Comparative Analysis of the Ultrastructure Between Discoid and Normal-Shaped Lateral Meniscus

Abstract

Background: Discoid lateral meniscus (DLM) has long been observed and diagnosed in clinic, whereas its etiology and pathology remain unclear. The aim of this study is to investigate the difference of ultrastructure as regard to cell function and protein expression profile between DLM and normal-shaped lateral meniscus (NLM). Methods and Results: By using the iIsobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analysis was performed to detect the difference of protein profiling between DLM and NLM. Expression of the representative differentially expressed proteins (DEPs) was validated by western blotting and immunohistochemistry (IHC) assays. Gene ontology (GO) enrichment analysis and predicted interaction network were conducted to investigate the biological processes involved. Meanwhile, meniscus cells were isolated from DLM and NLM tissues and processed for proliferation assay and gene expression analysis. Findings: 1314 proteins in total were identified in the inner 2/3 part of DLM and normal-shaped lateral meniscus (NLM) specimens. 21 of them were up-regulated while 4 were down-regulated in the DLM group. Western blotting and IHC assays validated increased Expression expression of fibronectin (FN), periostin (POSTN) and, type IV collagenase (MMP-2) and while decreased expression of hemoglobin subunit beta (HBB) were validated byin DLM compared to those in NLM. western blotting and immunohistochemistry (IHC) assays. Gene ontology (GO) enrichment analysis and predicted interaction network of the differentially expressed proteins (DEPs) indicated that the DEPs were primarily involved in the molecular function of binding and transporting, mediating biological processes such as extracellular matrix (ECM) remolding, response to stimulus and angiogenesis. Proliferation potential Meniscus cells fromof DLM cells was demonstrated to be higher proliferation potential than those fromthat of NLM cells. Compared to mRNA expression in NLM cells, eExpression level of collagen I and aggrecan mRNAs were higher while expressionthat of collagen II and osteocalcin (OC) mRNAs were lower in DLM cells than in NLM cells. No significant difference of mRNA expression of FN, integrin V, integrin 1, POSTN, MMP-2 and HBB was observed between two groups. Interpretation: Ultrastructure of DLM was quite different from that of NLM. Cell proliferation potential and mRNA expression of aggrecan and collagen I were increased in DLM, synthesis of DEPs such as FN, POSTN and MMP-2 were increased while HBB was decreased. In addition, biological processes such as ECM remolding, response to stimulus and angiogenesis had been abnormally activated in DLM. Funding Statement: This study was supported by the National Natural Science Foundations of China (81501852, 81472046, 81271942, 81802200, 81472045), by the Distinguished Youth Foundation of Peking Union Medical College Hospital (JQ201506), by the Beijing nova program (2016), by the Central Level Public Interest Program for Scientific Research Institute (No.13, 2015), by the Basic Scientific Research Business Project of Central Public-interest Scientific Institution of Chinese Academy of Medical Sciences (2015PT320013), and by the National Key R&D program of China (2018YFF0301105). Declaration of Interests: All the authors declare no conflicts of interests. Ethics Approval Statement: Written informed consents were received from all the patients prior to their participation in the study. This study was approved by the Ethical Committee of Clinical Investigation of PUMCH.