破骨细胞分化过程中microRNA表达谱及miR-148a的功能机制研究

发布时间：2018-04-30 17:57

本文选题：CD14+单核细胞 + 破骨细胞　；参考：《中南大学》2011年博士论文

【摘要】：第一部分人外周血单核细胞向破骨细胞分化过程中microRNA表达谱的研究目的：诱导人外周血单核细胞向破骨细胞分化,研究在人外周血单核细胞向破骨细胞分化过程中microRNA表达谱的变化。方法：采用密度梯度离心法从外周血分离单个核细胞贴壁培养,用免疫磁珠法分选出高纯度的CD14+外周血单核细胞(Peripheral Blood Mononuclear Cells, PBMCs);使用重组人巨噬细胞集落刺激因子(recombinant human-macrophage colony stimulating factor, rh-MCSF)和人核因子κB受体活化素配体(human receptor activator of nuclear factor-κB ligand, hRANKL)诱导CD14+PBMCs向破骨细胞分化,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase, TRAP)染色和骨吸收陷窝实验观察破骨细胞的分化成熟情况。建立成熟的诱导人外周血单核细胞向破骨细胞分化的细胞模型。应用miRNA微阵列技术,分析人外周血单核细胞向破骨细胞分化过程中miRNA表达谱的变化。通过qRT-PCR方法验证表达明显变化的miRNA在CD 14+PBMCs向破骨细胞分化过程中的表达情况。选取表达显著增高的miR-148a作为进一步的研究对象。结果：(1)采用密度梯度离心法成功从人外周血中分离出单核细胞,并利用MACS磁珠分选系统分选出高纯度的CD14+PBMCs; (2)rh-MCSF和hRANKL诱导C14+PBMCs培养至14天后,可见TRAP染色阳性的多核巨细胞形成,甲苯胺蓝染色可见骨吸收陷窝形成；(3)用miRNA microarray芯片检测CD14+PBMCs向破骨细胞分化过程中miRNA表达谱变化,发现差异表达的miRNA有27个,表达明显上调的有5个,分别是：hsa-miR-148a, hsa-miR-483, hsa-miR-223, hsa-miR-21, hsa-miR-214;表达明显下调的有4个,分别是：hsa-miR-155, hsa-miR-125a, hsa-miR-27b, hsa-miR-145以hsa-miR-148a表达上调最为明显。(4)对明显差异表达的miRNA进一步行实时定量RT-PCR验证,发现定量PCR结果与芯片结果一致,表明miRNA芯片结果是可靠的。结论：建立了成熟的诱导人CD 14+PBMCs向破骨细胞分化的细胞模型。应用microRNA芯片发现CD 14+PBMCs向破骨细胞分化过程中,hsa-miR-148a, hsa-miR-483, hsa-miR-223, hsa-miR-21, hsa-miR-214表达明显上调,而hsa-miR-155, hsa-miR-125a, hsa-miR-27b, hsa-miR-145表达明显下调。以hsa-miR-148a表达上调最为显著。第二部分miR-148a对破骨细胞分化的功能研究目的：研究hsa-miR-148a在CD14+PBMCs向破骨细胞分化过程中的作用。方法：用rh-MCSF和hRANKL诱导CD14+PBMCs向破骨细胞分化,Northern印迹杂交检测hsa-miR-148a在此过程中的表达模式。根据hsa-miR-148a前体序列设计引物,用pSilencer 4.1-CMV puro合成hsa-miR-148a表达载体pre-miR-148a。将pre-miR-148a转染PBMCs,构建pre-miR-148a过表达细胞模型,转染后加入rh-MCSF和hRANKL诱导分化, Northern杂交检测hsa-miR-148a表达变化,TRAP染色及骨吸收陷窝观察破骨细胞分化情况,实时定量PCR检测破骨细胞分化指标TRAP、NFATc 1 (nuclear factor of activated T cells c1,活化T细胞核因子c1)、OSCAR (osteoclast-associated receptor,破骨细胞相关受体)、CathK (Cathepsin K,组织激酶K) mRNA水平,Western印迹杂交检测TRAP蛋白表达水平。用2'-O-methyl修饰的反义寡核苷酸anti-miR-148a转染PBMCs,构建pre-miR-148a表达降低细胞模型,同样行TRAP染色及骨吸收陷窝实验,实时定量PCR检测TRAP、NFATc1、OSCAR、CathK mRNA水平,Western印迹杂交检测TRAP蛋白表达水平。结果：(1)rh-MCSF和hRANKL诱导CD14+PBMCs向破骨细胞分化过程中,随着诱导时间的延长,hsa-miR-148a表达逐渐增多。(2)用pSilencer 4.1-CMV puro成功构建hsa-miR-148a表达载体pre-miR-148a,能在细胞中高表达hsa-miR-148a。(3) hsa-miR-148a过表达能促进TRAP阳性多核巨细胞形成和骨吸收陷窝形成,提高破骨细胞分化指标TRAP、NFATc1、OSCAR、CathK mRNA水平,提高TRAP蛋白表达水平。(4)抑制hsa-miR-148a能抑制TRAP阳性多核巨细胞形成和骨吸收陷窝形成,降低TRAP、NFATc1、OSCAR、CathK mRNA水平,降低TRAP蛋白表达水平。结论：构建了hsa-miR-148a表达载体,过表达hsa-miR-148a可以促进CD14+PBMCs向破骨细胞分化,而抑制hsa-miR-148a则延缓CD14+PBMCs向破骨细胞分化。第三部分hsa-miR-148a调控破骨细胞分化的机制研究目的：预测并验证hsa-miR-148a作用的靶基因,阐明hsa-miR-148a调控CD14+PBMCs向破骨细胞分化的作用机制。方法：用TargetScan和PicTar等靶基因预测软件分析得到hsa-miR-148a作用的靶基因。将含有hsa-miR-148a结合位点的靶基因MAFB (V-maf musculoaponeurotic fibrosarcoma oncogene homolog B, V-maf肌腱膜纤维肉瘤癌基因同源物B)3'UTR片段克隆到pGL3载体的3'UTR区,构建野生型报告载体WT-pGL3-MAFB-3'UTR。同时我们用QuickChange site-directed mutagenesis试剂盒构建了含有突变靶位点的突变型报告载体MUT-pGL3-MAFB-3'UTR。将这两个载体分别与pre-miR-148a或miR-C共同转染PBMCs,检测荧光素酶活性变化以证实该靶基因是否为hsa-miR-148a作用的靶基因。PBMCs单独转染pre-miR-148a, Western杂交和实时定量PCR分别检测靶基因MAFB蛋白和mRNA水平的变化,明确hsa-miR-148a对靶基因的调控作用。PCR扩增MAFB CDS全长片段,连接到pcDNA3.1(+)载体构建野生型靶基因表达载体,同时构建了含有突变靶位点的突变型靶基因表达载体。将这两个载体分别与pre-miR-148a或miR-C共同转染PBMCs,实时定量PCR检测TRAP mRNA水平,靶基因蛋白表达用Western杂交检测。结果：(1)TargetScan和PicTar等靶基因预测软件预测MAFB为hsa-miR-148a作用的靶基因。(2)与转染miR-C对照组相比,pre-miR-148a和WT-pGL3-MAFB-3'UTR共转染组的荧光素酶活性受到明显的抑制,而pre-miR-148a和MUT-pGL3-MAFB-3'UTR共转染组的荧光素酶活性则无明显变化。(3)单独转染pre-miR-148a可以降低MAFB蛋白水平,而对MAFBmRNA水平无影响。(4)pre-miR-148a与WT-pcDNA3.1-MAFB共同转染能增加TRAP mRNA水平,降低MAFB蛋白表达水平；而pre-miR-148a与MUT-pcDNA3.1-MAFB共同转染不能增加TRAP mRNA水平,对MAFB蛋白水平没有影响。因此,我们认为hsa-miR-148a主要通过抑制MAFB蛋白表达来促进破骨细胞分化。结论：(1)构建了野生型和突变型MAFB 3'UTR荧光素酶报告基因载体、野生型和突变型MAFB表达载体。(2)通过靶基因预测软件预测并经荧光素酶报告基因检测证实MAFB为hsa-miR-148a作用的靶基因。(3)hsa-miR-148a在转录后水平抑制MAFB表达,促进破骨细胞分化。(4)MAFB是hsa-miR-148a调控破骨细胞分化过程中最重要的靶基因。
[Abstract]:Study on microRNA expression profile during the differentiation of peripheral blood mononuclear cells from the first part of human peripheral blood mononuclear cells

Objective : To induce differentiation of human peripheral blood mononuclear cells ( PBMC ) from peripheral blood mononuclear cells ( PBMC ) and to investigate the changes of microRNA expression profiles in human peripheral blood mononuclear cells ( PBMC ) .

Methods : Peripheral blood mononuclear cells ( PBMCs ) were isolated from peripheral blood by density gradient centrifugation , and high - purity CD14 + peripheral blood mononuclear cells ( PBMCs ) were separated by immunomagnetic beads . The expression of miRNA expression profiles in human peripheral blood mononuclear cells ( PBMC ) was analyzed by using miRNA microarray technology .

Results : ( 1 ) Monocyte was isolated from human peripheral blood by density gradient centrifugation , and high purity CD14 + PBMCs were separated by MACS magnetic bead sorting system .
( 3 ) The expression profiles of miRNA in CD14 + PBMCs were detected by miRNA microarray . There were 27 miRNA expression profiles , respectively : hsa - miR - 148a , hsa - miR - 483 , hsa - miR - 223 , hsa - miR - 21 , hsa - miR - 214 ; the expression of hsa - miR - 1 , hsa - miR - 27b , hsa - miR - 145 was up - regulated in hsa - miR - 148a .

Conclusion : The expression of hsa - miR - 148a , hsa - miR - 483 , hsa - miR - 223 , hsa - miR - 21 , hsa - miR - 214 was significantly up - regulated during the differentiation of CD 14 + PBMCs from CD 14 + PBMCs by microRNA chip , while hsa - miR - 155 , hsa - miR - 125a , hsa - miR - 27b and hsa - miR - 145 were downregulated .

The Functional Study of the Second Part of miR - 148a on Osteoclasts Differentiation

Objective : To study the role of hsa - miR - 148a in the differentiation process of CD14 + PBMCs .

Methods : The pre - miR - 148a expression vector pre - miR - 148a was transfected into PBMCs by using the antisense oligonucleotides of hsa - miR - 148a to construct pre - miR - 148a expression vector pre - miR - 148a .

Results : ( 1 ) The expression of hsa - miR - 148a increased gradually with the increase of induction time . ( 2 ) hsa - miR - 148a expression vector pre - miR - 148a was successfully constructed by ptimi4.1 - CMV puro .

Conclusion : hsa - miR - 148a expression vector was constructed . hsa - miR - 148a could be used to promote the differentiation of CD14 + PBMCs and inhibit hsa - miR - 148a .

Mechanism of Regulation of Osteoclasts Differentiation by the Third Part of hsa - miR - 148a

Objective : To predict and verify the target gene of hsa - miR - 148a , and to clarify the mechanism of hsa - miR - 148a regulation on the differentiation of CD14 + PBMCs to Osteoclasts .

Methods : The target gene of hsa - miR - 148a was obtained by target gene prediction software of TargetScan and PicTar . The target gene MAFB containing hsa - miR - 148a binding site was cloned into the 3 ' untranslated region of pGL3 vector . The expression vector of wild type reporter vector WT - pGL3 - MAFB - 3 ' was constructed .

Results : ( 1 ) The target gene predicted by TargetScan and PicTar and other target genes predicted MAFB as hsa - miR - 148a . ( 2 ) Compared with the transfected miR - C control group , the luciferase activity of the pre - miR - 148a and WT - pGL3 - MAFB - 3 ' untranslated group was significantly inhibited , whereas the pre - miR - 148a and WT - pcDNA3.1 - MAFB co - transfected group had no effect on the level of MAFB mRNA , and ( 4 ) pre - miR - 148a and WT - pcDNA3.1 - MAFB co - transfected the target gene , which can increase the TRAP mRNA level and reduce the expression level of MAFB protein ;
However , it was suggested that hsa - miR - 148a promoted the differentiation of Osteoclasts mainly by inhibiting the expression of MAFB protein .

Conclusion : ( 1 ) The expression vector of wild - type and mutant MAFB 3 ' utu luciferase reporter gene vector , wild type and mutant MAFB expression vector was constructed . ( 2 ) The target gene was predicted by the target gene prediction software and confirmed by luciferase reporter gene .

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329

【共引文献】