on heterotopic bone formation in vivo were systematically studied through gain and loss function.In addition,we also explored the role of SPRY4 in melatonin response and its specific molecular mechanism.In the third part,according to the previous microarray results,we studied miR-181b-5p,the most up-regulated miRNA in BMSCs of AIS patients.After enlarging the sample for validation,we have fully studied the role of miR-181b-5p in osteogenic differentiation of BMSCs in vitro and on heterotopic bone formation in vivo,and explored its target genes and molecular pathways.MethodsIn the first part,BMSCs from AIS patients and healthy donors were separated by density gradient centrifugation.Alkaline phosphatase(ALP)staining,ALP activity detection,Alizarin red staining and quantitative analysis were used to compare the osteogenic differentiation abilities of BMSCs from AIS patients and healthy donors.In the second part,SPRY4 was selected based on the analysis of microarray results and its expression changes in AIS patients were verified by RT-qPCR.To study the function of SPRY4 in osteogenic differentiation of BMSCs in vitro,siRNA knockdown and lentivirus overexpression were used to change SPRY4 expression.Besides,the model of heterotopic bone formation in vivo was established in mice,and the effect of SPRY4 on heterotopic bone formation was studied by HE staining and Masson staining.The changes of signaling pathway were studied by using its inhibitors and Western blot.After adding melatonin and other treatments in the medium,BMSCs were induced to osteoblasts.Then,the expression of key transcription factors and marker genes in osteogenic differentiation was detected by RT-qPCR and Western blot.Furthermore,the change of osteogenic differentiation abilities of BMSCs under different treatment conditions were studied by ALP staining and other methods mentioned above.In the third part,the expression change of miR-181b-5p in AIS patients were verified by RT-qPCR.miR-181b-5p mimics or inhibitors were transfected to study its function in osteogenic differentiation of BMSCs in vitro.Similarly,the effect of miR-181b-5p on heterotopic bone formation in vivo was studied by heterotopic bone formation model,HE staining and Masson staining.Finally,the structural and functional match between miR-181b-5p and its target gene SPRY4 was predicted by bio informatics software,and verified by double luciferase target assay,Western blot and siRNA knockdown.ResultsIn the first part,compared with healthy donors,the positive rate of ALP staining,ALP activity,and alizarin red staining of BMSCs in AIS patients were decreased,suggesting that the osteogenic differentiation ability of BMSCs in AIS patients was reduced.In the second part,we found that SPRY4 was significantly downregulated in BMSCs of AIS patients.Knockdown of SPRY4 impaired osteogenic differentiation of healthy BMSCs in vitro,while overexpression of SPRY4 enhanced osteogenic differentiation of BMSCs in AIS patients.Besides,SPRY4 promoted heterotopic bone formation of BMSCs in vivo.Mechanism studies have found that SPRY4 regulates osteogenic differentiation by activating the MEK-ERK1/2 pathway.Furthermore,melatonin treatment boosted osteogenic differentiation of BMSCs,whereas the promotional effects of melatonin on osteogenic differentiation were ablated by knocking down of SPRY4.Mechanism studies have found that SPRY4 was upregulated by melatonin exposure,and SPRY4 mediated melatonin response in a MEK-ERK1/2 dependent manner.Thus,loss of SPRY4 in BMSCs leads to reduced osteogenic differentiation,and these defects are further exacerbated under the influence of melatonin.In conclusion,SPRY4 plays a key role in regulating osteogenic differentiation and melatonin response.In the third part,we found that miR-181b-5p was remarkably upregulated in BMSCs of AIS patients.Overexpression of miR-181b-5p suppressed osteogenic differentiation of healthy BMSCs in vitro,while inhibition of endogenous miR181b-5p promoted osteogenic differentiation of BMSCs in AIS patients.In addition,miR-181b-5p impaired heterotopic bone formation of BMSCs in vivo.As predicted by the bioinformatics website,double luciferase target assay verified that miR-181b-5p directly bind to the 3’UTR of SPRY4.Overexpression of miR-181b-5p significantly downregulated the expression of SPRY4 protein,but had no significant effect on SPRY4 mRNA.Knockdown of the target gene SPRY4 blocked the role of miR-1 81 b-5p inhibitors in promoting osteogenic differentiation.Mechanism studies found that miR-181b-5p might be involved in osteogenic differentiation of BMSCs by inhibiting the phosphorylation of ERK1/2.ConclusionCompared with healthy donors,osteogenic differentiation of BMSCs was reduced in AIS patients in vitro.The lower expression of SPRY4 in AIS patients impaired the osteogenic differentiation of BMSCs by inhibiting the MEK-ERK1/2 signaling pathway and reducing the melatonin response.The expression of miR-181b-5p in BMSCs of patients with AIS was upregulated.miR-181b-5p inhibited the osteogenic differentiation of BMSCs by downregulating the expression of SPRY4 and inhibiting the phosphorylation of ERK1/2.The above studies indicate that BMSCs abnormalities in AIS patients can reduce BMD by regulating osteogenic differentiation,and may be involved in AIS pathogenesis.