miR-26a-5p/环腺苷酸反应元件结合蛋白1分子轴调控脂肪来源干细胞成骨分化的机制研究

Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells

目的探讨miR-26a-5p通过调控环腺苷酸反应元件结合蛋白1(cAMP response element binding protein 1,CREB1)对脂肪来源干细胞(adipose-derived mesenchymal stem cells,ADSCs)成骨分化的调控作用及其作用机制。方法取4只3~4周龄雌性C57BL/6小鼠脂肪组织,采用消化分离法分离培养细胞并传代。经细胞形态学观察以及流式细胞仪检测鉴定其为ADSCs后,取第3代细胞行成骨分化诱导。于诱导培养0、3、7、14 d行茜素红染色观察细胞钙沉积,ALP活性检测,实时荧光定量PCR检测miR-26a-5p和CREB1 mRNA表达,Western blot检测CREB1蛋白及其磷酸化(phospho-CREB1,p-CREB1)水平以及p-CREB1/CREB1。经starBase数据库预测miR-26a-5p和CREB1存在靶向结合位点后,取HEK-293T细胞行双荧光素酶报告基因实验验证靶向关系(以培养48 h后荧光素酶活性表示)。最后将miR-26a-p inhibitor(实验组)及对应阴性对照(对照组)转染至ADSCs中,成骨诱导培养7、14 d同上法行茜素红染色观察、ALP活性检测以及Western blot检测[目的蛋白包括CREB1、p-CREB1、Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、骨钙素(osteocalcin,OCN)]。结果经鉴定分离培养细胞为ADSCs。随成骨诱导培养时间延长,ADSCs钙化结节增多、ALP活性增加(P<0.05);细胞中miR-26a-5p相对表达量逐渐降低,而CREB1 mRNA及蛋白相对表达量、p-CREB1蛋白相对表达量升高,其中7、14 d与0 d差异有统计学意义(P<0.05);p-CREB1/CREB1各时间点间差异均无统计学意义(P>0.05)。通过starBase数据库预测发现miR-26a-5p和CREB1具有靶向结合序列,双荧光素酶报告基因实验显示过表达miR-26a-5p可明显抑制CREB1野生型荧光素酶活性(P<0.05)。成骨诱导7、14 d,与对照组相比,实验组细胞钙化结节数量、ALP活性以及CREB1、p-CREB1、OCN、RUNX2蛋白相对表达量均增加,差异均有统计学意义(P<0.05);两组p-CREB1/CREB1差异无统计学意义(P>0.05)。结论通过敲降miR-26a-5p上调CREB1及其磷酸化水平,能促进ADSCs成骨分化。

Objective To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells(ADSCs)by regulating cAMP response element binding protein 1(CREB1).Methods The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method.After morphological observation and identification by flow cytometry,the 3rdgeneration cells were subjected to osteogenic differentiation induction.At 0,3,7,and 14 days after osteogenic differentiation induction,the calcium deposition was observed by alizarin red staining,ALP activity was detected,miR26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR,and CREB1 protein and its phosphorylation(phospho-CREB1,p-CREB1)level were measured by Western blot.After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database,HEK-293T cells were used to conduct a dualluciferase reporter gene experiment to verify the targeting relationship(represented as luciferase activity after 48 hours of culture).Finally,miR-26a-p inhibitor(experimental group)and the corresponding negative control(control group)were transfected into ADSCs.Alizarin red staining,ALP activity,real-time fluorescent quantitative PCR(miR-26a-5p)and Western blot[CREB1,p-CREB1,Runt-related transcription factor 2(RUNX2),and osteocalcin(OCN)]were performed at 7 and 14 days after osteogenic induction culture.Results The cultured cells were identified as ADSCs.With the prolongation of osteogenic induction culture,the number of calcified nodules and ALP activity significantly increased(P<0.05).The relative expression of miR-26a-5p in the cells gradually decreased,while the relative expressions of CREB1 mRNA and protein,as well as the relative expression of p-CREB1 protein were increased.The differences were significant between 7,14 days and 0 day(P<0.05).There was no significant difference in p-CREB1/CREB1 between different time points(P>0.05).The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences,and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity(P<0.05).After 7 and 14 days of osteogenic induction,compared with the control group,the number of calcified nodules,ALP activity,and relative expressions of CREB1,p-CREB1,OCN,and RUNX2 proteins in the experimental group significantly increased(P<0.05).There was no significant difference in p-CREB1/CREB1 between the two groups(P>0.05).Conclusion Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.