| Objective:Homing of MSCs plays an important role in fracture repair.Non-coding mieroRNAs(mieroRNAs)are involved in homing of MSCs.Bushen Huoxue Decoction is a common prescription for the treatment of fracture in clinic.Previous studies have shown that the petroleum ether extract of Bushen Huoxue Decoction can promote the migration of MSCs in vitro.However,the underlying molecular mechanism remains unclear.In this study,differentially expressed microRNAs were screened by MSCs treated with petroleum ether extract of Bushen Huoxue Decoction.To elucidate the role of the key microRNAs in the migration of MSCs promoted by the petroleum ether extract of Bushen Huoxue Decoction.Methods:The MSCs of SD rats were cultured with the whole bone marrow adherent wall method,and the cell surface antigen flow cytometry was detected and induced into fat and osteoblast differentiation in the third generation MSCs.CCK-8 was used to detect the effect of petroleum ether extract of Bushen Huoxue Decoction on the proliferation of MSCs.Transwell experiment was used to observe the effect of petroleum ether of Bushen Huoxue Decoction on migration of MSCs.qRT-PCR was used to detect the expression of Wnt5a,JNK,PKC and CaMK?in MSCs treated with petroleum ether extract of Bushen Huoxue Decoction.Observation of the effect of siRNA silencing Wnt5a on migration of MSCs.Screening of differentially expressed microRNAs from petroleum ether extracts of Bushen Huoxue Decoction after interfering with MSCs by microarray technology.qRT-PCR was used to validate differentially expressed microRNAs.The function of microRNAs was verified by Transwell and scratch experiments after transfection of MSCs with microRNA mimics and inhibitors.Bioinformatics prediction software was used to predict target genes.Double luciferase reporter gene experiment and Western blot detection were used to identify target genes regulated by mieroRNAs.Results:Flow cytometry showed that the cultured Passage3 cells had high expression of CD90 and CD44(97.44%and 96.55%)and low expression of CD45 and CD34(0.14%and 0.21%).It successfully induces the formation of osteocytes and adipocytes,which is consistent with the characteristics of stem cells.The results of CCK-8 experiment showed that the intervention of petroleum ether of Bushen Huoxue Decoction in each concentration group(1?g/ml?10?g/ml and 100?g/ml)could promote the proliferation of MSCs.Transwell experiment showed that all concentration groups(1?g/ml?10?g/ml and 100?g/ml)treated with petroleum ether of Bushen Huoxue Decoction significantly promoted the migration of MSCs,and the optimum concentration of petroleum ether in Bushen Huoxue Decoction was 10?g/ml.The results of qRT-PCR showed that petroleum ether of Bushen Huoxue Decoction could promote the expression of Wnt5a,JNK,PKC and CaMK? in different concentration groups(1?g/ml?10?g/ml and 100?g/ml).After transfection of siRNA silencing Wnt5a,the migration ability of MSCs decreased significantly.Microarray screened 70 mieroRNAs with multiple difference greater than 2 after the intervention of Bushen Huoxue Decoction petroleum ether,of which 27 were highly expressed and 43 were low expressed.Twelve differentially.expressed mieroRNAs related to cell migration were verified by qRT-PCR.It was found that the expression of microRNAs was low expression.Transwell and scratch experiments showed that over-expression of miR-539 inhibited migration of MSCs,while low-expression of miR-539 promoted migration of MSCs.Target gene prediction and double fluorescein reporter gene test showed that Wnt5a was the regulatory target of miR-539.Western blot results showed that over-expression of miR-539 could inhibit the expression of Wnt5a protein.Conclusion:The petroleum ether extract of Bushen Huoxue Decoction can effectively promote the proliferation of MSCs.The petroleum ether extract of Bushen Huoxue Decoction can effectively promote the migration of MSCs,and its mechanism may be achieved by down-regulating the miR-539 and targeting the Wnt5a signaling pathway. |
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