Scleral miRNA profiles in adult and fetal eyes

Introduction: In human eyes, ocular enlargement/ growth, reflects active scleral extracellular matrix remodeling. miRNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences, and serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses miRNAs, some of which modulate genes regulating ocular growth. In this study, the scleral miRNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses. Results: Human sclera expressed several miRNAs. Microarray comparison of adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10^11), with increased expression of collagen specific mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 in fetal sclera subsequently confirmed (1.5 to 4 fold changes, p<0.01). For both adult and fetal samples, no significant differences in miRNA expression profiles of sclera from posterior and peripheral ocular regions were observed. Conclusion: This is the first study to catalogue miRNA expression in human sclera. The sclera expresses several miRNAs, some of which show age-related differential regulation, higher in rapidly growing fetal eyes, consistent with a role in ocular growth regulation. These findings may be useful for linking scleral miRNA expression with potential manipulation in disorders such as scleral ectasia/ axial myopia. Scleral samples from normal human fetal (24 wk) and normal adult donor eyes (n=4 to 6, each group) were obtained, and RNA extracted. Genome-wide miRNA profiling was performed using the Agilent miRNA microarray platform. miRNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms.

引言：在人体眼部中，眼球增大或生长反映了巩膜细胞外基质（extracellular matrix）的活跃重塑。微小RNA（microRNAs, miRNAs）是一类小型非编码RNA，通过与靶序列碱基配对调控基因表达，可作为信号网络的节点分子。我们提出如下假设：与多数人体组织一样，巩膜可表达miRNAs，其中部分可调控参与眼球生长的相关基因。本研究采用高通量微阵列（high-throughput microarray）与定量聚合酶链式反应（quantitative PCR）分析技术，对比了快速生长的人类胎儿眼球与状态稳定的成人供体眼球的巩膜miRNA表达谱。

结果：人类巩膜可表达多种miRNAs。通过微阵列对比成人与胎儿样本发现，大量miRNAs呈现差异表达（p<0.01，最小p值为6.5×10^11）；胶原特异性的mir-214、let-7c、let-7e、mir-103、mir-107及mir-98在胎儿巩膜中的表达上调后续得到验证（表达变化幅度为1.5至4倍，p<0.01）。无论成人还是胎儿样本，巩膜在后极部与周边眼区的miRNA表达谱均未观察到显著差异。

结论：本研究首次对人类巩膜中的miRNA表达进行了系统性编目。巩膜可表达多种miRNAs，其中部分呈现年龄相关的差异调控模式：在快速生长的胎儿巩膜中表达水平更高，这与其在眼球生长调控中发挥作用的假说相符。本研究结果可为关联巩膜miRNA表达与相关疾病的潜在干预手段提供参考，例如巩膜扩张症/轴性近视。
本研究获取了正常人类胎儿（24周龄）与正常成人供体眼球的巩膜样本（每组样本量n=4~6），并提取总RNA。全基因组miRNA表达谱分析采用安捷伦（Agilent）miRNA微阵列平台。miRNA靶基因预测通过Microcosm、TargetScan及PicTar算法完成。