miR-143/HDAC7 pathway 通过调控组蛋白乙酰化改变影响骨肉瘤转移特性的分子机制研究

The development of early metastasis is the major cause of death in osteosarcoma, and the mechanisms that orchestrate the multiple oncogenic insults required for metastatic activity of osteosarcoma are urgent in current studies. To date, epigenetic regulation, for example, histone acetylation has been suggested to participate in osteosarcoma development. However, we are still at the beginning of revealing the roles of histone deacetylases in osteosarcoma carcinogenesis and progression. Here, we focused on the roles of histone deacetylase (HDAC) 7, which is an important regulator of histone acetylation and epigenetic modification, in metastatic osteosarcoma. In our previous study， we found miR-143 was down-regulated in osteosarcoma cell lines and primary tumor samples, and the restoration of miR-143 reduced cell viability, promoted cell apoptosis and suppressed metastatic activity. Interestingly, we found that HDAC7 was over-expressed in metastatic osteosarcoma, and HDAC7 identified to be a potential novel target of miR-143. Additionally, And the overexpression of HDAC7 CDS without 3'UTR region partly rescued the influence of miR-143 on the osteosarcoma cell line U2OS. In our current proposal, we plan to test the hypothesis that the miR-143 regulates the metastatic ability of osteosarcoma cell lines through a HDAC7-dependent manner. Then we try to scan for the specific histone acetylation sites in the osteosarcoma cells genome and investigate their roles in osteosarcoma metastasis. Thus, our current proposal may find a novel mechanism of osteosarcoma metastasis through a mir-143/HDAC7 pathway and indicate its potential application in cancer therapy.

骨肉瘤早期转移是影响临床预后的关键因素，转移发生分子机制受广泛关注，组蛋白去乙酰化酶在肿瘤转移中的调控作用及其机制值得深入研究。本项目组在前期研究中发现了microRNA miR-143可以显著影响骨肉瘤细胞迁移能力，进一步研究发现该microRNA可以靶向调控HDAC7，且HDAC7过表达可以补救miR-143对迁移能力的影响，提示miR-143/HDAC7 pathway可能在骨肉瘤转移中发挥重要作用。本项目组拟首先通过基因过表达和干扰、细胞迁移和侵蚀能力检测、小动物活体成像等技术在体内外充分验证miR-143/HDAC7对骨肉瘤的迁移能力的影响，并进一步利用ChIP-on-chip技术分析miR-143通过HDAC7影响的特异性组蛋白乙酰化位点，从而系统地探讨microRNA及组蛋白乙酰化酶协同调控骨肉瘤转移的新的表观遗传学机制，并为骨肉瘤的临床诊断和分子靶向治疗提供新的研究靶点。

影响骨肉瘤预后的主要因素为骨肉瘤的早期转移，但是其机制和分子特征尚未清楚，缺乏早期诊断和预后评价指标。近年来，microRNA在肿瘤转移机制和早期诊断，基因治疗领域上的研究进展为这一问题的解决提供了新的思路。鉴于近年来骨肉瘤转移能力的重要发现，我们从芯片数据中选取差异低表达基因miR-133a、miR- 422a及高表达基因miR-148a进行功能学研究。通过细胞培养、RNA抽提、RT-PCR、转染、western blot检测、细胞增殖实验（MTT）、凋亡分析等一系列实验方法，得出结论如下：.1、miR-133a在骨肉瘤发生发展中发挥重要作用并可能作为新的骨肉瘤预后判断临床指标。miR-133a是一个新的骨肉瘤抑癌基因，对骨肉瘤发生发展有的抑制作用。miR-133a能够通过抑制凋亡抑制基因Bcl-xL和Mcl-1的表达进而抑制骨肉瘤细胞的增殖并诱导凋亡。miR-133a作为抑癌基因在骨肉瘤发生发展中的作用，并为骨肉瘤的预后判断和临床治疗提供了新的潜在靶点。.2、miR-148a在骨肉瘤细胞和临床样本中呈显著高表达；miR-148a显著高表达和骨肉瘤患者的临床分级及预后生存密切相关；miR-148a在体内外均可促进骨肉瘤细胞增殖；miR-148a可通过靶向抑制PTEN的表达，激活PI3K 信号转导通路。miR-148a可以靶向抑制抑癌基因PTEN的表达，从而激活PI3K信号通路，在骨肉瘤中起到促进细胞增殖的作用。.3、miR-422a由于组蛋白H3K9me3、H3K27me3的甲基化与H3K4me3的去甲基化引起在骨肉瘤中下调表达；miR-422a 通过抑制抗凋亡作用以及阻遏细胞在G1/G0期，从而抑制肿瘤的生长；miR-422a靶向Bcl2l2与Kras发挥抑制骨肉瘤生长的作用。. 通过此项研究我们可获取合适的肿瘤转移相关microRNA做为骨肉瘤转移的.早期诊断和预后评价指标，同时对microRNA调节的骨肉瘤转移机制进行深入研究、探讨，为骨肉瘤转移的早期诊断、临床治疗以及预后评价等领域提出新的研究方向。