RNA-seq and Microarray in Transcriptome Profiling of Anterior Cruciate Ligament Tears: Implications for Prognostic Biomarkers Discovery

Injury to anterior cruciate ligament (ACL) is common in young individuals and a frequent cause of functional instability and early onset of osteoarthritis. The healing potential of an injured ACL is known to decay over time. The molecular origin of this healing deficiency largely remains elusive but plausibly involves gene transcripts associated with tissue healing. To explore this possibility, we set out to identify transcript expression differences in injured ACL remnants recovered at the time of surgical reconstruction, via microarray (n=24) and RNA-seq (n=8) technologies in transcriptome profiling. We found that time-from-injury was an important determinant of changes in gene expression signatures predominately resulting in repression of several biological processes as identified by gene ontology. The most interesting observation was a time-dependent decline in the gene transcripts as well as the biological processes common to both microarray and RNA-seq analyses. Compared to acute tears, in chronic several important biological processes were namely extracellular matrix organization, angiogenesis, cell adhesion, wound healing, mesenchyme transition, and response to hypoxia. Furthermore, the cross-platform concordance in terms of differentially expressed transcripts or enriched pathways was linearly correlated (r=0.64). Microfluidic digital PCR confirmed the expression of selected differentially expressed transcripts. These intriguing findings suggest an initial attempt of the injured ACL to repair, which drops with time. These findings have implications for efforts to repair the ACL and may be relevant for its reconstruction. These findings also emphasize the utility of differentially expressed transcripts as prognostic biomarkers in patients with ACL injury. Overall design: Examination of transcript expression differences by time-from-injury in anterior cruciate ligament

前交叉韧带（anterior cruciate ligament, ACL）损伤在年轻群体中十分常见，亦是引发功能不稳与骨关节炎早发的常见病因。已知损伤ACL的愈合潜力随时间推移逐渐衰减，此类愈合缺陷的分子根源在很大程度上仍未明确，但推测可能涉及与组织愈合相关的基因转录本。为探究该可能性，我们借助转录组分析中使用的微阵列（microarray，n=24）与RNA测序（RNA-seq，n=8）技术，对手术重建时获取的损伤ACL残端的转录表达差异进行鉴定。研究发现，损伤至手术的时间间隔是基因表达特征变化的重要决定因素，该变化主要表现为经基因本体（gene ontology, GO）注释所鉴定的多项生物学过程受到抑制。最引人关注的结果是，微阵列与RNA-seq分析共同得到的基因转录本及生物学过程均随时间呈下降趋势。与急性撕裂伤相比，慢性损伤中显著富集的重要生物学过程包括细胞外基质组织、血管生成、细胞黏附、伤口愈合、间质转化以及缺氧应答。此外，差异表达转录本或富集通路的跨平台一致性呈线性相关（r=0.64）。微流控数字PCR验证了部分筛选出的差异表达转录本的表达水平。这些有趣的发现表明，损伤的ACL最初存在修复尝试，但该修复能力随时间推移逐渐减弱。本研究结果对ACL修复相关研究具有参考价值，也可能为其重建手术提供指导。此外，本研究还强调了差异表达转录本作为ACL损伤患者预后生物标志物的应用潜力。整体实验设计：基于损伤至手术的时间间隔，探究前交叉韧带的转录表达差异。