【作者】 李辉；

【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：查找新的小鼠成骨细胞特异性表达或优先表达的microRNA (miRNA),并对其在多个组织器官和细胞中的表达模式进行研究。方法：首先提取小鼠成骨细胞的小分子RNA,经过Poly(A);加尾并连接5’端接头后逆转录为cDNA,随后进行PCR扩增、回收,连接至pcDNA3.1 TOPO载体构建小分子RNA的cDNA文库,最后进行菌落PCR,得到的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miRNA。用Northern印迹杂交检测新的miRNA在小鼠成骨细胞、破骨细胞、骨、肝脏、心脏、肺、肾脏、大脑、脂肪、脾及骨骼肌的表达情况。用BMP-2诱导ST2细胞向成骨细胞分化,Northern印迹杂交检测miRNA在此过程中的表达模式。结果：共克隆得到162个小分子RNA,有68个为miRNAs,包括2个新的miRNAs,其中1个提交miRBase数据库后命名为miR-2861。miR-2861位于小鼠第2条染色体,在人类基因组中存在保守性。miR-2861在小鼠成骨细胞及骨高表达,在肝脏表达较低,在其他组织和破骨细胞不表达。miR-2861在ST2细胞不表达,BMP-2诱导ST2向成骨细胞分化12h后可以检测到miR-2861表达,并随着诱导分化时间的延长,表达逐渐增加。结论：首次构建了小鼠成骨细胞小分子RNA的cDNA文库。首次发现一个新的成骨细胞优先表达的miRNA-miR-2861。MiR-2861在小鼠与人之间具有保守性,其表达随着成骨细胞分化的进展而逐渐升高,提示其可能参与了成骨细胞分化的调控过程。目的：研究miR-2861在小鼠基质细胞ST2和原代骨髓基质细胞(BMSCs)向成骨细胞分化中的作用。方法：根据miR-2861前体序列设计引物,退火后连接至pSilencer 4.1-CMV puro载体,构建miR-2861的表达载体pre-miR-2861。将pre-miR-2861分别转染ST2细胞和骨髓基质细胞(bone marrow stromal cells, BMSCs),造成miR-2861过表达细胞模型,随后予300ng/ml BMP-2诱导分化,观察成骨细胞分化指标变化。碱性磷酸酶(alkaline phosphatase, ALP)活性用分光光度计检测对硝基苯酚释放获得,骨钙素(Osteocalcin, OC)用放射免疫测定法测定,细胞钙沉积量用邻甲酚酞络合铜比色法检测。Western杂交和实时定量PCR分别检测Runx2蛋白和mRNA表达的变化。用2’-O-methyl修饰的反义寡核苷酸anti-miR-2861瞬时转染ST2细胞和BMSCs,同样方法检测ALP活性、OC分泌以及细胞中钙沉积量变化,Runx2蛋白和mRNA表达分别用Western杂交和实时定量PCR检测。结果：(1)转染用pSilencer 4.1CMV puro构建的miR-2861表达载体pre-miR-2861能够在细胞中稳定地高表达miR-2861。(2)miR-2861过表达促进ST2和BMSCs向成骨细胞分化过程中的ALP活性增高和OC分泌,提高Runx2蛋白表达,而不影响Runx2 mRNA水平。ST2细胞转染pre-miR-2861能够增加细胞中的钙沉积量(3)抑制miR-2861降低ST2和BMSCs向成骨细胞分化过程中的ALP活性,减少OC分泌,降低BMP-2诱导引起的Runx2蛋白表达升高,但不影响Runx2 mRNA水平,转染ST2细胞5天后,钙沉积含量较对照组降低。结论：成功构建了miR-2861表达载体,过表达miR-2861可以促进ST2和BMSCs向成骨细胞分化,抑制miR-2861能够延缓ST2和BMSCs向成骨细胞分化。目的：预测并验证miR-2861作用的靶基因,阐明miR-2861促进成骨细胞分化的作用机制。方法：用多个靶基因预测软件分析得到miR-2861作用的靶基因。PCR扩增包含靶位点在内的靶基因编码序列(coding sequence, CDS),产物连接到pGL3载体,构建野生型靶基因CDS荧光素酶报告基因载体。以此为模板,用QuickChange site-directed mutagenesis试剂盒在靶位点中引入两个碱基突变,构建突变型靶基因CDS荧光素酶报告基因载体。将这两个载体分别与pre-miR-2861共同转染ST2细胞,检测荧光素酶活性变化以证实该靶基因是否为miR-2861作用的靶基因。ST2细胞单独转染pre-miR-2861,观察miR-2861对靶基因表达的影响,Western杂交和实时定量PCR分别检测靶基因蛋白和mRNA水平的变化,明确miR-2861对靶基因的调控作用。PCR扩增靶基因CDS全长cDNA,连接到pcDNA3.1(+)载体构建野生型靶基因表达载体,以此为模板,用QuickChange site-directed mutagenesis试剂盒在靶位点中引入两个碱基突变,构建突变型靶基因表达载体,这两个载体分别与pre-miR-2861或miR-C共同转染ST2细胞,加BMP-2诱导分化,观察ALP活性和靶基因蛋白表达,ALP活性用分光光度计检测对硝基苯酚释放获得,靶基因蛋白表达用Western杂交检测。结果：(1)软件预测HDAC5为miR-2861作用的靶基因。(2)与转染miR-C对照组相比,miR-2861过表达组HDAC5 CDS的荧光素酶活性显著下降,而转染突变型HDAC5 CDS的荧光素酶活性则无明显变化。(3)单独转染pre-miR-2861可以降低HDAC5蛋白水平,而对HDAC5 mRNA无影响。(4)pre-miR-2861与野生型HDAC5 CDS表达载体共转染可以增加ALP活性,降低HDAC5蛋白表达,而pre-miR-2861与突变型HDAC5 CDS表达载体共转染则不会产生这种作用。表明miR-2861在成骨细胞分化中的作用完全或主要是通过作用与HDAC5来完成的。结论：(1)成功构建了野生型和突变型HDAC5 CDS荧光素酶报告基因载体、野生型和突变型HDAC5表达载体。(2)软件预测并经荧光素酶报告基因检测证实HDAC5为miR-2861作用的靶基因。(3)miR-2861通过转录后水平抑制HDAC5表达,促进成骨细胞分化。(4)HDAC5是miR-2861在成骨细胞分化中最重要的靶基因。更多还原

【Abstract】 ObjectiveTo find novel microRNAs (miRNAs) specifically or preferentially expresses in mouse osteoblast, and explore their expression pattern in multiple mouse tissues and cells.MethodsFirstly, extract small RNAs from mouse primary osteoblast according to a protocol described in the main text. The small RNAs were polyadenylated by using poly(A) polymerase, and ligated with a 5’RNA adapter. Secondly, the cDNAs were obtained by reverse transcription of tailed and ligated RNA, and amplify the cDNAs by polymerase chain reaction (PCR). Thirdly, recover and purify the interested PCR products, insert them into pcDNA3.1 TOPO cloning vectors, and construct a cDNA library of small RNAs of mouse osteoblast by transformating competent cells. Lastly, Select positive clones containing interested fragments from the cDNA library by colony PCR for further sequencing, and bioinformatic analysis was performed with the RNAs whose size ranged from 19 to 26 nt. The expression of novel miRNA was examined by Northern blotting using total RNA from primary mouse osteoblasts in addition to total RNA prepared from primary mouse osteoclasts, bone, liver, heart, lung, kidney, brain, fat, spleen, and skeletal muscle. The novel miRNA expression profile of the osteogenic differentiation of stromal cell line ST2 was also detected by Northern blotting.ResultsA total of 162 clones were characterized by DNA sequencing and database searching. Of them, there are 68 miRNAs including 2 novel miRNAs, a new miRNA was termed "miR-2861" by miRBase and is located on chromosome 2 and conserved in the human sequence. Northern blotting analysis confirmed that miR-2861 was primarily expressed in osteoblasts. In mouse tissue, miR-2861 was preferentially expressed in bone and detected at lower levels in liver, but was not found in other tissues. In cultured bone cells, miR-2861 was detected in primary mouse osteoblasts, but not osteoclasts. Expression of miR-2861 in ST2 cells was detected after treatment with BMP2 for 12 hours and increased progressively after 24-48 hours of treatment.ConclusionIn this study, we firstly constructed a cDNA library of small RNAs from mouse osteoblasts, and firstly found a novel miRNA which was termed "miR-2861" by miRBase. MiR-2861 was conserved in the human sequence. MiR-2861 is preferentially expressed in osteoblast, and its expression level was increased progressively together with the process of osteogenic differentiation. Our results suggest miR-2861 may take part in the regulation process of osteogenic differentiation. ObjectiveTo investigate the role of miR-2861 in osteogenic differentiation of mouse ST2 stromal cells and primary mouse bone marrow stromal cells (BMSCs).MethodsThe expression vector of miR-2861 was constructed by linked the annealed primers designed according to the precursor sequence of miR-2861 to the pSilencer 4.1CMV puro vector. The expression vector was named "pre-miR-2861". The parameters of osteoblast differentiation were measured after transfection of ST2 and BMSCs with pre-miR-2861. Osteoblastic differentiation was induced by the addition of 300ng/ml BMP2. Alkaline phosphatase (ALP) activity was measured by spectrophotometric measurement of P-nitrophenol release, osteocalcin (OC) secretion was detected by radioimmunoassay, and total calcium deposition was quantified using o-cresolphthalein complexone colorimetric method. Runx2 protein and mRNA expression were detected by Western-blot and quantitative real-time PCR (qRT-PCR) respectively. Then, we transient transfected BMP2-induced ST2 cells and BMSCs with 2’-O-methyl antisense inhibitory oligoribonucleotides (anti-miR-2861). ALP activity, OC secretion, calcium deposition, Runx2 protein and mRNA expression were measured as described above.Results(1)Stable and high expression of miR-2861 was obtained in cells after transfection with the expression vector of miR-2861 constructed by using pSilencer 4.1CMV puro vector. (2) Compared with control cells, the levels of ALP activity and OC secretion were increased by transfection of pre-miR-2861. Transfection of miR-2861 promoted calcium accumulation in cells. Runx2 protein levels were also enhanced by transfection of miR-2861, whereas Runx2 mRNA levels showed no considerable change. (3) Treatment with anti-miR-2861 attenuated the levels of ALP activity and OC secretion and reduced the increase in Runx2 protein induced by BMP2, but it did not influence the level of Runx2 mRNA. Decreased calcium accumulation was observed at day 5 of ST2 cell culture after transfection with anti-miR-2861.ConclusionThe expression vector of miR-2861 was successfully constructed. MiR-2861 overexpression promoted osteoblast differentiation of ST2 cells and BMSCs. Inhibition of miR-2861 attenuated osteogenic differentiation of ST2 cells and BMSCs. ObjectiveTo predict and identify the target gene of miR-2861, elucidate the mechanism of miR-2861 promoting osteoblast differentiation.MethodsPredict the target gene of miR-2861 by using multiple miRNA target prediction software tools. To create wild type (WT) target gene coding sequence (CDS) luciferase reporter vector, a segment of the mouse target gene CDS including the putative target site was PCR amplified from mouse genomic DNA. The product was then inserted into the downstream of the stop codon in the pGL3-Control Firefly Luciferase reporter vector, resulting in WT target gene CDS reporter vector. The QuickChange site-directed mutagenesis kit was used to introduce two point mutations into the seed region of WT target gene CDS reporter vector, resulting in mutant (MUT) target gene CDS reporter vector. These two reporter vectors were cotransfected with pre-miR-2861 to ST2 cells, and the luminescent signal was quantified by Dual Luciferase Reporter Assay System. To directly test the validity of the putative target, ST2 cells were transfected with pre-miR-2861. The mRNA and protein levels of target gene were measured by qRT-PCR and Western blotting. The full length CDS of mouse target gene was amplified and linked to pcDNA3.1(+) vector, resulting in WT target gene expression vecor. The QuickChange site-directed mutagenesis kit was used to introduce two point mutations into the seed region of WT target gene expression vector, resulting in MUT target gene expression vector. We cotransfected the WT or MUT target gene expression vector, with pre-miR-2861 or miR-C into BMP2-induced ST2 cells to determine the effects on ALP activity and target gene protein expression. ALP activity was measured by spectrophotometric measurement of P-nitrophenol release. Target gene protein expression was detected by Western-blot.Results(1) HDAC5 is the target gene predicted by miRNA target prediction software tools. (2) Compared with control, overexpression of miR-2861 suppressed the luciferase activity of the HDAC5 CDS reporter gene. Mutation of 2 nucleotides within the putative target site in the HDAC5 CDS abolished this repression, confirmed HDAC5 is the target gene of miR-2861. (3) Relative to the control, overexpression of miR-2861 downregulated endogenous HDAC5 protein. By contrast, no change in HDAC5 mRNA levels was noted. (4) Induction of ALP activity by pre-miR-2861 was rescued by the mutant HDAC5 CDS construct. Western blotting showed that the mutant HDAC5 CDS construct was also able to rescue the pre-miR-2861-induced downregulation of HDAC5 protein. These results suggested that miR-2861 exert its role in osteoblast differentiation by perfectly or primarily effect on HDAC5.Conclusion(1) We have succeed constructed WT and MUT HDAC5 CDS Luciferase reporter vector, and WT and MUT HDAC5 expression vector. (2) The fact that HDAC5 is target gene of miR-2861, have been predicted by miRNA target prediction software tools and identified by Luciferase reporter vector measurement. (3) miR-2861 promotes osteoblast differentiation by repressing HDAC5 expression at the post-transcriptional level. (4) HDAC5 is the most important target of miR-2861 in osteoblast differentiation.更多还原