骨关节炎(OA)是第二常见的关节炎,但OA的致病机制和调节机制仍不完全清楚。这里,我们的目的是使用蛋白质组学方法,在啮齿动物OA模型中鉴定与microRNA-1(miR-1)治疗OA相关的机制.首先,N=18SpragueDawley(SD)大鼠接受假手术(n=6)或ACL横切(n=12),随后每隔一周随机分配ACL横断组,以关节内给药50µL安慰剂(对照组)或miR-1agomir,模拟内源性miR-1(实验组)。经过八周的重塑,收集关节软骨组织并对MMP-13进行免疫组织化学染色。第二,N=30Col2a1-cre-ERT2/GFPf1/fl-RFP-miR-1转基因小鼠随机分配给任一安慰剂(对照组,N=15)或他莫昔芬,miR-1表达的诱导剂(实验组,N=15),在间隔五天后接受内侧半月板(DMM)手术破坏之前。经过八周的重塑,收集关节软骨组织并进行差异蛋白质组学分析。具体来说,采用串联质量标记(TMT)定量蛋白质组学分析鉴定组间差异表达蛋白(DEP),使用实时定量聚合酶链反应(RT-qPCR)技术对选定的DEP进行验证。免疫组织化学检测MMP-13在实验组大鼠中表达显著降低,小鼠组织匀浆的蛋白质组学分析表明,在3526种鉴定的蛋白质中,实验组中差异表达(相对上调和下调)345份。蛋白质Fn1,P4ha1,P4ha2,Acan,F2,Col3a1,Fga,Rps29、Rpl34和Fgg是十大最相关的蛋白质,这意味着miR-1可能调控涉及这些蛋白质的表达网络。在这十种蛋白质中,通过RT-qPCR选择三个用于进一步验证:已知与OA相关的Fn1的转录物,实验组表现出相对上调,而P4ha1和Acan的转录物表现出相对下调。因此,这些蛋白质可能代表OA调控机制中的关键miR-1靶标,并可能提供有关miR-1在OA背景下的治疗机制的其他见解。
Osteoarthritis (OA) is the second-commonest arthritis, but pathogenic and regulatory mechanisms underlying OA remain incompletely understood. Here, we aimed to identify the mechanisms associated with microRNA-1 (miR-1) treatment of OA in rodent OA models using a proteomic approach. First, N = 18 Sprague Dawley (SD) rats underwent sham surgery (n = 6) or ACL transection (n = 12), followed at an interval of one week by randomization of the ACL transection group to intra-articular administration of either 50 µL placebo (control group) or miR-1 agomir, a mimic of endogenous miR-1 (experimental group). After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and immunohistochemically stained for the presence of MMP-13. Second, N = 30 Col2a1-cre-ERT2 /GFPf1/fl -RFP-miR-1 transgenic mice were randomized to intra-articular administration of either placebo (control group, N = 15) or tamoxifen, an inducer of miR-1 expression (experimental group, N = 15), before undergoing surgical disruption of the medial meniscus (DMM) after an interval of five days. After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and underwent differential proteomic analysis. Specifically, tandem mass tagging (TMT) quantitative proteomic analysis was employed to identify inter-group differentially-expressed proteins (DEP), and selected DEPs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) technology. Immunohistochemically-detected MMP-13 expression was significantly lower in the experimental rat group, and proteomic analyses of mouse tissue homogenate demonstrated that of 3526 identified proteins, 345 were differentially expressed (relative up- and down-regulation) in the experimental group. Proteins Fn1, P4ha1, P4ha2,
Acan, F2, Col3a1, Fga, Rps29, Rpl34, and Fgg were the *top ten most-connected proteins, implying that miR-1 may regulate an expression network involving these proteins. Of these ten proteins, three were selected for further validation by RT-qPCR: the transcript of Fn1, known to be associated with OA, exhibited relative upregulation in the experimental group, whereas the transcripts of P4ha1 and
Acan exhibited relative downregulation. These proteins may thus represent key miR-1 targets during OA-regulatory mechanisms, and may provide additional insights regarding therapeutic mechanisms of miR-1 in context of OA.