Profiling microRNA expression during fracture healing.
Profiling microRNA expression during fracture healing.
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DOI：
10.1186/s12891-016-0931-0
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发表时间：
2016-02-16
影响因子：
2.3
通讯作者：
Kurosaka M
中科院分区：
文献类型：
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作者：
Waki T;Lee SY;Niikura T;Iwakura T;Dogaki Y;Okumachi E;Oe K;Kuroda R;Kurosaka M
microRNA（miRNA）的发现揭示了一种新型的基因表达调控机制。越来越多的证据表明，miRNA调节软骨细胞、成骨细胞和破骨细胞的分化和功能，表明miRNA是骨形成、吸收、重塑和修复的关键调节因子。我们推测某些miRNAs的功能及其表达模式的改变可能在骨折愈合过程中起着至关重要的作用。在70只大鼠的股骨中建立了标准愈合骨折和在骨折部位通过骨膜烧灼产生的不愈合骨折，其中一半被分配到标准愈合骨折组，一半被分配到不愈合组。在骨折后第3、7、10、14、21和28天，从骨折部位新产生的组织中提取包括miRNA的总RNA。在骨折后第14天，用来自每组的miRNA样品进行微阵列分析。为了进一步分析，我们从微阵列数据中选择了标准愈合骨折组中高度上调的五种miRNA。在上述每个时间点对miRNA样本进行实时PCR，以比较标准愈合骨折和未愈合骨折之间所选miRNA的表达水平，并研究其时程变化。第14天的微阵列和实时聚合酶链反应（PCR）分析显示，与未愈合的骨折相比，5种miRNA，miR-140- 3 p，miR-140- 5 p，miR-181 a-5 p，miR-181 d-5 p和miR-451 a在标准愈合的骨折中显著高表达。实时PCR分析进一步揭示，在标准愈合骨折中，所有五种miRNAs的表达在第14天达到峰值，此后下降。我们的研究结果表明，使用微阵列和实时PCR分析确定的五个miRNAs可能在骨折愈合过程中发挥重要作用。这些发现为进一步了解骨折愈合的分子机制提供了有价值的信息，并可能导致基于miRNA的组织工程策略的发展，以促进骨折愈合。
The discovery of microRNA (miRNA) has revealed a novel type of regulatory control for gene expression. Increasing evidence suggests that miRNA regulates chondrocyte, osteoblast, and osteoclast differentiation and function, indicating miRNA as key regulators of bone formation, resorption, remodeling, and repair. We hypothesized that the functions of certain miRNAs and changes to their expression pattern may play crucial roles during the process of fracture healing. Standard healing fractures and unhealing fractures produced by periosteal cauterization at the fracture site were created in femurs of seventy rats, with half assigned to the standard healing fracture group and half assigned to the nonunion group. At post-fracture days 3, 7, 10, 14, 21, and 28, total RNA including miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture day 14. For further analysis, we selected highly up-regulated five miRNAs in the standard healing fracture group from the microarray data. Real-time PCR was performed with miRNA samples at each time point above mentioned to compare the expression levels of the selected miRNAs between standard healing fractures and unhealing fractures and investigate their time-course changes. Microarray and real-time polymerase chain reaction (PCR) analyses on day 14 revealed that five miRNAs, miR-140-3p, miR-140-5p, miR-181a-5p, miR-181d-5p, and miR-451a, were significantly highly expressed in standard healing fractures compared with unhealing fractures. Real-time PCR analysis further revealed that in standard healing fractures, the expression of all five of these miRNAs peaked on day 14 and declined thereafter. Our results suggest that the five miRNAs identified using microarray and real-time PCR analyses may play important roles during fracture healing. These findings provide valuable information to further understand the molecular mechanism of fracture healing and may lead to the development of miRNA-based tissue engineering strategies to promote fracture healing.