长非编码核糖核酸LncRNA-Dnm3os调节细胞生长与软骨发育的作用与机制

发布时间：2019-05-16 03:34

【摘要】：Dnm3os是由位于1号染色体上,嵌合在Dynamin 3基因内含子中一个独立转录单位所编码的长非编码RNA(long noncoding RNA,or lncRNA)。包括出自本实验室在内的以往数据显示,Dnm3os所携带的两个微型RNA之一,miR-214,可以与原癌基因N-Ras形成一个调控环路,通过加速细胞从分裂期逃逸,从而促进成肌母细胞沿肌纤维方向或在BMP的作用下沿骨方向定向分化。我博士论文研究发现miR-214虽然在胚胎发育中不具必要的作用,但是却可以促进成年肌肉的损伤修复,在胚胎成纤维细胞中也具有抑制细胞增殖的作用。我还进一步发现miR-214调节细胞生长的作用还体现在抑制儿童中常见的软组织恶性肿瘤,横纹肌肉瘤(rhabdomyosarcma,RMS)发生方面。这种肿瘤主要是由肌肉前体细胞的恶性增殖与低分化所造成的,因此与miR-214的作用原理有密切关系。我发现miR-214在人横纹肌肉瘤细胞系中明显低表达,在人胚胎型横纹肌肉瘤细胞系(RD)中过表达miR-214可以显著抑制其增殖,退出细胞周期而开始肌分化。裸鼠体内荷瘤实验也证实了miR-214抑制RD细胞在体移植成瘤的能力。通过对N-ras 3’端非转录区域(3’-UTR)中miR-214结合位点的突变分析,我再一次证实了miR-214和N-ras调节环路在细胞生长与RMS发生中的重要作用。。Noonan综合征是一种常见的常染色体显性遗传疾病,患者具有特定的面部表征,通常身材矮小,先天性心脏缺陷,以及认知障碍等临床症状。由于导致Noonan综合征的已知遗传因素都撁集在RAS/MAPK信号通路,因而这一综合症也称为RAS病(RASopathies)。通过与美国明尼苏达州儿童医院合作,我们发现了一位由母系遗传而来的Noonan综合征病童。没有发现RAS/MAPK通路基因突变,但是比较基因组杂交(comparative genomic hybridization,CGH)显示1号染色体1q24.3区域内一个690kb狭小片段缺失,涵盖DYNAMIN3,嵌其中的LncRNA-Dnm3os,以及PIGC,clorf105,clorf9以及FSALG 4个基因。利用qPCR,我发现该家系受影响的成员外周血中NS特征性基因的表达明显上升;过表达Dnm3os可以抑制NS特征性基因的表达。虽然生物信息学分析6个Noonan综合症致病基因是miR-214的靶基因,但是miR-214敲除小鼠并没有出现相应症状,提示Dnm3os全片段的缺失与Noonan综合症发生密切相关。结合已经发表的LncRNA-Dnm3os敲除小鼠表型数据,我们推测LncRNA-Dnm3os在骨骼软骨内钙化发育过程中起关键性作用。进一步实验发现,在软骨内钙化成骨过程中LncRNA-Dnm3os促进软骨细胞的增殖抑制其分化,维持增殖性软骨细胞的自我更新,为软骨细胞分化提供足够的细胞来源,而且这功能的发挥不依赖于镶嵌其中的miR-199a_2和mi R-214。以上结果表明,LncRNA-Dnm3os在骨骼软骨内钙化发育过程中起关键性作用,从而使患者表现出典型的综合症。以上发现为临床诊断治疗提供新的理论依据。
[Abstract]:Dnm3os is a long non-coding RNA (long noncoding RNA,or lncRNA). Encoded by an independent transcriptional unit located on chromosome 1 and chimeric in the intron of Dynamin 3 gene. Previous data, including from our laboratory, show that miR-214, one of the two miniature RNA carried by Dnm3os, can form a regulatory loop with proto-oncogene N-Ras by accelerating the escape of cells from mitotic phase. In order to promote the differentiation of myoblasts along the direction of muscle fiber or in the direction of bone under the action of BMP. My doctoral study found that miR-214 does not play a necessary role in embryonic development, but it can promote the repair of adult muscle injury and inhibit cell proliferation in embryonic fibroblasts. I further found that the role of miR-214 in regulating cell growth is also reflected in the inhibition of rhabdomyosarcoma (rhabdomyosarcma,RMS), a common soft tissue malignant tumor in children. This tumor is mainly caused by malignant proliferation and poor differentiation of muscle progenitor cells, so it is closely related to the principle of miR-214. I found that the expression of miR-214 in human rhabdomyosarcoma cell line was significantly low. Overexpression of miR-214 in human embryonic rhabdomyosarcoma cell line (RD) could significantly inhibit its proliferation, withdraw from the cell cycle and begin muscle differentiation. Tumor-bearing experiments in nude mice also confirmed the ability of miR-214 to inhibit the tumorigenesis of RD cells in vivo. The mutation of miR-214 binding site in N-ras 3 'terminal non-transcriptional region (3'-UTR) was analyzed. I have once again confirmed the important role of miR-214 and N-ras regulatory loops in cell growth and RMS. Noonan syndrome is a common autosomal dominant genetic disease with specific facial representation and usually short stature. Congenital heart defects, as well as cognitive impairment and other clinical symptoms. Because the known genetic factors leading to Noonan syndrome are concentrated in the RAS/MAPK signaling pathway, the syndrome is also known as RAS disease (RASopathies). In partnership with Minnesota Children's Hospital, we found a maternal child with Noonan syndrome. No RAS/MAPK pathway gene mutation was found, but the comparative genomic hybrid (comparative genomic hybridization,CGH) showed the deletion of a narrow 690kb fragment in the 1q24.3 region of chromosome 1, covering LncRNA-Dnm3os, and PIGC,clorf105, embedded in DYNAMIN3,. Clorf9 and FSALG four genes. By using qPCR, I found that the expression of NS characteristic gene in peripheral blood of affected members of the family was significantly increased, and overexpression of Dnm3os could inhibit the expression of NS characteristic gene. Although bioinformatics analysis of six genes causing Noonan syndrome is the target gene of miR-214, there are no corresponding symptoms in miR-214 knockout mice, suggesting that the deletion of the whole fragment of Dnm3os is closely related to the occurrence of Noonan syndrome. Based on the published phenotypic data of LncRNA-Dnm3os knockout mice, we speculate that LncRNA-Dnm3os plays a key role in the development of calcification in skeletal cartilage. It was further found that LncRNA-Dnm3os promoted the proliferation and inhibition of the differentiation of cartilage cells, maintained the self-renewal of proliferating cartilage cells, and provided sufficient cell source for the differentiation of cartilage cells in the process of intra-cartilage calcification and osteogenesis. Moreover, the function of this function does not depend on the mosaic of miR-199a_2 and miR- 214. These results suggest that LncRNA-Dnm3os plays a key role in the development of calcification in skeletal cartilage, which makes the patients show typical syndrome. The above findings provide a new theoretical basis for clinical diagnosis and treatment.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R725.9

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