| Objective To investigate the effct of miR-139-5p/CXCR4 on the migration ability of BMSCs;to preliminarily investigate the role of TMP to promote BMSCs’ migration through miR-139-5p/CXCR4,and to further elucidate TMP promoting BMSCs migration to the ischemic periphery to improve cerebral ischemia by regulating miR-139-5p/CXCR4.Methods 1.BMSCs were isolated and cultured in vitro from 4-week-old male SD rats,and the cell purity was determined by flow cytometry.2.miR-139-5p mimic、miR-139-5p inhibitor were transfected into BMSCs using Lipofectamine 3000,BMSCs were divided into Control group,miR-139-5p mimic group,miR-139-5p mimic NC group,miR-139-5p inhibitor and miR-139-5p inhibitor NC group,the migration ability of BMSCs was detected by cell scratch and Transwell assay.The protein expression of CXCR4 was detected by Western blot.3.The target gene prediction software and dual-luciferase assay were used to detect whether miR-139-5p was targeted to CXCR4.4.MiR-139-5p inhibitor and siRNA-CXCR4 were cotransfected,BMSCs were divided into Control group,inhibitor group,inhibitor NC group,inhibitor+siRNA-CXCR4 group,inhibitor+siRNA-NC group,the migration ability of BMSCs was detected by cell scratch assay and Transwell assay.5.BMSCs were treated with different concentrations of TMP(50 μM,100 μM),and cell migration was detected by scratch assay,miR-139-5p and CXCR4 mRNA expression was detected by RT-qPCR,and CXCR4 protein expression was detected by Western blot.6.BMSCs were transfected with miR-139-5p mimic and simultaneously treated with 100 μM TMP,BMSCs were divided into Control group,TMP group,TMP+miR-139-5p mimic group,and TMP+miR-139-5p mimic NC group,cell migration was examined using scratch and Transwell assays,and CXCR4 expression was detected by Western blot.7.The right middle cerebral artery occlusion(MCAO)model was established in SD rats,and BrdU-labeled BMSCs,TMP-BMSCs,TMP-miR-139-5p mimicBMSCs,and TMP-miR-139-5p mimic NC-BMSCs were transplanted into the tail vein,and the neurobehavioral function of rats in each group was detected by Longa score,the cerebral infarction volume of rats in each group was detected by TTC staining,the number of BrdU+BMSCs in the ischemic peripheral area was detected by immunofluorescence,and the protein expression of CXCR4 in the ischemic peripheral area of rats in each group was detected by Western blot.Results 1.Isolated BMSCs were identified by flow cytometry with high purity,and the positive rates of CD90,CD29,CD45 and CD31 were 98.67%,98.52%,2.95%and 1.00%respectively.2.RT-qPCR results showed that miR-139-5p miRNA content was significantly increased in the mimic group(P<0.01),and the inhibitor group had the opposite effect(P<0.01).Cell scratch assay showed that the wound healing speed was inhibited in the mimic group(P<0.01),and transwell assay showed that the number of migrating cells in the mimic group decreased(P<0.01),and inhibitor group had the opposite effect.WB results showed that CXCR4 protein expression decreased in the mimic group(P<0.05)and increased in the inhibitor group(P<0.01).3.The results predicted by PicTar and miRDB software as well as the results of dual luciferase experiments all suggested that CXCR4 was the target gene of miR139-5p.4.BMSCs were co-transfected with miR-139-5p inhibitor and siRNA-CXCR4,and compared with inhibitor group,scratch healing rate of BMSCs in the inhibitor+siRNACXCR4 group was slowed down(P<0.01)and the number of migrating cell was decreased(P<0.01).5.RT-qPCR results showed that miR-139-5p was down-regulated(P<0.01)and CXCR4 mRNA expression was up-regulated(P<0.05)in BMSCs treated with 100 μM TMP.WB results showed that CXCR4 protein expression was up-regulated in TMP group(P<0.05).Cell scratch results showed that TMP promoted scratch healing in BMSCs(P<0.01).6.Compared with the TMP group,scratch results showed that the scratch healing speed of miR139-5p mimic+TMP group was slowed down(P<0.01),and Transwell results showed that the number of BMSCs migrated in miR-139-5p mimic+TMP group was reduced(P<0.01);WB results showed that CXCR4 expression of miR-139-5p mimic+TMP group was downregulated(P<0.01).7.TTC staining results showed that compared with MCAO group,the infarct volume in the BMSCs group was reduced(P<0.01),the infarct volume in the TMPBMSCs group was further reduced compared with the BMSCs group(P<0.01),while the infarct volume in the TMP-mimic-BMSCs group was larger than that in the TMP-BMSCs group(P<0.05).Compared with MCAO group,the Longa score of TMP-BMSCs group was downregulated(P<0.01),while the score of TMP-mimic-BMSCs group was higher than that of TMPBMSCs group(P<0.05).BrdU immunofluorescence results showed that the number of BrdU+cells in TMP-BMSCs group was significantly higher than that in BMSCs group(P<0.01),and the number of BrdU+cells in TMP-mimic-BMSCs group was less than that in TMP-BMSCs group(P<0.01).WB results showed that the expression of CXCR4 protein in MCAO group was up-regulated(P<0.05),the expression of CXCR4 protein in TMP-BMSCs group was higher than that in BMSCs group(P<0.01),and the expression of CXCR4 protein in TMPmimic-BMSCs group was lower than that in TMP-BMSCs group(P<0.05).Conclusion MiR-139-5p down-regulates CXCR4 expression to inhibit BMSCs migration.TMP promotes BMSCs migration through miR-139-5p/CXCR4.TMP promotes BMSCs migration to improve cerebral ischemia injury through miR-139-5p/CXCR4. |
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