Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
Department of Orthopaedic Surgery, Hyogo Prefectural Kakogawa Medical Center, Kakogawa, 675-8555, Japan.
J Orthop Surg Res. 2020 Apr 7;15(1):135. doi: 10.1186/s13018-020-01658-x.
MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate gene expression. There is increasing evidence that some miRNAs are involved in the pathology of diabetes mellitus (DM) and its complications. We hypothesized that the functions of certain miRNAs and the changes in their patterns of expression may contribute to the pathogenesis of impaired fractures due to DM.
In this study, 108 male Sprague-Dawley rats were divided into DM and control groups. DM rats were created by a single intravenous injection of streptozotocin. Closed transverse femoral shaft fractures were created in both groups. On post-fracture days 5, 7, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was conducted with miRNA samples from each group on post-fracture days 5 and 11. The microarray findings were validated by real-time polymerase chain reaction (PCR) analysis at each time point.
Microarray analysis revealed that, on days 5 and 11, 368 and 207 miRNAs, respectively, were upregulated in the DM group, compared with the control group. The top four miRNAs on day 5 were miR-339-3p, miR451-5p, miR-532-5p, and miR-551b-3p. The top four miRNAs on day 11 were miR-221-3p, miR376a-3p, miR-379-3p, and miR-379-5p. Among these miRNAs, miR-221-3p, miR-339-3p, miR-376a-3p, miR-379-5p, and miR-451-5p were validated by real-time PCR analysis. Furthermore, PCR analysis revealed that these five miRNAs were differentially expressed with dynamic expression patterns during fracture healing in the DM group, compared with the control group.
Our findings will aid in understanding the pathology of impaired fracture healing in DM and may support the development of molecular therapies using miRNAs for the treatment of impaired fracture healing in patients with DM.
微小 RNA（miRNA）是一类小的非编码 RNA 分子，可调节基因表达。越来越多的证据表明，某些 miRNA 参与了糖尿病（DM）及其并发症的病理过程。我们假设某些 miRNA 的功能及其表达模式的变化可能有助于 DM 导致的骨折愈合受损的发病机制。
本研究将 108 只雄性 Sprague-Dawley 大鼠分为 DM 组和对照组。DM 大鼠通过单次静脉注射链脲佐菌素建立。两组均造成闭合性股骨骨干横断骨折。在骨折后第 5、7、11、14、21 和 28 天，从骨折部位新生成的组织中提取 miRNA。在骨折后第 5 和 11 天，对每组的 miRNA 样本进行 miRNA 芯片分析。通过实时聚合酶链反应（PCR）分析在每个时间点验证微阵列结果。
微阵列分析显示，与对照组相比，DM 组在第 5 天和第 11 天分别有 368 个和 207 个 miRNA 上调。第 5 天上调最明显的前 4 个 miRNA 是 miR-339-3p、miR-451-5p、miR-532-5p 和 miR-551b-3p。第 11 天上调最明显的前 4 个 miRNA 是 miR-221-3p、miR376a-3p、miR-379-3p 和 miR-379-5p。在这些 miRNA 中，miR-221-3p、miR-339-3p、miR-376a-3p、miR-379-5p 和 miR-451-5p 通过实时 PCR 分析得到验证。此外，PCR 分析显示，与对照组相比，在 DM 组骨折愈合过程中，这 5 个 miRNA 的表达具有动态表达模式，存在差异。
我们的研究结果将有助于理解 DM 导致的骨折愈合受损的病理过程，并可能支持使用 miRNA 进行分子治疗，为 DM 患者的骨折愈合受损治疗提供支持。
