Suppression of SHH Expression in Arthritis by Butea monosperma

描述（由申请人提供）：骨关节炎（OA）是最常见的肌肉骨骼疾病，唯一有效的治疗方法是手术关节置换。MicroRNA（miRNA）是一类调控基因表达的非编码RNA。特定的miRNAs在OA发病机制中的作用尚未确定。我们的初步结果表明，Hsa-MIR-323 B-5 p（miR-323 b-5 p），没有已知的功能，在IL-1？刺激的软骨细胞中下调了几倍。计算机模拟分析将Sonic Hedgehog（SHH）mRNA鉴定为miR-323 b-5 p的靶标。SHH信号与MMP-13的表达和软骨退变相关，抑制SHH可减轻OA的严重程度。Butea monosperma（Lam）在印度广泛分布，Butea monosperma花的水提取物（BME）用于治疗关节炎。我们的初步结果表明，IL-1？刺激OA软骨细胞中SHH的表达。值得注意的是，BME调节SHH信号传导基因的表达，并阻断SHH诱导的软骨外植体中MMP-13的表达，并且miR-323 b-5 p通过直接靶向mRNA中的编码区来抑制SHH蛋白表达。我们建议在体外和体内使用描述良好的测定和OA的临床前动物模型来测试BME的软骨保护活性。我们的基本假设是“BME通过调节人软骨细胞中miR-323 b-5 p的表达，通过SHH蛋白表达的转录后调节，抑制IL-1？诱导的OA软骨分解代谢作用”。该假说的推论是“BME的生物活性成分可通过调节特异性miRNA的表达在OA中发挥其软骨保护作用，所述特异性miRNA在体内负调节SHH和其他分解代谢因子的表达”。具体目标1：测定（a）BME对IL-1 β诱导的SHH及其受体PTCH 1表达的影响;和（B）BME对体外人OA软骨细胞和软骨外植体中SHH诱导的PTCH 1、GLI-1和MMP-13表达的影响。使用微阵列分析，我们还将（c）鉴定其他miRNAs 其表达在人OA软骨细胞中受IL-1调节，并生物信息学地确定额外的miRNA是否靶向SHH mRNA;和（d）使用报告基因测定验证新鉴定的额外的miRNA与SHH mRNA的相互作用。具体目标2：我们将分析IL-1对miR-323 b-5 p表达改变的软骨细胞中mRNA表达谱（转录组）的影响，并确定表达改变的基因是否也是miR-323 b-5 p的靶基因。具体目标3：使用兔OA模型，我们将表征疾病诱导和进展期间关节中SHH和miRNA的表达谱。将对关节软骨进行肉眼和组织学评价。将分析滑液和血清的（a）炎性细胞因子水平（IL-1、TNF-α、IL-6）;（B）分泌的MMP-2、MMP-9、MMP-13的表达;以及（c）在给予两种不同剂量的BME和异丁酸甘油酯的动物中，我们将测定BME组分的水平我们还将检查BME和异丁酸甘油酯消耗对miR-323 b-5 p表达水平的影响，MMP-13和SHH mRNA和蛋白在关节中的表达与疾病的诱导和进展相关。

DESCRIPTION (provided by applicant): Osteoarthritis (OA) is the most common musculoskeletal disorder and the only effective treatment is surgical joint replacement. MicroRNAs (miRNA) are a class of non-coding RNAs regulating gene expression. Role of specific miRNAs in OA pathogenesis is yet to be defined. Our preliminary results showed that Hsa-MIR-323B-5P (miR-323b- 5p), with no known function, was downregulated several fold in IL-1¿-stimulated chondrocytes. In silico analysis identified Sonic Hedgehog (SHH) mRNA as a target of miR-323b-5p. SHH signaling is associated with the expression of MMP-13 and cartilage degeneration and inhibition of SHH attenuates the severity of OA. Butea monosperma (Lam) is widely distributed in India and water extract of Butea monosperma flowers (BME) is used to treat arthritis. Our preliminary results show that IL-1¿ stimulates the expression of SHH in OA chondrocytes. Of note, BME modulated the SHH signaling genes expression and blocked the SHH-induced expression of MMP-13 in cartilage explants and that miR-323b-5p inhibited SHH protein expression by directly targeting coding region in the mRNA. We propose to test the cartilage protective activity of BME in vitro and in vivo using well described assays and a preclinical animal model of OA. Our basic hypothesis is that "BME suppresses the IL-1¿-induced cartilage catabolic effects in OA via post-transcriptional regulation of SHH protein expression by modulating the expression of miR-323b-5p in human chondrocytes". A corollary of this hypothesis is that "bioactive constituents of BME may exert their cartilage protective effects in OA by modulating the expression of specific miRNAs that negatively regulate the expression of SHH and other catabolic factors in vivo". Specific Aim-1: Determine (a) the effect of BME on IL-1¿- induced expression of SHH and its receptor PTCH1; and (b) the effect of BME on the SHH-induced expression of PTCH1, GLI-1, and MMP-13 in human OA chondrocytes and cartilage explants in vitro. Using microarray profiling we will also (c) identify additional miRNAs whose expression is modulated by IL-1¿ in human OA chondrocytes and bioinformatically determine if additional miRNAs target SHH mRNA; and (d) validate the interactions of newly identified additional miRNAs with SHH mRNA using reporter assays. Specific Aim-2: We will analyze the effect of IL-1¿ on the mRNA expression profile (Transcriptome) in chondrocytes with altered miR-323b-5p expression and determine whether the genes with altered expression are also targets of miR-323b-5p. Specific Aim-3: Using a rabbit model of OA we will characterize the expression profile of SHH and of the miRNAs in the joints during disease induction and progression. The articular cartilage will be evaluated macroscopically and histologically. Synovial fluid and serum will be analyzed for (a) levels of inflammatory cytokines (IL-1¿, TNF-¿, IL-6); (b) expression of secreted MMP-2, -9,-13; and (c) in animals given two different doses of BME and Isobutrin we will determine the levels of BME constituents (Butein, Butrin, Isobutrin) by LC/MS. We will also examine the effect of BME and Isobutrin consumption on the expression levels of miR-323b-5p, MMP-13 and SHH mRNA and protein in the joints and correlate with disease induction and progression.