MicroRNA-99a regulates early chondrogenic differentiation of rat mesenchymal stem cells by targeting the BMPR2 gene
MicroRNA-99a regulates early chondrogenic differentiation of rat mesenchymal stem cells by targeting the BMPR2 gene
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DOI：
10.1007/s00441-016-2416-8
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发表时间：
2016-10-01
影响因子：
3.6
通讯作者：
Li, Guitao
中科院分区：
文献类型：
--
作者：
Zhou, Xiaozhong;Wang, Jing;Li, Guitao
骨髓间充质干细胞（MSCs）是关节软骨再生的候选细胞，因为它们具有软骨分化的潜力。骨髓间充质干细胞易于获得并在体外扩增。调节软骨形成的特定microRNAs（miRNAs）尚未被鉴定，涉及的机制仍有待确定。miRNA通过结合靶mRNA以减少蛋白质合成来调节生物过程。在这项研究中，我们发现，miR-99 a和miR-125 b-3 p的表达增加的骨髓间充质干细胞（rMSCs）来源于挪威棕色大鼠（Rattus norvegicus）的早期软骨分化。MiR-99 a敲低促进蛋白多糖沉积，并在早期软骨形成分化期间增加ACAN和COL 2A 1的表达。miR-99 a敲低促进rMSCs的早期软骨分化。双荧光素酶报告基因测定显示，miR-99 a靶向骨形态发生蛋白（BMP）2型受体（BMPR 2）3 '-UTR中的假定结合位点。过表达miR-99 a可降低BMPR 2蛋白的表达水平。在rMSCs向软骨细胞分化的早期，总p38和p-p38的表达在第7天和第14天增加。在rMSCs早期软骨分化过程中，miR-99 a过表达后总p38和p-p38蛋白水平降低。BMPR 2沉默逆转了miR-99 a抑制对rMSCs早期软骨分化过程中蛋白多糖沉积和ACAN、COL 2A 1、总p38和p-p38蛋白表达的影响。总之，在大鼠MSC中的这些体外研究的发现支持miR-99 a通过在早期直接靶向BMPR 2基因作为早期软骨形成分化的负调节剂的作用。
Mesenchymal stem cells (MSCs) are candidates for the regeneration of articular cartilage as they possess the potential for chondrogenic differentiation. MSCs are easily obtained and expanded in vitro. Specific microRNAs (miRNAs) that regulate chondrogenesis have yet to be identified and the mechanisms involved remain to be defined. The miRNAs regulate biological processes by binding target mRNA to reduce protein synthesis. In this study, we show that expression of miR-99a and miR-125b-3p were increased during early chondrogenic differentiation of MSCs (rMSCs) derived from the Norwegian brown rat (Rattus norvegicus). MiR-99a knockdown promoted proteoglycan deposition and increased the expression of ACAN and COL2A1 during early chondrogenic differentiation. MiR-99a knockdown promoted early chondrogenic differentiation of rMSCs. A dual-luciferase reporter gene assay showed that miR-99a targeted a putative binding site in the 3'-UTR of bone morphogenetic protein (BMP) receptor type 2 (BMPR2). Overexpression of miR-99a reduced the expression levels of BMPR2 protein. The expression of total p38 and p-p38 increased at 7 and 14 days during early chondrogenic differentiation of rMSCs. Reduction in levels of total p38 and p-p38 protein followed miR-99a overexpression during early chondrogenic differentiation of rMSCs. BMPR2 silencing reversed the effects of miR-99a inhibition on proteoglycan deposition and protein expression of ACAN, COL2A1, total p38 and p-p38 during early chondrogenic differentiation of rMSCs. In conclusion, the findings of these in vitro studies in rat MSCs support a role for miR-99a as a negative regulator of early chondrogenic differentiation by directly targeting the BMPR2 gene at an early stage.