| Part Ⅰ: Effect of miR1-2 on promoting the differentiation of BMSCs into cardiomyocytesObjectiveThe heart is a non-renewable terminal organ.For end-stage heart disease,transplantation of the induced bone marrow-derived mesenchymal stem cells(BMSCs)can repair the damaged heart and improve heart function.However,the ability of BMSCs to differentiate into cardiomyocytes is limited,and the current induction methods often add exogenous chemicals such as 5-azacytine(5-aza),etc,but this method can not significantly improve the efficiency of BMSCs differentiating into cardiomyocytes.In addition,even in the case of best induction time and dose,5-aza still can not avoid damaging cells.miR1-2 is an endogenous small molecule,mainly related to the development of the myocardium.Moreover,research showed that micro RNAs are molecules that can determine the fate of stem cell differentiation.Therefore,the aim of this study was to investigate whether miR1-2 promoted the differentiation of BMSCs into cardiomyocytes,and whether the differentiation rate and the safety of the cells were higher than that of the traditional 5-aza induction method.Methods1 BMSCs were cultured in vitro.The BMSCs were divided into normal control group,miR1-2 mimics group,miR1-2 mimics negative control(miR1-2 mimics NC)group and 5-aza group2 The expression of miR1-2 in BMSCs was detected by quantitative real time polymerase chain reaction(qrt-PCR)technique at 24 h,48h and 72 h after 5-aza transfection.miR1-2 mimics sequences were transfected into BMSCs by lipofectamine2000.The expression of miR1-2 was detected by qrt-PCR technique at 24 h,48h and 72 h after transfection3 The expression of GATA4,NKx2.5 and cTnI m RNA in each group were detected by qrt-PCR at 48 h and 72 h after treatment.4 The expression of cTnI protein in each group were detected by western blotting(WB)technique at 72 h after treatment5 Flow cytometry was used to detect the percentage of apoptosis at 48 h and 72 h after treatment.Results1 The level of miR1-2 in BMSCs treated with 5-aza did not change at 24 h after treatment(P>0.05),but the level of miR1-2 increased at 48 h and 72 h after treatment,which were 1.71 times and 3.17 times as much as the normal control group respectively(P<0.01).2 The level of miR1-2 in BMSCs was significantly increased after transfection with miR1-2 mimics,which was 8539,1433 and 690 times of that in the control group at 24 h,48h and 72 h after transfection(P<0.001).3 The expressions of GATA4,Nkx2.5 and cTnI were significantly increased after 48 h treatment with 5-aza(P<0.01),at the same time,The expressions of Nkx2.5 and cTnI were significantly increased after 72 h treatment with 5-aza.After transfection of miR1-2 mimicss at 48 h,the expression of GATA4,Nkx2.5and cTnI significantly increased also compared to the control(P<0.01),and they increased with time,after 72 h of treatment,the expression of these genes in miR1-2 group were significantly higher than those of the 5-aza group and the control group(P<0.01).4 The levels of cTnI protein in BMSCs treated with 5-aza and miR1-2 mimics were higher than the control group after treatment for 72h(P<0.001).When the miR1-2 mimics group was compared with the 5-aza group,the expression level of cTnI was also higher significantly than that of the 5-aza group(P<0.05).5 After treated with 5-aza and miR1-2 mimics for 48 h,both did not cause significant apoptosis of BMSCs immediately(P>0.05).However,with time passing by,5-aza gradually induced cell apoptosis and the apoptosis rate significantly increased at 72 h to 16.88%,which was 2.48 times of that of the normal control group(P<0.001).However,there was no significant increase of apoptotic rate after transfected with miR1-2 mimics for 72 h,when comparing to the normal control group(P>0.05).ConclusionsmiR1-2 mimics could induce the BMSCs to differentiate into cardiomyocytes as evidenced by the findings that the expression of GATA4,NKx2.5,cTnI were significantly increased.Moreover,the expression level of m RNA and protein were both higher than the traditional 5-aza induction method.Also,the effect of miR1-2 mimics on the apoptosis of BMSCs was significantly lower than that of 5-aza.All of these results suggested that miR1-2 could induce the differentiation of BMSCs into cardiomyocytes,and this method may be more effective and less cytotoxic than that of 5-aza.Part Ⅱ: miR1-2 promot the differentiation of BMSCs into cardiomyocytes through Wnt signaling pathwayObjectiveThe heart is one of the earliest embryonic developmental organs.Its development depends on the orderly function of various cardiac developmental factors,signal channel molecules and so on.Wnt signaling pathway is an important signal pathway in cardiac development.It participates in the process of cardiac four-chamber heart formation,cardiac cyclization,cardiac development and valve formation,suggesting that this signal pathway may be also involved in the process of miR1-2-induced differentiation of BMSCs into myocardial cells.Therefore,the purpose of this study is to explore whether the mechanism of miR1-2-induced differentiation of BMSCs into cardiomyocytes is related to the activation of Wnt pathway.Methods1 BMSCs were cultured in vitro and were divided into normal control group,miR1-2 mimics group,miR1-2 mimics negative group,Wnt signal pathway inhibitor(LGK-974)+ miR1-2 mimics combination group2 qrt-PCR was used to detect the m RNA levels of WNT signaling pathway molecules such as β-catenin,Wnt11,JNK,and TCF in each group.3 WB was used to detect the proteins expression of Wnt signaling pathway such as β-catenin and JNK.4 WB was used to detect the proteins expression of GATA4,NKx2.5 and cTnI after treatment with Wnt signal pathway inhibitor LGK-974 and miR1-2 mimics.Results1 The m RNA levels of Wnt signaling pathway molecules: β-catenin,Wnt11,JNK and TCF in miR1-2 mimics group were higher significantly than those in the control group at 24 h,48h and 72 h after transfection(P<0.05).After the mimics group was added into Wnt signal pathway inhibitor LGK-974,the levels of β-catenin,Wnt11,JNK,and TCF were reduced significantly,Comparing to group treated with miR1-2 mimics solely(P<0.05).2 The proteins expression of Wnt/β-catenin signaling pathway after transfection with miR1-2 mimics was higher than those in the control group(P<0.05).After the mimics group was added into Wnt signal pathway inhibitor LGK-974,the protein expression of Wnt/β-catenin signaling pathway were evidently reduced,compared with group treated with mimics solely,the difference was statistically significant(P<0.01).3 After using Wnt/β-catenin signaling pathway inhibitor LGK-974 to inhibit Wnt/β-catenin signaling pathway,the proteins expression levels of GATA4,NKx2.5 and cTnI were significantly lower than those in the simple mimics group untreated with LGK-974 inhibitor(P<0.05).ConclusionIn the process of miR1-2 promoting the differentiation of BMSCs into the myocardium,the Wnt/β-catenin signaling pathway were activated.After Wnt signaling pathway inhibitor LGK-974 was used to inhibit Wnt/β-catenin signaling pathway,the protein expression levels of GATA4,NKx2.5 and cTnI in BMSCs induced by miR1-2 mimics were significantly reduced,suggesting that miR1-2 prompted the differentiation of BMSCs into myocardium through Wnt/β-catenin signaling pathway. |
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