环状RNA CDR1as促进小鼠骨髓间充质干细胞成骨分化和成血管相关基因表达

Circular RNA CDR1as promotes osteogenic differentiation and angiogenesis related genes expression in mouse bone marrow mesenchymal stem cells

目的 探讨环状RNA小脑变性相关蛋白1的反义转录物(antisense to the cerebellar degeneration-related protein 1 transcript,CDR1as)在Balb/C小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)中过表达与低表达后对其成骨和成血管相关基因的影响。方法 体外培养及鉴定BMSCs,将含过表达与沉默circRNA CDR1as基因的慢病毒(lentiviruses,LV)载体及对照组慢病毒分别转染至小鼠BMSCs并筛选稳定的细胞株,分为circRNA CDR1as过表达组、过表达对照组、circRNA CDR1as低表达组和低表达对照组。各组成骨诱导14、21 d后分别进行茜素红染色和碱性磷酸酶染色;qRT-PCR检测目的基因circRNA CDR1as、成骨分化特异标志物碱性磷酸酶(alkaline phosphatase,ALP)、Runt相关基因2(runt-related transcription factor 2,RUNX2)、骨钙素(osteocalcin,OCN)、骨桥素(osteopontin,OPN)、锌指结构转录因子(osterix,OSX)、Ⅰ型胶原蛋白(collagen-I,COL-1),以及成血管特异标志物血管内皮生长因子(vascular endothelial grown factor,VEGF)、血管生成素-1(angiogenin-1,Ang-1)的mRNA表达水平。结果 茜素红和ALP染色均显示:过表达实验组钙沉淀和ALP染色区域多于过表达对照组;而低表达实验组钙沉淀和ALP染色区域少于低表达对照组,随着成骨诱导天数的增加,各组的钙沉淀和ALP染色面积也增大。qRT-PCR结果显示:过表达实验组BMSCs中circRNA CDR1as、ALP、RUNX2、OCN、OPN、OSX、COL-1、VEGF和Ang-1的mRNA表达水平显著升高(P<0.001);低表达实验组BMSCs中circRNA CDR1as、ALP、RUNX2、OCN、OPN、OSX、COL-1、VEGF和Ang-1的m RNA表达水平显著降低(P<0.001)。结论 过表达circRNA CDR1as基因可促进BMSCs的成骨分化及血管生成的能力;低表达circRNA CDR1as基因可抑制BMSCs的成骨分化及血管生成的能力。

Objective To investigate the effects of over expression and low expression of antisense transcripts of circular RNA cerebellar degeneration associated protein 1(CDR1as) in Balb/C mouse bone marrow mesenchymal stem cells(BMSCs) on factors related to osteogenesis and angiogenesis. Methods BMSCs were cultured and identified in vitro. The lentiviral(LV) vector containing the overexpressed and silenced circRNA CDR1as genes and the control lentivirus were respectively transfected into mouse BMSCs, and stable cell lines were screened. The cells were divided into the circRNACDR1as over expression group and the over expression control group, and the CircRNACDR1as low expression group and the low expression control group. The components were stained with Alizarin Red S and alkaline phosphatase after 14 and 21 days of osteoinduction;qRT-PCR was used to detect the target genes circRNA CDR1as, osteogenic differentiation markers alkaline phosphatase(ALP), runt-related transcription factor 2(RUNX2), osteocalcin(OCN), osteopontin(OPN), osterix(Osx), collagen I(COL-1), and the mRNA expression levels of vascular endothelial grown factor(VEGF) and angiogenin-1(Ang-1). Results The results of alizarin red staining and alkaline phosphatase staining showed that the extracellular matrix calcium precipitation and ALP staining area of the over expression experimental group was greater than its control group,and those of the low expression experimental group was less than its control group. As the number of days of osteogenic induction increased,the calcium precipitation and ALP staining in each group also increased. RT-PCR results showed that the mRNA expression levels of circRNA CDR1as, ALP,RUNX2, OCN, OPN, OSX, COL-1, VEGF and Ang-1 in the over expression experimental group BMSCs were significantly increased(P<0.001). In the low expression experimental group, the mRNA expression levels of circRNA CDR1as,ALP, RUNX2, OCN, OPN, OSX, COL-1, VEGF and Ang-1 in BMSCs were significantly reduced(P<0.001). Conclusion Over expression of the circRNA CDR1as gene promotes the osteogenic differentiation and angiogenesis of BMSCs. Low expression of the circRNA CDR1as gene inhibits the osteogenic differentiation and angiogenesis of BMSCs.