Circ＿0000020 Regulates The Proliferation And Osteogenic Differentiation Of HBMSCs Through The MiR-182-5p/RUNX2 Molecular Axis

Background and purposeOsteoporosis is an age-related disease of the skeletal system.It is characterized by decreased bone strength and increased risk of fractures.Its severity is second only to cardiovascular disease,and its incidence is linear with the increase in the global elderly population.Related.According to statistics,one-third of women over the age of 50 and one-fifth of men are at risk of osteoporotic fractures.With age,bone homeostasis is gradually destroyed and leads to senile osteoporosis.In individuals with osteoporosis,hip and spine fractures may occur due to minor or even no trauma,and may cause pain,dysfunction,and even death.These complications seriously affect the health and quality of life of patients,and may shorten their lifespan.Therefore,the early diagnosis,prevention and interventional treatment of osteoporosis are of great significance.Bone formation involves the proliferation of osteoblasts and the mineralization of the extracellular matrix.Bone is mainly surrounded by matrix and fibers secreted by osteoblasts to form osteoid.Bone tissue is formed after calcium salts are deposited in the osteoid,so osteoblasts are the main cell type involved in bone formation.They are derived from bone marrow mesenchymal stem cells(BMSC)and are regulated by many factors in the body.In bone marrow,BMSCs are not only hematopoietic stem cells,but also have the potential for self-renewal and multidirectional differentiation.Osteogenesis is closely related to the differentiation direction of BMSCs.Decreased differentiation of BMSCs into osteoblasts and increased adipogenic differentiation are important mechanisms for the pathogenesis of senile osteoporosis.Therefore,the key factors to determine the direction of differentiation of BMSCs may provide new ideas for the research and treatment of senile osteoporosis.However,the mechanism by which BMSCs differentiate into osteoblasts is still unclear.Many self-regulation(for example,transcription factors that control the expression of genes involved in this process)and environmental(for example,secretory and chemical factors,extracellular matrix,and cell-cell interaction)factors affect the differentiation of BMSCs.In the past few decades,circular RNAs(circRNAs)have been at the forefront of life science research.They include a class of non-coding RNAs(ncRNAs)that are different from linear RNAs because they do not have 5’-end caps or 3’-end caps.The end is poly(A)tail and forms a ring structure with covalent bond.They are mainly produced by exons and exist in almost all organisms.The closed-loop structure of circRNA makes it resistant to exonuclease(RNase)degradation and is more stable than linear RNA.Circular RNA has spatial,temporal and disease specificity,is expressed in various cells and tissues,and may be used as a biomarker and therapeutic target.One of the roles of circRNA is that it acts as a sponge for miRNA binding,which means that circRNA "absorbs" its target miRNA by pairing with the target mRNA.MicroRNA(miRNA)contains 20-22 nucleotides and belongs to the ncRNA family.They usually bind in the 3’non-coding region(3’UTR)of the target gene to regulate gene transcription.circRNA usually regulates the transcription of mRNA through sponge miRNA,thereby participating in the occurrence and development of many diseases.A new circRNA circ＿0000020 was previously found to positively regulate the osteogenic differentiation of rat BMSCs,which has attracted our attention,but how it plays a role in the regulation of osteoporosis is little known.In order to explore the function and mechanism of circ＿0000020 in the pathogenesis of osteoporosis,as well as to find the combination of miRNA and possible miRNA/mRNA pathways,we designed and conducted this research.This topic includes the following two parts:The first part:the influence of circ＿0000020 on the proliferation and osteogenic differentiation of hBMSCs;the second part:the study of the molecular mechanism of circ＿0000020 in the proliferation and osteogenic differentiation of hBMSCs.Part I The effect of circ＿0000020 on the proliferation and osteogenic differentiation of hBMSCsMethods1.Use real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)to detect the expression of circ＿0000020 in the bone tissue of patients with osteoporosis;2.The stability of circ＿0000020 is determined by qRT-PCR after treatment with RNase R and reverse transcription with Random primers or oligo(dT)18 primers;3.qRT-PCR method to analyze the expression changes of circ＿0000020 and osteopontin(OPN)and osteocalcin(OCN)related to osteogenic markers in hBMSCs osteogenic differentiation;4.Transfect circ＿0000020 shRNA or shRNA control into hBMSCs,and use qRTPCR to determine the transfection effect;5.Thiazole blue(MTT)analysis of the effect of knocking down circ＿0000020 on cell proliferation;6.Flow cytometry analysis of the effect of knocking down circ＿0000020 on cell apoptosis;7.Western blot analysis of the effect of knocking down circ＿0000020 on the expression of bone OPN and OCN proteins,which are related to osteogenic markers;8.Use alkaline phosphatase(ALP)kit to detect the effect of knocking down circ＿0000020 on ALP activity;9.Alizarin red staining method was used to determine the effect of knocking down circ＿0000020 on the formation of mineralized nodules.Results1.The expression of circ＿0000020 is significantly down-regulated in bone tissue of patients with osteoporosis;2.circ＿0000020 has a ring structure and high stability;3.The expression of circ＿0000020 gradually increased with the prolongation of the induction time of hBMSCs osteogenic differentiation;4.The expression of osteogenic differentiation markers OPN and OCN gradually increased with the prolonged induction time of hBMSCs osteogenic differentiation;5.Transfection of circ＿0000020 shRNA can effectively knock down the expression of circ＿0000020 in hBMSCs;6.Knockdown of circ＿0000020 can significantly inhibit the proliferation of hBMSCs and induce apoptosis of hBMSCs;7.Knock down circ＿0000020 to inhibit the expression of osteogenic differentiation markers OPN and OCN;8.Knock down circ＿0000020 to inhibit ALP activity and the formation of mineralized nodules.PartⅡ Study on the molecular mechanism of circ＿0000020 in hBMSCs proliferation and osteogenic differentiationMethods1.Using bioinformatics online databases starBase v3.0,circinteractome and circbank to predict miRNAs that have a targeting relationship with circ＿0000020;2.Use dual luciferase reporter gene experiment,RIP experiment and RNA pulldown experiment to verify the relationship between circ＿0000020 and miR-182-5p;3.qRT-PCR to detect the expression of miR-182-5p in the bone tissue of patients with osteoporosis and the osteogenic differentiation of hBMSCs;4.Pearson correlation analysis of the expression correlation between circ＿0000020 and miR-182-5p;5.qRT-PCR detects the effect of knocking down circ＿0000020 on the expression of miR-182-5p;6.Co-transfect circ＿0000020 shRNA and miR-182-5p inhibitors in hBMSCs;7.MTT and flow cytometry analysis simultaneously inhibit the effects of circ＿0000020 and miR-182-5p on the proliferation and apoptosis of hBMSCs;8.qRT-PCR,ALP activity detection and alizarin red staining analysis simultaneously inhibit the effects of circ＿0000020 and miR-182-5p on the osteogenic differentiation of hBMSCs;9.Use starBase v3.0 database to predict target genes that have a targeted binding relationship with miR-182-5p;10.The dual luciferase reporter gene experiment,RIP experiment and RNA pulldown experiment verify the relationship between miR-182-5p and RUNX2;11.qRT-PCR and Western blot to detect the expression of RUNX2 in bone tissue of patients with osteoporosis;12.qRT-PCR analysis of RUNX2 mRNA expression changes in hBMSCs osteogenic differentiation;13.Pearson correlation analysis of the correlation between the expression of miR182-5p and RUNX2 mRNA;14.Western blot to detect the effect of overexpression or knockdown of miR-182-5p on RUNX2 protein expression;15.Transfect RUNX2 shRNA or shRNA control into hBMSCs;16.MTT,flow cytometry,Western blot,ALP activity detection and Alizarin Red staining to analyze the effect of knocking down RUNX2 on the proliferation,apoptosis and osteogenic differentiation of hBMSCs;17.Co-transfect RUNX2 overexpression plasmid and miR-182-5p mimics in hBMSCs;18.MTT,flow cytometry,Western blot,ALP activity detection,and Alizarin Red staining to analyze the effects of simultaneous overexpression of miR-182-5p and RUNX2 on the proliferation,apoptosis and osteogenic differentiation of hBMSCs.19.Western blot analysis of the effect of knocking down circ＿0000020 or knocking down both circ＿0000020 and miR-182-5p on the expression of RUNX2.Results1.The database starBase v3.0,circinteractome and circbank predict and screen out miR-182-5p that has a targeting relationship with circ＿0000020;2.The dual luciferase reporter gene experiment,RIP and RNA pulldown experiments verified the targeted binding relationship between circ＿0000020 and miR-182-5p;3.The expression of miR-182-5p is significantly up-regulated in the bone tissue of patients with osteoporosis,while its expression is down-regulated in the osteogenic differentiation of hBMSCs;4.The expression of circ＿0000020 and miR-182-5p has an obvious negative correlation;5.Knockdown of circ＿0000020 can up-regulate the expression of miR-182-5p;6.Inhibition of miR-182-5p can reverse the inhibition of the proliferation and osteogenic differentiation of hBMSCs by knocking down circ＿0000020,and reduce the apoptosis of hBMSCs;7.StarBase v3.0 database prediction combined with literature search to screen out RUNX2 as the target gene of miR-182-5p;8.The dual luciferase reporter gene experiment,RIP and RNA pulldown experiments verified that RUNX2 is the direct target gene of miR-182-5p;9.RUNX2 is down-regulated in bone tissue of patients with osteoporosis;10.The expression of RUNX2 mRNA gradually increases with the induction of osteogenic differentiation of hBMSCs;11.The expression of miR-182-5p and RUNX2 mRNA showed a significant negative correlation;12.Overexpression of miR-182-5p can inhibit the expression of RUNX2,while inhibiting miR-182-5p can promote the expression of RUNX2;13.Knockdown of RUNX2 can hinder the proliferation and osteogenic differentiation of hBMSCs,and induce cell apoptosis;14.Overexpression of RUNX2 can effectively reverse the inhibition of overexpression of miR-182-5p on the proliferation and osteogenic differentiation of hBMSCs,and effectively reduce the apoptosis of hBMSCs.15.Knockdown of circ＿0000020 inhibits the expression of RUNX2 by targeting miR182-5p.Conclusions1.circ＿0000020 is down-regulated in the bone tissue of patients with osteoporosis,but up-regulated in the osteogenic differentiation of hBMSCs.2.Knockdown of circ＿0000020 can inhibit the proliferation and osteogenic differentiation of hBMSCs,and promote the apoptosis of hBMSCs.3.circ＿0000020 can regulate the proliferation and osteogenic differentiation of hBMSCs through the miR-182-5p/RUNX2 molecular axis.