突变RUNX2调控的miRNAs参与颅骨锁骨综合征发病机制的研究

Cleidocranial dysplasia (CCD; OMIM# 119600) is a rare congenital disorder, which usually has an autosomal dominant mode of inheritance. The main cause of CCD is heterozygous mutations in RUNX2, the homology gene of Runx2. The clinical manifestations of CCD are mainly related to the bones and teeth. The typical features of patients with CCD include hypoplasia or aplasia of clavicles, patent fontanels, wormian bones, short status and several other changes. Multiple supernumerary teeth are present, the development and eruption of permanent teeth are severely delayed, cellular cementum is virtually absent and acellular cementum is partially hyperplastic. Runx2 is a master transcription factor involved in development of bone and tooth. Homozygous mutants die of respiratory failure shortly after birth. Analysis of their skeletons revealed an absence of osteoblasts and bone. Heterozygous mice showed specific skeletal abnormalities that are characteristic of the human heritable skeletal disorder, cleidocranial dysplasia (CCD). .MicroRNAs (miRNA or miR) are endogenous, small, noncoding 22nt RNAs that regulate gene expression by binding to the 3’untranslated region (3’UTR) of mRNAs to repress translation or induce mRNA cleavage. microRNAs are involved in the regulation of a variety of biological events, including organ development, virus defense, hematopoiesis, organogenesis, cell proliferation and apoptosis, fatty metabolism and so on. Gene expression regulation is a complex biological process, which is controlled by various factors including proteins and RNAs in multiple levels. As key regulators of gene expression, transcription factors (TFs) and miRNAs are able to co-regulate the expression of targets in forms of feed-forward loops (FFLs) and feedback loops (FBLs) .These regulatory loops serve as important motifs in gene regulatory networks and play critical roles in multiple biological processes and different diseases. Major progress has been made in bioinformatics and experimental study for the TF and miRNA co-regulation in recent years..Since Runx2 is an important transcription factors, how does it regulate miRNAs during the development of bone and teeth? Does a co-regulate network exist between Runx2 and miRNAs? If the function of mutation of Runx2 was disrupted, how will it influence the whole network? To elucidate these questions, we choose three point mutation sites which were detected in CCD patients and constructed three expression vectors. We overexpressed them in normal ameloblast-like LS8 cells and MC3T3-E1 pre-osteoblasts, respectively. Using miRNA expression microarrays, we identified the different expression profiles between wild type and mutated Runx2 groups and try to verify the roles of the representative miRNAs (eg. MiRNA-185-5p) involved in bone and dental enamel development. We also attempt to establish the co-regulate network between Runx2 and miRNAs and give the new clues for the diagnosis and therapy of related disease.

RUNX2是在骨和牙齿的发育过程中起至关重要作用的转录因子，其突变可导致罕见的常染色体显性遗传病颅骨锁骨发育不全综合征（CCD）。miRNAs是基因转录后的重要调控分子，在多种生命过程中起重要的调控作用，转录因子和miRNA之间往往可通过相互作用更精准地调节基因表达。作为重要的转录因子，RUNX2在调控骨和牙齿发育中如何与miRNAs共同协调靶基因？如果发生突变而功能受损，相应的调控网络又有何种变化？运转机制如何？我们选取了位于不同结构域的3种点突变，以小鼠同源序列为背景构建质粒，在前成骨细胞MC3T3-E1和成釉细胞LS8中过表达，比较不同突变分子引起的miRNAs表达谱变化，并挑选显著变化的代表性miRNAs（如miRNA-185-5p）在体内外进行功能和机制研究，为深入了解RUNX2与miRNAs相互调节的机制，揭示骨和牙齿发育及CCD表型形成的机制及相关疾病的诊断治疗提供新线索。

RUNX2是骨和牙齿的发育的主导转录因子，其突变可导致罕见的常染色体显性遗传病颅骨锁骨发育不全综合征（Cleidocranial dysplasia，CCD）。miRNAs是重要的基因转录后调控分子，在多种生命过程中发挥调控作用，RUNX2突变后是否可以造成miRNA的表达改变，进而影响骨及牙齿发育未见深入研究。本项目成功构建了三个Runx2突变质粒，证明突变分子可抑制成釉细胞成釉能力和前成骨细胞的成骨分化和矿化能力；采用过表达突变Runx2体系，通过miRNA芯片筛选得到了Runx2突变后成釉细胞和成骨细胞中的miRNA差异表达谱，其中miR-185-5p在成釉细胞和前成骨细胞中都出现了高倍上调表达，生物信息学预测及体外实验证明Dlx2为其靶基因，miR-185-5p可通过降低Dlx2表达而抑制成釉细胞成釉分化和前成骨细胞成骨分化及矿化功能；利用CRISPR/Cas9技术构建了mmu-miR-185 基因全身敲除（KO）小鼠，发现KO小鼠生长和发育未见异常，骨骼和牙齿无自发异常表型；但KO原代颅骨细胞增殖能力高于WT小鼠，其成骨分化和矿化能力增加；KO小鼠BMSCs成骨向分化能力增加，成骨晚期矿化水平增加。KO小鼠软骨细胞增殖能力高于WT细胞，但软骨细胞成骨分化与WT小鼠无明显差异。在小鼠双侧卵巢切除骨质疏松模型中， KO小鼠骨量流失少于野生鼠。通过尾静脉注射miR-185-5p类似物， KO小鼠的骨质疏松抑制现象消失。证实Bgn为其靶基因，MiR-185敲除引起小鼠骨组织及BMSCs中BGN表达上升，进而激活BMP/Smad信号通路，促进骨生成。在单层皮质骨缺损模型中，KO小鼠骨髓腔新生骨增加多于WT小鼠。本项目还选取了单独在成釉细胞中出现表达下调的miRNA-1963进行体外实验，生物信息学预测及实验证明Smoc2是其靶基因，miRNA-1963可通过抑制Smoc2表达抑制成釉分化。本项目也证明了参与骨形成和骨稳态的重要基因Pth是miR-185-5p的靶基因。以上结果表明，突变Runx2可以通过调控多种miRNAs的表达影响骨和牙齿的形成，而miRNAs也可通过多种靶基因形成复杂的分子网络参与和影响多种组织器官发育及稳态，为揭示CCD的病因机制提供了新的依据；鉴于骨形成及稳态维持是机体终生动态过程，本项目结果也为骨质疏松症等代谢性骨病的治疗干预提供了新线索。