成骨不全症血清中骨相关microRNAs的初步筛查

Screening of bone-related microRNAs in serum of patients with osteogenesis imperfect

摘要:

旨在初步筛选出成骨不全症 (Osteogenesis imperfecta，OI) 患者血清中差异表达的骨相关microRNAs (miRNAs)，通过探讨其在该疾病中的可能作用，为进一步研究成骨相关miRNA的功能及miRNA在成骨不全症诊断中的应用奠定基础。首先用geNorm和NormFinder等程序挑选出适于血清miRNAs定量检测的内参基因，然后利用实时荧光定量PCR技术对miRanda，Targetscan和Pictar软件以及文献报道筛选出的骨形成相关miRNAs进行定量检测，最后用配对t检验统计学方法筛查出差异表达的骨相关miRNAs。结果显示6个候选内参基因在不同年龄、性别和用药情况的成骨不全症患者和健康个体血清中的表达稳定性良好 (表达稳定值M<1.5)，后经标准化因子配对差异 (Pairwise variations) 分析确定最适内参数目为4个 (配对差异值V4/5 = 0.133<0.15)，分别是miR-16、Let-7a、snRNAU6、miR-92a。通过在16个成骨不全症患者以及8个健康对照个体的血清中进一步检测miR-16、Let-7a、snRNAU6和miR-92a，发现其表达稳定性依然良好 (M<1.5)。对100余种骨相关miRNAs进行实时荧光定量检测，发现11种miRNAs在成骨不全症患者血清中有差异表达 (P<0.05)，并且生物信息学分析显示这些差异表达的miRNAs 很可能在成骨不全症的发生发展中起重要作用。以上实验结果表明成骨不全症患者血清中存在多种差异表达的骨相关miRNAs，而且这些miRNAs很可能成为一种用于成骨不全症血清学检查及诊断的生物标志物。

Abstract:

We screened differential expression bone-related microRNAs (miRNAs) in serum of patients with osteogenesis imperfect (OI). First, we selected the reference gene (s) fit for quantitative detection of serum miRNAs by using geNorm and several other programmes. Then real-time fluorescent quntitative PCR was used to detect the expression level of bone-related miRNAs gained by means of miRanda, Targetscan and Pictar softwares caculation and reading literature. Then, the results were analyzed with the matched t test. All 6 candidate reference genes had a stable expression level in serum of healthy controls and patients with different characters, and the optimal number of reference genes is 4 (miR-16, let-7a, snRNAU6, miR-92a) after Pairwise Variations analysis (V4/5=0.133<0.15). For validating the universality of expression stability, we detected the relative expression value of miR-16, let-7a, snRNAU6 and miR-92a in another 8 healthy controls and 16 patients with OI and the result revealed that the expression of 4 genes remained stable (M<1.5). After measuring serum levels of more than 100 bone-related miRNAs in patients with real-time qPCR, 11 miRNAs showed differential expression, and bioinformatic analysis suggested these altered expressional mioRNAs had possibilities to participate in the process of OI. So the experiment indicated that there existed many differential expression bone-related miRNAs in serum of patients with OI, and these miRNAs had potentials to be promising biomarkers for serologic tests and diagnosis of OI.