Mir-134/487b/668基因簇与PTEN介导的信号通路在恶性纤维组织细胞瘤中的作用及机制研究

Malignant fibrous histiocytoma (MFH) is a soft tissue tumor of unknown etiology. In our preliminary study, we found that there was an extra marked chromosome in MFH cells, which were formed by chromosome 14q translocation; Microarray analysis showed mir134/487b/668 gene cluster showed high expression and they located in the long arm of chromosome 14. Literatures have confirmed that the target gene MAGI2 of mir-134/487b serves as scaffold protein of PTEN, and we also found that PTEN expression was low in MFH. We hereby put forward the following hypothesis: extra marked chromosome was formed by the long arm of chromosome 14 translocation, so mir-134/487b/668 gene cluster, which located in it, showed abnormal expression, its target gene MAGI2 inhibits expression of PTEN, so Triggering PTEN-induced signal pathway abnormalities, which can cause tumorigenesis. For the hypothesis, we use MFH cell line, RNA interferencing technology, transfect gene cluster fragments mimetics and inhibitors to MFH cells, detect MAGI2/PTEN signal networks changes, observe MFH cell function changes in vivo and in vitro, and find mir-134/487b/668 gene cluster function and mechanism on MFH.

恶性纤维组织细胞瘤（MFH）是一种病因不明的肿瘤。申请者前期研究发现MFH存在由14号染色体长臂转位形成的额外标记染色体；芯片分析显示mir134/487b/668 基因簇高表达且位于14号染色体长臂。文献证实mir-134/487b的靶基因是PTEN的支架蛋白MAGI2；mir-668的靶基因预测为PTEN，我们也发现PTEN在MFH呈低表达。据此提出以下假设：MFH由于14号染色体长臂转位形成额外染色体，可能导致位于其上的mir-134/487b/668基因簇高表达，靶向抑制支架蛋白MAGI2及PTEN基因，从而引发PTEN介导的多条信号通路异常，导致MFH的发生。针对假设申请者拟利用MFH细胞系，运用RNA干扰技术双向调节基因簇表达，检测MAGI2/PTEN信号网络变化，体内外观察MFH细胞功能变化，探讨mir-134/487b/668基因簇在MFH的功能及作用机制。

课题组对前期实验基因芯片检测数据进行了系统分析，建立了MiroRNA-Pathway网络，找到差异表达的microRNA（7个microRNA及12个相关靶基因）。课题组采用ALDH免疫标记恶性纤维组织细胞瘤（MFH）细胞，流式细胞仪分选出MFH中的ALDH+细胞与ALDH-细胞，运用Real-time PCR对上述的miRNA和靶基因进行了验证。结果显示ALDH+与ALDH-细胞中12个靶基因中有4个（MAP3K1，PIK3R3，NRAS，AKT3）变化显著，而检测的MIRNA均呈现明显变化。针对PTEN，MAGI2以及软组织肉瘤相关信号通路的关键因子FAK，NF-kb，ERK，AKT进行了蛋白水平检测，发现PTEN，p-AKT，EKR的蛋白表达降低。课题组针对差异表达miRNA，运用RNA干扰技术，分别制作了miRNA的模拟物和抑制剂，采用Lipofectamin2000转染MFH细胞，观察其对细胞增殖侵袭能力的影响。结果显示mir-206对肿瘤细胞的增殖活性具有抑制作用，而其他miRNA，包括Mir-134/487b/668基因簇对MFH细胞增殖的影响不明显。所有miRNA对细胞侵袭能力没有明显抑制。细胞耐药实验采用两种化疗药物多西他赛和吉西他滨分别对MFH细胞处理，获得相应的耐药细胞株。运用real-time PCR检测耐药细胞中关键miRNA的表达变化。结果发现多西他赛耐药细胞中有6个miRNA下调超过0.5倍，1个miRNA上调超过2倍。然而在吉西他滨耐药细胞中只有3个miRNA变化明显。这可能是细胞对不同药物产生的耐药机制不同有关。结论：mirR-206在MFH细胞中呈高表达，并对肿瘤细胞增殖活性具有抑制作用。在多西他赛耐药细胞中miR-134，miR-206，miR-302e，miR-340-5p，miR-487b-3p，miR-767-5p都有明显下调，可能参与多西他赛耐药过程，具有靶向调控潜力。MFH的病理过程可能与PI3K/AKT信号通路和ERK/MAPK信号通路的异常有关。