一个新克隆的microRNA在甲状旁腺发育及甲状旁腺腺瘤发病中的作用及机制研究

We cloned a novel tissue specific expressing miRNA(temporarily named miR-A) in parathyroid gland in early and middle embryo. The exprssion of miR-A decreased in early embryo and postnatal, whereas miR-A was highly expressed in parathyroid adenoma. Therefore, we speculated that miR-A promoted the development of parathyroid gland in early and middle embryo. Moreover it was associated with the mechanism of development of parathyroid adenoma. The aim of our study is to prove this hypothesis. First, we induce the parathyroid precursor cells to form mature cells by culture mediums without calcium which are consist of serum. During this stage, by regulating the expression of miR-A through gene transfection strategy, we observe the changes in parathyroid cells differentiation and verify the target gene of miR-A is ZEB1 and further clarify the function of miR-A in parathyroid cells differentiation. Second, in order to analyze the function of miR-A in parathyroid development and discuss if highly expressed miR-A result in ZEB1 down-regulating which help to reveal the mechanism of onset of parathyroid adenoma, we construct over/low-expressed miR-A animal models in different stages by interfering different embryo stages and postnatal mice with agomirs/antagomirs. This research contribute to clarify the mechanism of parathyroid development and enable to find preclusive and therapeutic approaches for the parathyroid adenoma.

我们首次克隆了一个新的甲状旁腺特异性表达的miRNA（暂命名为miR-A），其在胚胎早期及出生后甲状旁腺表达下降，而在胚胎中晚期及甲状旁腺瘤中高丰度表达。故推测miR-A与胚胎中晚期甲状旁腺分化发育及高分化甲状旁腺瘤发病相关。本课题拟证实此设想。首先诱导甲状旁腺前体细胞向成熟细胞分化，在此过程中运用基因转染策略调控miR-A表达，观察miR-A表达改变对甲状旁腺细胞分化的影响，并验证ZEB1为miR-A靶基因，阐明miR-A在甲状旁腺细胞分化过程中的作用。在体内，用agomirs/antagomirs干预不同胚胎期小鼠，构建miR-A过表达或缺失动物模型，解析miR-A在甲状旁腺发育中的作用。进而在NOD/SCID鼠甲状旁腺瘤模型中促进或抑制miR-A表达，探讨miR-A在高分化腺瘤发生中的机制。本研究有利于阐明甲状旁腺发育机理，为甲状旁腺瘤的防治拓展新思路。

microRNA是在真核生物和一些病毒中发现的小的非编码RNA分子（含有约22个核苷酸），其在RNA沉默和基因表达的转录后调节中起作用。已有文献报导甲状腺、甲状旁腺和胸腺有近似的胚胎期起源，来自第三咽囊和第四咽囊。我们选取SD大鼠的甲状旁腺、甲状腺、胸腺做miRNA基因芯片，分析其miRNA表达谱。根据miRNA的差异表达,明确哪些miRNA在甲状旁腺生长发育过程中上调或者下调，并筛选出人与大鼠同源性的miRNA，代入人的甲状旁腺标本中行RT-qPCR验证表达情况。由于伦理学的限制，我们无法从临床上获得人类正常甲状旁腺，故选取甲状旁腺增生标本作为对照组。. 经鉴定，miRNA-21-5p在人类甲状旁腺瘤标本中较甲状旁腺增生标本表达量增高，通过软件预测下游靶基因，选取TIMP-3作为miRNA-21-5p的靶基因。. 基质金属蛋白酶组织抑制因子3(TIMP-3)是组织金属蛋白酶抑制物家族成员,它能与基质金属蛋白酶(MMPs)形成复合物,下调MMPs，降解细胞外基质的活性,抑制肿瘤血管生成,从而抑制癌细胞的侵袭与转移。已有文献证实TIMP-3在胃癌，乳腺癌等癌症转归中起作用。在免疫组化切片中，观察到人类甲状旁腺增生组TIMP-3的含量高于甲状旁腺瘤组。证明了甲状旁腺瘤的发生机制之一是因为miRNA-21-5p的上调，使靶基因Timp-3的活性降低。这提示我们可能通过抑制miRNA-21-5p的上调，从而保持TIMP-3的活性，这为甲状旁腺瘤的治疗提供了新的途径。