Role And Mechanism For MiR-590-3p In Osteogenic Differentiation Of Human Mesenchymal Stem Cells

This study aimed to find potential bio-marker for osteogenic differentiation.With bio-informatics study and qRT-PCR assay verification,we identified miR-590-3p as target.The miR-590-3p over-expressed cell-line were constructed with lenti-virus transfection,with empty vector control and transient transfected miR-590-3p inhibition cell-line.MiR-590-3p expression was related with cell proliferation and cell cycle regulation,osteogenic differentiation and wnt/p-catenin signaling pathway activities.Dual luciferase assay was employed to study the mechanism of miR-590-3p's effect.The mechanism of MicroRNA-590-3p could influence the level of osteogenic differentiation was related to Wnt/?-catenin signaling pathway through binding to 3'UTR of APC mRNA,which could provide some useful references for alveolar bone regeneration and bone tissue engineeringChapter 1 Bio-informatics study on microRNA expression profile in osteogenic differentiation of human mesenchymal stem cells.Objective:Finding novel target through bio-informatics study in GEO gene expression data and validate with qRT-PCR.Method:non-coding RNA expression data was retrieved from GEO database,PCA analysis was used to identify potential targets,which was then screened with independent samples t test.Few microRNAs were chosen after initial screening,qRT-PCR verification of their expression during osteogenic induction were carried out to find the potential target.Then GO analysis and pathway analysis were used to find its potential target.Result:GSE7429 was chosen for further study.Several microRNAs including miR-130a,miR-142-3p and miR-590-3p.qRT-PCR assay indicated that these miRNAs elevated as human mesenchymal stem cells undergone osteogenic differentiation,among them miR-590-3p increased significantly.GO analysis and pathway analysis indicated APC,FZD1,Wnt5a in wnt signaling pathway was involved in miR-590-3p's action.Conclusion:Using bioinformatics study and experiment validation,we found miR-590-3p as potential biomarker in osteogenic differentiation,and hypothesized that wnt signaling pathway was involved.Chapter 2 construction of miR-590-3p over-expression/inhibition cell line and biological effect evaluationObjective:To construct miR-590-3p over-expressed/inhibition human mesenchymal stem cell,evaluate the effect of miR-590-3p on cell proliferation and cell cycle regulation.Method:.The miR-590-3p over-expressed cell-line were constructed with lenti-virus transfection,with empty vector control and transient transfected miR-590-3p inhibition cell-line were compared by qRT-PCR to detect the level of expression for miR-590-3p in three groups of cells.Then MTT assay was carried out to study cell proliferation activities,CyclinDl expression and phosphorylated pRb were evaluated in all three groups of cells.Result:Cell proliferation,CyclinD1 expression and phosphorylated pRb level in miR-590-3p hMSC were all superior comparing with vector control and miR-590-3p-inh hMSC.Conclusion:MiR-590-3p expression was related with cell proliferation and cell cycle regulation,which was essential for mesenchymal stem cell differentiation.Chapter 3:Mechanism for miR-590-3p regulating osteogenic differentiation of human mesenchymal stem cells.Objective:Wnt signaling pathway is important part for osteogenic differentiation regulating mechanisms.MiR-590-3p was proposed as potentially related with Wnt signaling pathway.This chapter is intended for evaluating role and mechanisms for miR-590-3p in osteogenic differentiation.Method:qRT-PCR assays for various biomarker of osteogenic differentiation were carried out in miR-590-3p hmsc,vector control hmsc,miR-590-3p-inh hmsc.Alizarin red staining and quantification were employed to study mineralized deposition.?-catenin/TCF/LEF level was evaluated with TOP/FOP assay,intranuclear ?-catenin was further tested with western blot study.Dual luciferase was used to demonstrate specific binding relationship between miR-590-3p and APC 3'UTR.Result:MiR-590-3p level was shown to be positively related with level of biomarker for osteogenic differentiation.TOP/FOP and intra-nuclear level of?-catenin was also influenced by miR-590-3p.Dual luciferase plasmid with wild type APC 3' UTR potential binding sequence shown varied luciferase activities due to different miR-590-3p levels,but such effect could not be observed in cells transfected with mutated APC 3'UTR plasmid.Conclusion:MicroRNA-590-3p could influence the level of osteogenic differentiation,such action was related to Wnt/?-catenin signaling pathway through binding to 3'UTR of APC mRNA.