The Effect Of MiR-153-3p Regulates Osteogenic Differentiation Of BMSCs In High Glucose Microenvironment

Objective:To explore the effects of high glucose environment on the proliferation,apoptosis and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).And also to explore the effect of mi R-153-3p on osteogenic differentiation of BMSCs in high glucose microenvironment and its potential mechanism.Methods:Primary BMSCs of SD rats were isolated and cultured in vitro,and identified by morphological observation,osteogenic adipogenesis induction and flow cytometry.Under the condition of osteogenic induction medium,different concentrations(5 m M;25m M)of glucose were used to induce BMSCs,and CCK-8 was used to detect cell proliferation.Westernblot(WB)and q RT-PCR were used to detect the changes of expression in osteogenesis-related genes Runx2,BSP,collagen I and ALP.The early osteogenesis of BMSCs was detected by ALP activity,and the osteogenic differentiation was detected by ALP staining,alizarin red staining and semi-quantitative analysis.Besides,q RT-PCR was used to detect the expression level of mi R-153-3p on the 0,3,7 and 14 days during the process of osteogenesis induction,and to infer the correlation between the expression of mi R-153-3p and osteogenesis in high glucose environment.Groups(HG,HG+mimic control,HG+mi R-153-3pmimic)and groups(NG,HG,HG+inhibitorcontrol,HG+mi R-153-3pinhibitor)respectively realized the over-expression and silencing of the gene mi R-153-3p.The software(mi RBase,Target Scan)was used to predict the potential target Runx2 of mi R-153-3p,and it was found that there was a potential binding site between them in 3’-UTR region.The wild-type and mutant luciferase reporter plasmid vectors carrying 3’-UTR of Runx2 and mi R-153-3pmimic or its negative control were transferred into 293 T cells,respectively,and the OD value was measured by fluorescent enzyme-labeled instrument 48 h later.At the same time,the m RNA and protein expression level of Runx2 were further detected in BMSCs.Finally,the reversion experiment further verified whether mi R-153-3p inhibited the osteogenic differentiation of BMSCs by inhibiting the expression of endogenous Runx2.Results:BMSCs were successfully isolated and cultured in vitro,and the cells adhered to the wall and grew like spindle and fiber,which had the potential of osteogenesis and adipogenesis to induce directional differentiation.At the same time,the surface markers of stem cells were identified by flow cytometry,and the cells met the identification standards of BMSCs.High glucose can inhibit the proliferation and osteogenic differentiation potential of BMSCs and promote the expression level of apoptosis-related genes.The expression of gene mi R-153-3p gradually increased in the process of osteogenic differentiation,and it was time-dependent,suggesting that mi R-153-3p may continue to play a role in the process of osteogenic differentiation in the high glucose microenvironment and has a positive correlation with osteogenesis.Overexpression of mi R-153-3p inhibits the osteogenic differentiation potential of BMSCs,but silencing mi R-153-3p will alleviate this inhibitory effect.The results of Luciferase assay preliminarily confirmed that the 3’-UTR region of Runx2 has a target binding site of mi R-153-3p,which has negative regulatory effect.Molecular studies further confirmed that overexpression of mi R-153-3p in BMSCs can significantly inhibit the m RNA and protein expression level of endogenous Runx2,while silencing mi R-153-3p can up-regulate the expression level of Runx2,indicating that mi R-153-3p inhibits Runx2 not only at the transcription level,but also at the translation level.The experimental results show that silencing mi R-153-3p can enhance the osteogenic differentiation of BMSCs under high glucose conditions,while silencing Runx2 can weaken this effect,indicating that mi R-153-3p can inhibit the osteogenic differentiation of BMSCs by targeting 3’-UTR of Runx2.In conclusion,our study found that mi R-153-3p is the key regulator of high glucose affecting osteogenic differentiation of BMSCs,and it may play a negative regulatory role by inhibiting the expression of Runx2.Conclusions:Our results indicate that high glucose induced mi R-153-3p can inhibit the osteogenic differentiation of BMSCs,and silencing mi R-153-3p can alleviate the osteogenic disorder induced by high glucose,which may be related to the down-regulation of the expression of the transcription factor Runx2.This study provides experimental support at the cellular level for mi R-153-3p as a new therapeutic target for diabetes osteoporosis.