【作者】 刘唯；

【导师】 罗湘杭；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的：探寻新的小鼠成骨细胞表达的微小核糖核酸(microRNA, miRNA),并研究其在小鼠不同组织和细胞中的表达模式。方法：提取小鼠成骨细胞小分子RNA,经Poly(A)加尾、连接5’端接头后逆转录为cDNA,进行PCR扩增,回收PCR产物后连接至pcDNA3.1 TOPO载体构建cDNA文库,进行菌落PCR,阳性克隆测序后选取19-26nt大小的RNA进行生物信息学分析确定新的miRNAs。通过Northern blot检测新的miRNA在小鼠成骨细胞、破骨细胞、骨组织、肝脏、心脏、肾脏、肺、脾、大脑、脂肪及骨骼肌的表达,并进一步检测其在BMP-2诱导的小鼠基质细胞ST2成骨分化过程中的表达模式。结果：(1)共克隆得到162个小分子RNA序列,68个为miRNA分子,明确2个为新克隆的miRNA,其中一个命名为miR-X,其在小鼠与人类之间具有保守性,位于小鼠第二染色体。(2) miR-X在小鼠成骨细胞、骨组织及肝脏中均有表达,其中成骨细胞中表达最高,其他组织及破骨细胞不表达。(3)在BMP-2诱导的ST2细胞成骨分化过程中,miR-X随时间延长其表达量不断增加。结论：通过构建小鼠成骨细胞小分子RNA的cDNA文库的方法,新克隆了一个在小鼠与人类存在保守性的新miRNA--miR-X。miR-X在小鼠成骨细胞、骨组织、肝脏中表达,其中成骨细胞中表达最高,并且在小鼠成骨细胞分化过程中的表达呈时间依赖特性。目的：研究miR-X在小鼠基质细胞ST2成骨分化中的作用并预测及实验验证miR-X的靶基因。方法：根据miR-X前体序列设计引物,经退火处理后与线性化的pSilencer 4.1CMV puro载体连接,构建miR-X表达载体pre-miR-X。将pre-miR-X或2’-O-methyl修饰的反义寡核苷酸anti-miR-X分别转染BMP-2诱导分化的ST2细胞(300ng/ml),造成miR-X在成骨分化过程中的过表达或功能抑制。转染48小时后,采用分光光度计检测对硝基苯酚释放来反映碱性磷酸酶(alkaline phosphatase, ALP)活性,放射免疫测定法测定骨钙素(osteocalcin, OC)分泌,采用Western blot和实时定量PCR检测转录因子runt相关转录因子2 (runt related transcription factor 2, Runx2)蛋白和mRNA表达的变化。采用多种靶基因预测软件预测miR-X的靶基因。PCR扩增含靶位点的靶基因编码区(coding sequence, CDS),产物连接pGL3载体,构建野生型Hoxa2CDS荧光素酶报告基因载体,采用定点突变技术构建突变型Hoxa2CDS荧光素酶报告基因载体,分别与miR-X表达载体共转染BMP-2诱导的ST2细胞,通过荧光素酶活性变化证实Hoxa2是否为miR-X靶基因。将miR-X表达载体单独转染ST2细胞,采用Western blot和实时定量PCR检测Hoxa2蛋白和mRNA水平的变化,验证miR-X对Hoxa2的调控作用。结果：(1)miR-X过表达促进BMP-2诱导的ST2细胞成骨分化过程中的ALP活性增高和OC分泌,并增加Runx2 mRNA和蛋白的表达。(2)抑制miR-X的作用降低了ST2成骨分化过程中的ALP活性及OC分泌的上升程度,并部分抑制Runx2 mRNA和蛋白的表达。(3)Rnas22预测Hoxa2为miR-X的靶基因。(4)与对照组相比,共转染野生型Hoxa2 CDS荧光素酶报告基因载体及miR-X表达载体的ST2细胞荧光素酶活性显著下降,而共转染突变型Hoxa2 CDS荧光素酶报告基因载体及miR-X表达载体的ST2细胞荧光素酶活性无明显变化。(5)转染miR-X表达载体的ST2细胞Hoxa2蛋白水平下降,但Hoxa2 mRNA表达无明显变化。结论：miR-X在转录后水平抑制靶基因Hoxa2,增加转录因子Runx2的表达,从而促进BMP-2诱导的ST2细胞的成骨分化。目的：探索两个新克隆的miRNA, miR-X和miR-2861之间的关系及其与转录因子Runx2的相互作用。方法：通过BLAST等生物信息学方法寻找miR-X与miR-2861在基因组上的定位,分析两者之间的位置关系,采用Mfold软件预测初级转录本(primary microRNA, pri-miRNA)的二级结构。通过国外公布的人类及小鼠转录起始位点(TSS)预测文库,根据第四位赖氨酸三甲基化的H3组蛋白(H3K4me3)在TSS位点周围的富集与启动子CpG岛为主要标识,预测miR-X/miR-2861表达簇的TSS。运用TF-seach转录因子结合位点预测软件分析miR-X/miR-2861表达簇的TSS上游2kb内的DNA序列,预测可能的Runx2结合位点。通过染色体免疫沉淀法(ChIP),采用Runx2抗体(anti-Runx2)验证Runx2与所预测结合位点的相互关系。通过PCR扩增Ⅱ型Runx2 cDNA并亚克隆至pSG5载体,构建Runx2表达载体pSG5-Runx2,转染ST2细胞,并将体外合成的特异性阻断Runx2蛋白表达的小干扰RNAsiRNA-Runx2,转染BMP-2诱导的ST2细胞,分别采用Northern blot检测miR-X和miR-2861的表达,研究Runx2对miR-X与miR-2861表达的调控作用。结果：(1) miR-X与miR-2861在小鼠第2染色体上的基因定位仅相差69bp,两者以miR-X/miR-2861表达簇的形式存在。(2)确定miR-X/miR-2861表达簇的转录起始位点位于小鼠第2染色体10,104,622。(3)预测Runx2在]miR-X/miR-2861表达簇启动子的转录结合位点在其TSS上游2kb。(4) ChIP证实Runx2结合于所预测的转录因子结合位点。(5)与对照组细胞相比,转染Runx2表达载体的ST2细胞中,miR-X与miR-2861的表达增强。而转染siRNA-Runx2的BMP-2诱导ST2细胞miR-X与miR-2861的表达降低。结论：miR-X与miR-2861在基因组中成簇存在,共同转录。转录因子Runx2通过与miR-X/miR-2861表达簇的启动子结合,促进表达簇的转录,并与miR-X和miR-2861形成正反馈调控环路,协同调控成骨分化。更多还原

【Abstract】 Objective:To explore the novel microRNAs (miRNAs) which express in mouse osteoblasts and identify their expression profiles in different tissues and cells of mouse.Methods:Extracted small RNAs from primary mouse osteoblasts. The small RNAs were polyadenylated by using poly (A) polymerase, and ligated with a 5’RNA adapter. Then the cDNAs were obtained by reverse transcription of tailed and ligated RNAs, and amplified by polymerase chain reaction (PCR). The purified PCR products of interest were inserted into pcDNA3.1 TOPO cloning vectors and the recombinant vectors were transformed into competent cells to construct a cDNA library of small RNAs of mouse osteoblast. At last, selected positive clones containing interested fragments from the cDNA library by colony PCR for further sequencing, and analyzed the RNAs sized from 19 to 26 nt by bioinformatic methods. Northern blot was performed with total RNA extracted respectively from primary mouse osteoblast cells, primary mouse osteoclast cells, mouse bone, liver, lung, heart, kidney, brain, fat, spleen and skeletal muscle. The expression pattern of the novel miRNA in the osteoblast differentiation of mouse stromal cells ST2 was also determined by Northern blot.Results:(1) 162clones were identified by DNA sequencing and database searching. Of them, there are 68 miRNAs including 2 novel miRNAs. One of the two novel miRNAs was termed "miR-X", which was located on chromosome 2 and conserved in the human sequence. (2) miR-X was expressed in osteoblasts, bone and liver, representing a highest level in osteoblasts. But it was not detected in osteoclasts and other tissues. (3) Expression of miR-X in osteoblast differentiation of BMP-2 induced ST2 cells was increased progressively with the time.Conclusion:We cloned a novel miRNA termed "miR-X", which was conserved in human sequence, by constructing a cDNA library of small RNAs from mouse osteoblast. miR-X was detected in mouse osteoblasts, bone and liver, representing a highest level in osteoblasts and the expression of miR-X in osteoblast differentiation of BMP-2 ST2 cells represented a time-depended pattern. Objective:To investigate the role of miR-X in osteoblast differentiations of mouse stromal cells ST2 and identify the target gene of miR-X.Method:The primers designed according to pre-miRNA-X sequence were annealed and linked to the linearized pSilencer 4.1 CMV puro vector to construct miR-X expression vector termed "pre-miR-X". In order to make gain or loss of function analysis, pre-miR-X vectors or 2’-O-methyl antisense inhibitory oligoribonucleotides (anti-miR-X) were transfected into BMP-2 induced ST2 cells (300ng/ml) respectively.48 hours after transfection, alkaline phosphatase (ALP) activity was evaluated by spectrophotometric measurement of P-nitrophenol release and osteocalcin (OC) secretion was detected by radioimmunoassay. Runt related transcription factor 2 (Runx2) proteins and mRNA expression were determined by western blot and real-time quantitative PCR respectively. Multiple miRNA target prediction software tools were used to predict the target gene of miR-X. The coding sequence (CDS) of homeobox A2 (Hoxa2) containing target binding sites was amplified by PCR and the products were linke to pGL3 vectors to construct Wild type (WT) luciferase reporter vector. The mutant type (MUT) luciferase reporter vector was produced by site-directed mutagenesis. The two vectors were cotransfected with miR-X expression vectors into BMP-2 induced ST2 cells respectively. The luciferase activity was quantified by Dual Luciferase Reporter Assay System to validate whether Hoxa2 is the target gene of miR-X. To directly test the effects of miR-X on Hoxa2 gene, ST2 cells were transfected with miR-X expression vectors. The mRNA and protein levels of Hoxa2 were measured by qRT-PCR and western blot.Results:(1) miR-X overexpression increased levels of ALP activity and OC secretion and also promoted Runx2 protein and mRNA expression in osteoblast differentiation of BMP-2 induced ST2 cells. (2)The inhibition of miR-X attenuated the increase of ALP activity and OC secretion and also partly reduced the increase of Runx2 protein and mRNA levels. (3) Rna 22 predicted that Hoxa2 was the target gene of miR-X。(4) Compared with control, overexpression of miR-X suppressed the luciferase activity of the Hoxa2 CDS reporter gene, while 3 mutations of nucleotides within the putative target site in the Hoxa2 CDS abolished this repression, confirming that Hoxa2 is the target gene of miR-X. (5) Transfection of miR-X expression vector reduced the level of Hoxa2 protein, but did not change Hoxa2 mRNA level.Conclusion:miR-X increased Runx2 expression by repressing Hoxa2 at the post-transcriptional level, and further promoted osteoblast differentiation. Obejective:To explore the correlation of the two newly-cloned miRNAs miR-X and miR-2861 and the interaction between them and transcription factor Runx2.Method:Searched the gene loci of miR-X and miR-2861 by BLAST and analyzed the correlation of the two miRNAs. Mfold program was used to predict the secondary struction of primary microRNA (pri-miRNA). Based on histone H3 trimethylated at lysine 4(H3K4me3) enriched loci and promoter CpG islands, we predicted the TSS of miR-X/ miR-2861 cluster, according to the newly published library of candidate transcription start site (TSS) both in human and mouse. In order to predict the potential binding site for Runx2, the TF-Search prediction program was used to search examined 2 kb of the miR-X/miR-2861 cluster TSS upstream regions. To validate whether Runx2 could physically associate with the binding sites, chromatin immunoprecipitation (ChIP) analysis, using Runx2 antibody, was performed with extracts from BMP2-induced ST2 cells. Runx2 typeⅡcDNA was amplified by PCR and subcloned into pSG5 vector to construct Runx2 expression vector named pSG5-Runx2. To determine the function of Runx2 on miR-X and miR-2861 expression, pSG5-Runx2 was tranfected into ST-2 cells and siRNA-Runx2, which specifically knockdown the effect of Runx2, was transfected into BMP-2 induced ST2 cells, then the expression of miR-X and miR-2861 were detected by Northern blot.Results:(1) The distance between the site of miR-X and miR-2861 was just 69bp and the two miRNAs existed as miR-X/miR-2861 cluster. (2) Predicted TSS of miR-X/miR-2861 was located on the mouse Chromosome 2:10,141,622. (3) A potential binding site for Runx2 resided just upstream of the miR-X/miR-2861 cluster. (4) ChIP results comfirmed that Runx2 bound to the miR-X/miR-2861 cluster promoter through the putative binding sites. (5) Compared to the control, the expression of miR-X and miR-2861 were increased in ST2 cells tranfected with Runx2 expression vector. While the expression of miR-X and miR-2861 in ST2 cells transfected with siRNA-Runx2 were reduced.Conclusion:miR-X and miR-2861 were clustered and transcribed together. Transcription factor Runx2 increased the transcription of miR-X and miR-2861 by binding the promoter of miR-X/miR-2861 cluster. miR-X and miR-2861 represent a regulatory feedback loop with Runx2, suggesting a coordinated control of osteoblast differentiation.更多还原