诱导hBMSCs成骨分化中微小RNA和靶基因表达水平的变化

CHANGES OF MICRO RNA AND TARGET GENE EXPRESSION LEVELS IN OSTEOGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS

目的明确萎缩性骨不连组织中表达上调的数种微小RNA(micro RNA,miRNA)与其相应靶基因mRNA、蛋白在hBMSCs成骨分化过程中的表达变化趋势和生物学功能。方法取自体髂骨植骨手术患者的髂骨骨髓血,采用密度梯度离心法分离培养hBMSCs。取第4代hBMSCs以成骨诱导培养液诱导成骨分化,分别提取0、12 h,1、2、4、7、14 d时的细胞总RNA和蛋白,进行miRNA的实时定量PCR(quantitative real-time PCR,qRT-PCR)、相应靶基因mRNA的qRT-PCR和蛋白Western blot检测。结果诱导hBMSCs成骨分化时,成骨性靶基因碱性磷酸酶(alkalinephosphatase liver/bone/kidney,ALPL)、PDGF-α多肽(PDGF-αpolypeptide,PDGF-A)和BMP-2的mRNA和蛋白表达在多数时间点同对照(0 h)相比增加(BMP-2在12 h和1 d时下降),1～7 d变化最为显著。不同时间点的miRNA、靶基因mRNA和蛋白表达水平存在差异,其中hsa-miRNA-149*和hsa-miRNA-654-5p miRNA含量的变化趋势与各自靶基因ALPL和BMP-2的mRNA及蛋白表达水平总体上成负相关(P<0.05),hsa-miRNA-221与其靶基因PDGF-A的变化趋势无明显负相关关系(P>0.05)。结论诱导hBMSCs成骨分化过程中,hsa-miRNA-149*和hsa-miRNA-654-5p对其相应靶基因ALPL和BMP-2的mRNA及蛋白存在密切调控关系。

Objective To clarify the trends of expression levels of several up-regulated micro RNA（miRNA） in tissues of atrophic bone nonunion and mRNAs and proteins of their related target genes in osteogenic differentiation of human bone marrow mesenchymal stem cells（hBMSCs）,and to explore their biological functions.Methods The hBMSCs were isolated from bone marrow of iliac bone by gradient centrifugation,and cultured.Osteogenic culture medium was used for osteogenic differentiation of the 4th generation of hBMSCs.The changes of corresponding miRNAs,mRNA and protein expression levels of related target genes were observed at 0 hour,12 hours,1 day,2 days,4 days,7 days,and 14 days,by quantitative real-time PCR and Western blot.Results In the process of hBMSCs osteogenic differentiation,the mRNA and protein expression levels of osteoblastic target genes [alkaline phosphatase liver/bone/kidney（ALPL）,bone morphogenetic protein 2（BMP-2）,and platelet-derived factor alpha polypeptide（PDGF-A）] at most time points increased significantly when compared with the values at 0 hour except that of BMP-2 decreased at 12 hours and 1 day,with maximum changes at 1 to 7 days.The miRNA expression levels,mRNA and protein expression levels changed significantly at different time points,while the trends of hsa-miRNA-149＊ and hsa-miRNA-654-5p changes were negatively correlated with the trends of ALPL and BMP-2 mRNA and protein expression changes respectively（P 0.05）.There was no obviously negative correlation between the trends of hsa-miRNA-221 change and PDGF-A change（P 0.05）.Conclusion In the osteogenic differentiation process of hBMSCs,hsa-miRNA-149＊ and hsa-miRNA-654-5p are closely related with the mRNA and protein regulation of ALPL and BMP-2,respectively.