微小RNA-608在人骨髓间充质干细胞成骨与成脂肪分化动态平衡中的作用

The effect of microRNA-608 in the dynamic balance of osteogenesis and adipogenesis differentiation of human bone marrow mesenchymal stem cells

目的检测微小RNA(miRNA,miR)-608在人骨髓间充质干细胞(hBMSCs)向成骨细胞和成脂肪细胞分化过程中的表达变化以及对hBMSCs向成骨细胞和成脂肪细胞分化的调控作用机制.方法利用实时荧光定量聚合酶链反应(FQ-PCR)分别检测hBMSCs向成骨细胞以及成脂肪细胞分化过程中miR-608的表达变化.细胞计数试剂盒(CCK-8)和克隆形成实验检测miR-608对hBMSCs增殖的影响.利用生物信息学软件分析miR-608的靶基因.利用双荧光素酶报告基因检测miR-608与靶基因的结合.利用蛋白质印迹法(Western blot)检测miR-608对靶基因蛋白水平的表达变化.利用茜素红染色和油红O染色检测miR-608对hBMSCs向成骨细胞和成脂肪细胞分化的功能的影响.应用SPSS 17.0统计软件分析,数据以均值±标准差(Mean±SD)表示.两样本均数比较采用独立样本t检验,多样本均数比较采用单因素方差分析.结果 FQ-PCR检测显示在hBMSCs向成骨细胞分化第3天miR-608表达量下降,与对照组(1.00±0.11)比较,成骨诱导分化组(0.20±0.09)中miR-608的表达显著性降低,差异有统计学意义(t=9.479,P<0.01).FQ-PCR检测发现在hBMSCs向成脂肪细胞分化第3天表达量升高,与对照组(1.00±0.09)比较,成骨诱导分化组(4.36±0.55)中miR-608的表达显著性降低,差异有统计学意义(t=10.442,P<0.05).CCK-8和克隆形成实验检测发现miR-608抑制hBMSCs的增殖.生物信息学软件分析miR-608与Runt相关基因2(Runx2)以及音猬因子(SHH)3'端非编码区(3'UTR)区有结合位点.双荧光素酶报告基因检测可见与miR-608 mimics NC组(1.00±0.22)比较,miR-608 mimcs组(0.21±0.11)荧光素酶活性显著降低,差异有统计学意义(t=5.542,P<0.05).Western blot检测发现miR-608可以抑制Runx2和SHH蛋白水平的表达.茜素红染色检测显示较miR-608 mimics NC组(1.00±0.14)比较,miR-608 mimcs组(0.32±0.12)茜素红染色显著降低,差异有统计学意义(t=6.387,P<0.05).油红O染色检测发现与miR-608 mimics NC组(19.20±2.34)比较,miR-608 mimcs组(32.12±6.25)油红O细胞阳性数目显著升高,差异有统计学意义(t=3.353,P<0.05).结论 miR-608在hBMSCs向成骨细胞分化过程中表达降低,而向成脂肪细胞分化过程中升高.miR-608抑制hBMSCs的增殖.miR-608通过靶向Runx2和SHH mRNA 3'UTR区调控Runx2和SHH蛋白水平的表达.miR-608抑制hBMSCs向成骨细胞分化而促进hBMSCs向脂肪细胞分化.

Objective To detect the expression of miR-608 in human bone marrow mesenchymal stem cells (hBMSCs) during differentiation into osteoblasts and adipocytes, and the regulatory mechanism of hBMSCs in differentiation into osteoblasts and adipocytes. Methods Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of miR-608 during the differentiation of hBMSCs into osteoblasts and adipocytes. Cell counting kit-8 (CCK-8) and clone formation assay were used to detect the effect of miR-608 on the proliferation of hBMSCs. The target gene of miR-608 was analyzed by bioinformatics software. Double luciferase reporter gene was used to detect the binding of miR-608 to target gene. Western blotting was used to detect the expression changes of the target gene protein of miR-608. Alizarin red staining and oil red O staining were used to detect the effect of miR-608 on the differentiation of hBMSCs into osteoblasts and adipocytes. Results FQ-PCR showed that the expression of miR-608 decreased significantly on the 3rd day of differentiation from hBMSCs to osteoblasts. Compared with the control group (1.00±0.11), the expression of miR-608 in osteogenic differentiation group (0.20±0.09) was significantly lower (t=9.479, P<0.01). FQ-PCR showed that the expression of miR-608 increased on the 3rd day of differentiation from hBMSCs to adipocytes. Compared with the control group (1.00±0.09), the expression of miR-608 in osteogenic differentiation group (4.36±0.55) decreased significantly (t=10.442, P<0.05), and the difference was statistically significant. CCK-8 and clonogenic assay showed that miR-608 inhibited the proliferation of hBMSCs. Bioinformatics software analyzed the binding sites of miR-608 with related transcription factor-2 (Runx2) and SHH 3’untranslated regions (3’UTR). The double luciferase reporter gene assay showed that the luciferase activity in the group of miR-608 mimics (0.21±0.11) was significantly lower than that in the group of miR-608 mimics NC (1.00±0.22)(t=5.542, P<0.05). Western blotting analysis showed that miR-608 could inhibit the expression of Runx2 and SHH proteins. Alizarin red staining showed that the alizarin red staining was significantly lower in the miR-608 mimics group (0.32±0.12) than in the miR-608 mimics NC group (0.32±0.12)(t=6.387, P<0.05). Oil red O staining showed that the positive number of oil red O cells in the group of miR-608 mimics (32.12±6.25) was significantly higher than that in the group of miR-608 mimimics NC (19.20±2.34)(t=3.353, P<0.05). Conclusion The expression of miR-608 decreased during the differentiation of hBMSCs into osteoblasts, but increased during the differentiation of hBMSCs into adipocytes. miR-608 inhibits the proliferation of hBMSCs. miR-608 regulates the expression of Runx2 and SHH proteins by targeting Runx2 and SHH mRNA 3’UTR regions. Mi-608 inhibits the differentiation of hBMSCs into osteoblasts and promotes the differentiation of hBMSCs into adipocytes.