一个新的调节成骨细胞分化的microRNA的克隆及其功能研究
|
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。
Objective
To find novel microRNAs (miRNAs) specifically or preferentially expresses in mouse osteoblast, and explore their expression pattern in multiple mouse tissues and cells.
Methods
Firstly, 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.
Results
A 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.
Conclusion
In 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.
Objective
To investigate the role of miR-2861 in osteogenic differentiation of mouse ST2 stromal cells and primary mouse bone marrow stromal cells (BMSCs).
Methods
The 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.
Conclusion
The 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.
Objective
To predict and identify the target gene of miR-2861, elucidate the mechanism of miR-2861 promoting osteoblast differentiation.
Methods
Predict 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.
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。
Objective
To find novel microRNAs (miRNAs) specifically or preferentially expresses in mouse osteoblast, and explore their expression pattern in multiple mouse tissues and cells.
Methods
Firstly, 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.
Results
A 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.
Conclusion
In 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.
Objective
To investigate the role of miR-2861 in osteogenic differentiation of mouse ST2 stromal cells and primary mouse bone marrow stromal cells (BMSCs).
Methods
The 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.
Conclusion
The 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.
Objective
To predict and identify the target gene of miR-2861, elucidate the mechanism of miR-2861 promoting osteoblast differentiation.
Methods
Predict 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.
引文
[1]Rodriguez A, Griffiths-Jones S, Ashurst JL, et al. Identification of mammalian microRNA host genes and transcription units. Genome Res,2004, 14(10A):1902-1910
[2]Zeng Y. Principles of microRNA production and maturation. Oncogene,2006, 25(46):6156-6162
[3]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993, 75(5):843-854
[4]Lim LP, Glasner ME, Yekta S, et al. Vertebrate microRNA genes. Science, 2003,299(5612):1540
[5]Lai EC, Tomancak P, Williams RW, et al. Computational identification of Drosophila microRNA genes. Genome Biol,2003,4(7):R42
[6]Lim LP, Lau NC, Weinstein EG, et al. The microRNAs of Caenorhabditis elegans. Genes Dev,2003,17(8):991-1008
[7]Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature,2000, 403(6772):901-906
[8]Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature,2005, 436(7048):214-220
[9]Kloosterman WP, Lagendijk AK, Ketting RF, et al. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol,2007,5(8):e203
[10]Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs. Science,2001,294(5543):853-858
[11]Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res,2004, 32(Database issue):D 109-111
[12]Griffiths-Jones S, Grocock RJ, van Dongen S, et al. miRBase:microRNA sequences, targets and gene nomenclature. Nucleic Acids Res,2006,34(Database issue):D140-144
[13]Griffiths-Jones S, Saini HK, van Dongen S, et al. miRBase:tools for microRNA genomics. Nucleic Acids Res,2008,36(Database issue):D154-158
[14]Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res,2003,31(13):3406-3415
[15]Ro S, Park C, Song R, et al. Cloning and expression profiling of testis-expressed piRNA-like RNAs. RNA,2007,13(10):1693-1702
[16]Ro S, Park C, Sanders KM, et al. Cloning and expression profiling of testis-expressed microRNAs. Dev Biol,2007,311(2):592-602
[17]Berezikov E, van Tetering G, Verheul M, et al. Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis. Genome Res,2006,16(10):1289-1298
[18]Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science,2001,294(5543):862-864
[19]Lagos-Quintana M, Rauhut R, Yalcin A, et al. Identification of tissue-specific microRNAs from mouse. Curr Biol,2002,12(9):735-739
[20]Lagos-Quintana M, Rauhut R, Meyer J, et al. New microRNAs from mouse and human. RNA,2003,9(2):175-179
[21]Houbaviy HB, Murray MF and Sharp PA. Embryonic stem cell-specific MicroRNAs. Dev Cell,2003,5(2):351-358
[22]Kim J, Krichevsky A, Grad Y, et al. Identification of many microRNAs that copurify with polyribosomes in mammalian neurons. Proc Natl Acad Sci U S A,2004, 101(1):360-365
[23]Fu H, Tie Y, Xu C, et al. Identification of human fetal liver miRNAs by a novel method. FEBS Lett,2005,579(17):3849-3854
[24]Ruby JG, Jan C, Player C, et al. Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell,2006, 127(6):1193-1207
[25]Long JE, Chen HX. Identification and characteristics of cattle microRNAs by homology searching and small RNA cloning. Biochem Genet,2009, 47(5-6):329-343
[26]Oskowitz AZ, Lu J, Penfornis P, et al. Human multipotent stromal cells from bone marrow and microRNA:regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci U S A,2008,105(47):18372-18377
[27]Li Z, Hassan MQ, Jafferji M, et al. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem,2009, 284(23):15676-15684
[28]Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A, 2008,105(37):13906-13911
[29]Chen H, Shalom-Feuerstein R, Riley J, et al. miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro. Biochem Biophys Res Commun,2010,394(4):921-927
[30]Chen JF, Mandel EM, Thomson JM, et al. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet,2006, 38(2):228-233
[31]Esau C, Davis S, Murray SF, et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab,2006,3(2):87-98
[32]Chen CZ, Li L, Lodish HF, et al. MicroRNAs modulate hematopoietic lineage differentiation. Science,2004,303(5654):83-86
[1]Hime GR, Somers WG. Micro-RNA mediated regulation of proliferation, self-renewal and differentiation of mammalian stem cells. Cell Adh Migr,2009, 3(4):425-432
[2]Friedman JM, Jones PA. MicroRNAs:critical mediators of differentiation, development and disease. Swiss Med Wkly,2009,139(33-34):466-472
[3]Shi Y, Jin Y. MicroRNA in cell differentiation and development. Sci China C Life Sci,2009,52(3):205-211
[4]Sheedy FJ, O'Neill LA. Adding fuel to fire:microRNAs as a new class of mediators of inflammation. Ann Rheum Dis,2008,67 Suppl 3:iii50-55
[5]Krutzfeldt J, Stoffel M. MicroRNAs:a new class of regulatory genes affecting metabolism. Cell Metab,2006,4(1):9-12
[6]Tsitsiou E, Lindsay MA. microRNAs and the immune response. Curr Opin Pharmacol,2009,9(4):514-520
[7]Shenouda SK, Alahari SK, MicroRNA function in cancer:oncogene or a tumor suppressor? Cancer Metastasis Rev,2009,28(3-4):369-378
[8]Chen JF, Mandel EM, Thomson JM, et al. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet,2006, 38(2):228-233
[9]Chen CZ, Li L, Lodish HF, et al. MicroRNAs modulate hematopoietic lineage differentiation. Science,2004,303(5654):83-86
[10]Tuddenham L, Wheeler G, Ntounia-Fousara S, et al. The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Lett,2006, 580(17):4214-4217
[11]Mizuno Y, Yagi K, Tokuzawa Y, et al. miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun,2008,368(2):267-272
[12]Luzi E, Marini F, Sala SC, et al. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res,2008,23(2):287-295
[13]Mizuno Y, Tokuzawa Y, Ninomiya Y, et al. miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b. FEBS Lett,2009,583(13):2263-2268
[14]Li Z, Hassan MQ, Jafferji M, et al. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem,2009, 284(23):15676-15684
[15]Banerjee C, Javed A, Choi JY, et al. Differential regulation of the two principal Runx2/Cbfal n-terminal isoforms in response to bone morphogenetic protein-2 during development of the osteoblast phenotype. Endocrinology,2001, 142(9):4026-4039
[16]Osyczka AM, Leboy PS. Bone morphogenetic protein regulation of early osteoblast genes in human marrow stromal cells is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling. Endocrinology, 2005,146(8):3428-3437
[17]Kim YJ, Kim HN, Park EK, et al. The bone-related Zn finger transcription factor Osterix promotes proliferation of mesenchymal cells. Gene,2006, 366(1):145-151
[18]Xiao G, Jiang D, Ge C, et al. Cooperative interactions between activating transcription factor 4 and Runx2/Cbfal stimulate osteoblast-specific osteocalcin gene expression. J Biol Chem,2005,280(35):30689-30696
[19]Bialek P, Kern B, Yang X, et al. A twist code determines the onset of osteoblast differentiation. Dev Cell,2004,6(3):423-435
[20]Lee KS, Hong SH, Bae SC. Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene,2002,21 (47):7156-7163
[21]Etheridge SL, Spencer GJ, Heath DJ, et al. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem Cells,2004,22(5):849-860
[22]Levy JB, Schindler C, Raz R, et al. Activation of the JAK-STAT signal transduction pathway by oncostatin-M cultured human and mouse osteoblastic cells. Endocrinology,1996,137(4):1159-1165
[23]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993, 75(5):843-854
[24]Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature,2000, 403(6772):901-906
[25]Krichevsky AM, Sonntag KC, Isacson O, et al. Specific microRNAs modulate embryonic stem cell-derived neurogenesis. Stem Cells,2006, 24(4):857-864
[26]Wang Q, Huang Z, Xue H, et al. MicroRNA miR-24 inhibits erythropoiesis by targeting activin type Ⅰ receptor ALK4. Blood,2008,111(2):588-595
[27]Fontana L, Pelosi E, Greco P, et al. MicroRNAs 17-5p-20a-106a control monocytopoiesis through AML1 targeting and M-CSF receptor upregulation. Nat Cell Biol,2007,9(7):775-787
[28]Anderson C, Catoe H, Werner R. MIR-206 regulates connexin43 expression during skeletal muscle development. Nucleic Acids Res,2006,34(20):5863-5871
[29]McCarthy JJ. MicroRNA-206:the skeletal muscle-specific myomiR. Biochim Biophys Acta,2008,1779(11):682-691
[30]Wong CF, Tellam RL. MicroRNA-26a targets the histone methyltransferase Enhancer of Zeste homolog 2 during myogenesis. J Biol Chem,2008, 283(15):9836-9843
[31]Lee SO, Masyuk T, Splinter P, et al. MicroRNA15a modulates expression of the cell-cycle regulator Cdc25A and affects hepatic cystogenesis in a rat model of polycystic kidney disease. J Clin Invest,2008,118(11):3714-3724
[32]张菊,关恒云,张鹏举,等.miRNA let-7al真核表达载体的构建及对肺癌细胞增殖的影响.中国病理生理杂志,2009,125(8)：1495-1500
[33]赵健,闫康,高杰,等.miR-21真核表达载体的构建及其活性鉴定.细胞与分子免疫学杂志,2009,25(11)：1047-1052
[34]Aubin JE, Liu F, Malaval L, et al. Osteoblast and chondroblast differentiation. Bone,1995,17(2 Suppl):77S-83S
[35]Levy MM, Joyner CJ, Virdi AS, et al. Osteoprogenitor cells of mature human skeletal muscle tissue:an in vitro study. Bone,2001,29(4):317-322
[36]Owen TA, Aronow M, Shalhoub V, et al. Progressive development of the rat osteoblast phenotype in vitro:reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol,1990,143(3):420-430
[37]Komori T. Regulation of bone development and maintenance by Runx2. Front Biosci,2008,13:898-903
[38]Otto F, Thornell AP, Crompton T, et al. Cbfal, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell,1997,89(5):765-771
[39]Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfal results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell, 1997,89(5):755-764
[40]Shui C, Spelsberg TC, Riggs BL, et al. Changes in Runx2/Cbfal expression and activity during osteoblastic differentiation of human bone marrow stromal cells. J Bone Miner Res,2003.18(2):213-221
[41]Huang J, Zhao L, Xing L, et al. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells,2010, 28(2):357-364
[42]Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A, 2008,105(37):13906-13911
[1]Carthew RW, Sontheimer EJ. Origins and Mechanisms of miRNAs and siRNAs. Cell,2009,136(4):642-655
[2]John B, Enright AJ, Aravin A, et al. Human MicroRNA targets. PLoS Biol, 2004,2(11):e363
[3]Lewis BP, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalian microRNA targets. Cell,2003,115(7):787-798
[4]Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 2005,120(1):15-20
[5]Krek A, Grun D, Poy MN, et al. Combinatorial microRNA target predictions. Nat Genet,2005,37(5):495-500
[6]Lytle JR, Yario TA, Steitz JA. Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5'UTR as in the 3'UTR. Proc Natl Acad Sci U S A, 2007,104(23):9667-9672
[7]Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res,2009, 19(7):1175-1183
[8]Tay Y, Zhang J, Thomson AM, et al. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature,2008, 455(7216):1124-1128
[9]Zhou X, Duan X, Qian J, et al. Abundant conserved microRNA target sites in the 5'-untranslated region and coding sequence. Genetica,2009,137(2):159-164
[10]Qin W, Shi Y, Zhao B, et al. miR-24 regulates apoptosis by targeting the open reading frame (ORF) region of FAF1 in cancer cells. PLoS One,2010, 5(2):e9429
[11]Miranda KC, Huynh T, Tay Y, et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell,2006,126(6):1203-1217
[12]Didiano D, Hobert O. Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions. Nat Struct Mol Biol,2006,13(9):849-851
[13]Javed A, Afzal F, Bae JS, et al. Specific residues of RUNX2 are obligatory for formation of BMP2-induced RUNX2-SMAD complex to promote osteoblast differentiation. Cells Tissues Organs,2009,189(1-4):133-137
14] Weissman AM. Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol,2001,2(3):169-178
[15]Hershko A. Ubiquitin:roles in protein modification and breakdown. Cell, 1983,34(1):11-12
[16]Ciechanover A, Orian A, Schwartz AL. The ubiquitin-mediated proteolytic pathway:mode of action and clinical implications. J Cell Biochem Suppl,2000, 34:40-51
[17]Zhao M, Qiao M, Harris SE, et al. Smurfl inhibits osteoblast differentiation and bone formation in vitro and in vivo. J Biol Chem,2004,279(13):12854-12859
[18]Zhao M, Qiao M, Oyajobi BO, et al. E3 ubiquitin ligase Smurfl mediates core-binding factor alphal/Runx2 degradation and plays a specific role in osteoblast differentiation. J Biol Chem,2003,278(30):27939-27944
[19]Kaneki H, Guo R, Chen D, et al. Tumor necrosis factor promotes Runx2 degradation through up-regulation of Smurfl and Smurf2 in osteoblasts. J Biol Chem, 2006,281(7):4326-4333
[20]Jeon EJ, Lee KY, Choi NS, et al. Bone morphogenetic protein-2 stimulates Runx2 acetylation. J Biol Chem,2006,281(24):16502-16511
[21]Schroeder TM, Kahler RA, Li X, et al. Histone deacetylase 3 interacts with runx2 to repress the osteocalcin promoter and regulate osteoblast differentiation. J Biol Chem,2004,279(40):41998-42007
[22]Schroeder TM, Westendorf JJ. Histone deacetylase inhibitors promote osteoblast maturation. J Bone Miner Res,2005,20(12):2254-2263
[23]Cho HH, Park HT, Kim YJ, et al. Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors. J Cell Biochem, 2005,96(3):533-542
[24]Luzi E, Marini F, Sala SC, et al. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res,2008,23(2):287-295
[25]Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A, 2008,105(37):13906-13911
[26]Huang J, Zhao L, Xing L, et al. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells,2010, 28(2):357-364
[27]Kapinas K, Kessler CB, Delany AM. miR-29 suppression of osteonectin in osteoblasts:regulation during differentiation and by canonical Wnt signaling. J Cell Biochem,2009,108(1):216-224
[28]Itoh T, Nozawa Y, Akao Y. MicroRNA-141 and-200a are involved in bone morphogenetic protein-2-induced mouse pre-osteoblast differentiation by targeting distal-less homeobox 5. J Biol Chem,2009,284(29):19272-19279
[29]Li Z, Hassan MQ, Jafferji M, et al. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem,2009, 284(23):15676-15684
[30]Mizuno Y, Tokuzawa Y, Ninomiya Y, et al. miR-210 promotes osteoblastic differentiation through inhibition of AcvRlb. FEBS Lett,2009,583(13):2263-2268
[1]Berezikov E, Guryev V, van de Belt J, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell,2005,120(1):21-24
[2]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993, 75(5):843-854
[3]Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature,2000, 403(6772):901-906
[4]Lau NC, Lim LP, Weinstein EG, et al. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science,2001, 294(5543):858-862
[5]Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science,2001,294(5543):862-864
[6]Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs. Science,2001,294(5543):853-858.
[7]Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res,2004, 32(Database issue):D109-111
[8]Griffiths-Jones S,Grocock RJ, van Dongen S, et al. miRBase:microRNA sequences, targets and gene nomenclature. Nucleic Acids Res,2006,34(Database issue):D 140-144
[9]Ambros V, Bartel B, Bartel DP, et al. A uniform system for microRNA annotation. RNA,2003,9(3):277-279
[10]Zhang BH, Pan XP, Cox SB, et al. Evidence that miRNAs are different from other RNAs. Cell Mol Life Sci,2006,63(2):246-254
[11]Pan X, Zhang B, San Francisco M, et al. Characterizing viral microRNAs and its application on identifying new microRNAs in viruses. J Cell Physiol,2007, 211(1):10-18
[12]Kim VN, Nam JW. Genomics of microRNA. Trends Genet,2006, 22(3):165-173
[13]Cullen BR. Transcription and processing of human microRNA precursors. Mol Cell,2004,16(6):861-865
[14]Cai X, Hagedorn CH, Cullen BR. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA,2004, 10(12):1957-1966
[15]Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase Ⅱ. EMBO J,2004,23(20):4051-4060
[16]Borchert GM, Lanier W, Davidson BL. RNA polymerase Ⅲ transcribes human microRNAs. Nat Struct Mol Biol,2006,13(12):1097-1101
[17]Han J, Lee Y, Yeom KH, et al. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell,2006,125(5):887-901
[18]Han J, Pedersen JS, Kwon SC, et al. Posttranscriptional crossregulation between Drosha and DGCR8. Cell,2009,136(1):75-84
[19]Denli AM, Tops BB, Plasterk RH, et al. Processing of primary microRNAs by the Microprocessor complex. Nature,2004,432(7014):231-235
[20]Gregory RI, Yan KP, Amuthan G, et al. The Microprocessor complex mediates the genesis of microRNAs. Nature,2004,432(7014):235-240
[21]Han J, Lee Y, Yeom KH, et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev,2004,18(24):3016-3027
[22]Du Z, Lee JK, Tjhen R, et al. Structural and biochemical insights into the dicing mechanism of mouse Dicer:a conserved lysine is critical for dsRNA cleavage. Proc Natl Acad Sci U S A,2008,105(7):2391-2396
[23]Zeng Y, Cullen BR. Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. Nucleic Acids Res,2004,32(16):4776-4785
[24]Yi R, Qin Y, Macara IG, et al. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev,2003,17(24):3011-3016
[25]Lund E, Guttinger S, Calado A, et al. Nuclear export of microRNA precursors. Science,2004,303(5654):95-98
[26]Bohnsack MT, Czaplinski K and Gorlich D. Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA,2004,10(2):185-191
[27]Shibata S, Sasaki M, Miki T, et al. Exportin-5 orthologues are functionally divergent among species. Nucleic Acids Res,2006,34(17):4711-4721
[28]Forstemann K, Tomari Y, Du T, et al. Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol,2005,3(7):e236
[29]Hutvagner G, McLachlan J, Pasquinelli AE, et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science,2001,293(5531):834-838
[30]Kurihara Y, Watanabe Y. Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci U S A,2004, 101(34):12753-12758
[31]Park MY, Wu G, Gonzalez-Sulser A, et al. Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci U S A,2005,102(10):3691-3696
[32]Lee Y, Hur I, Park SY, et al. The role of PACT in the RNA silencing pathway. EMBO J,2006,25(3):522-532
[33]Tay Y, Zhang J, Thomson AM, et al. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature,2008, 455(7216):1124-1128
[34]Lytle JR, Yario TA, Steitz JA. Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5'UTR as in the 3'UTR. Proc Natl Acad Sci U S A, 2007,104(23):9667-9672
[35]Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res,2009, 19(7):1175-1183
[36]Ruby JG, Jan CH, Bartel DP. Intronic microRNA precursors that bypass Drosha processing. Nature,2007,448(7149):83-86
[37]Okamura K, Hagen JW, Duan H, et al. The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila. Cell,2007,130(1):89-100
[38]Berezikov E, Chung WJ, Willis J, et al. Mammalian mirtron genes. Mol Cell, 2007,28(2):328-336
[39]Lau PW, MacRae IJ. The molecular machines that mediate microRNA maturation. J Cell Mol Med,2009,13(1):54-60
[40]Carthew RW, Sontheimer EJ. Origins and Mechanisms of miRNAs and siRNAs. Cell,2009,136(4):642-655
[41]Hornstein E, Mansfield JH, Yekta S, et al. The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development. Nature,2005,438(7068):671-674
[42]Bruno I, Wilkinson MF. P-bodies react to stress and nonsense. Cell,2006, 125(6):1036-1038
[43]Miyoshi K, Okada TN, Siomi H, et al. Characterization of the miRNA-RISC loading complex and miRNA-RISC formed in the Drosophila miRNA pathway. RNA, 2009,15(7):1282-1291
[44]Eulalio A, Behm-Ansmant I, Schweizer D, et al. P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol Cell Biol,2007, 27(11):3970-3981
[45]Schwarz DS, Hutvagner G, Du T, et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell,2003,115(2):199-208
[46]Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell,2003,115(2):209-216
[47]Hutvagner G. Small RNA asymmetry in RNAi:function in RISC assembly and gene regulation. FEBS Lett,2005,579(26):5850-5857
[48]MacRae IJ, Ma E, Zhou M, et al. In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci U S A,2008,105(2):512-517
[49]Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs:how many mechanisms? Trends Cell Biol,2007,17(3):118-126
[50]Kozak M. Faulty old ideas about translational regulation paved the way for current confusion about how microRNAs function. Gene,2008,423(2):108-115
[51]Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate translation. Science,2007,318(5858):1931-1934.
[52]Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res,2003,31(13):3406-3415
[53]Luciano DJ, Mirsky H, Vendetti NJ, et al. RNA editing of a miRNA precursor. RNA,2004,10(8):1174-1177
[54]Yang W, Chendrimada TP, Wang Q, et al. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases. Nat Struct Mol Biol,2006,13(1):13-21
[55]Yang Z, Ebright YW, Yu B, et al. HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2'OH of the 3'terminal nucleotide. Nucleic Acids Res,2006,34(2):667-675
[56]Wang JF, Zhou H, Chen YQ, et al. Identification of 20 microRNAs from Oryza sativa. Nucleic Acids Res,2004,32(5):1688-1695
[57]Berezikov E, Cuppen E, Plasterk RH. Approaches to microRNA discovery. Nat Genet,2006,38 Suppl:S2-7
[58]Lim LP, Lau NC, Weinstein EG, et al. The microRNAs of Caenorhabditis elegans. Genes Dev,2003,17(8):991-1008
[59]Wang X, Zhang J, Li F, et al. MicroRNA identification based on sequence and structure alignment. Bioinformatics,2005,21(18):3610-3614
[60]Lim LP, Glasner ME, Yekta S, et al. Vertebrate microRNA genes. Science, 2003,299(5612):1540
[61]Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet,2005,37(7):766-770
[62]Huang TH, Fan B, Rothschild MF, et al. MiRFinder:an improved approach and software implementation for genome-wide fast microRNA precursor scans. BMC Bioinformatics,2007,8:341
[63]Berninger P, Gaidatzis D, van Nimwegen E, et al. Computational analysis of small RNA cloning data. Methods,2008,44(1):13-21
[64]Creighton CJ, Reid JG, Gunaratne PH. Expression profiling of microRNAs by deep sequencing. Brief Bioinform,2009,10(5):490-497
[65]Hafner M, Landgraf P, Ludwig J, et al. Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods,2008, 44(1):3-12
[66]Addo-Quaye C, Miller W, Axtell MJ. CleaveLand:a pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics,2009, 25(1):130-131
[67]Hackenberg M, Sturm M, Langenberger D, et al. miRanalyzer:a microRNA detection and analysis tool for next-generation sequencing experiments. Nucleic Acids Res,2009,37(Web Server issue):W68-76
[68]Friedlander MR, Chen W, Adamidi C, et al. Discovering microRNAs from deep sequencing data using miRDeep. Nat Biotechnol,2008,26(4):407-415
[69]Bonnet E, Wuyts J, Rouze P, et al. Evidence that microRNA precursors, unlike other non-coding RNAs, have lower folding free energies than random sequences. Bioinformatics,2004,20(17):2911-2917
[70]Washietl S, Hofacker IL, Stadler PF. Fast and reliable prediction of noncoding RNAs. Proc Natl Acad Sci U S A,2005,102(7):2454-2459
[71]Washietl S, Hofacker IL, Lukasser M, et al. Mapping of conserved RNA secondary structures predicts thousands of functional noncoding RNAs in the human genome. Nat Biotechnol,2005,23(11):1383-1390
[72]Sewer A, Paul N, Landgraf P, et al. Identification of clustered microRNAs using an ab initio prediction method. BMC Bioinformatics,2005,6:267
[73]Xue C, Li F, He T, et al. Classification of real and pseudo microRNA precursors using local structure-sequence features and support vector machine. BMC Bioinformatics,2005,6:310
[74]Pfeffer S, Sewer A, Lagos-Quintana M, et al. Identification of microRNAs of the herpesvirus family. Nat Methods,2005,2(4):269-276
[75]Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression in zebrafish embryonic development. Science,2005,309(5732):310-311
[76]Kloosterman WP, Wienholds E, de Bruijn E, et al. In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods,2006,3(1):27-29
[77]Wheeler G, Ntounia-Fousara S, Granda B, et al. Identification of new central nervous system specific mouse microRNAs. FEBS Lett,2006,580(9):2195-2200
[78]Tuddenham L, Wheeler G, Ntounia-Fousara S, et al. The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Lett,2006, 580(17):4214-4217
[79]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods,2001, 25(4):402-408
[80]Tardif G, Hum D, Pelletier JP, et al. Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes. BMC Musculoskelet Disord,2009,10:148
[81]Benes V, Castoldi M. Expression profiling of microRNA using real-ime quantitative PCR, how to use it and what is available. Methods,2010,50(4):244-249
[82]Schmittgen TD, Jiang J, Liu Q, et al. A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res,2004,32(4):e43
[83]Jiang J, Lee EJ, Gusev Y, et al. Real-time expression profiling of microRNA precursors in human cancer cell lines. Nucleic Acids Res,2005,33(17):5394-5403
[84]Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res,2005,33(20):e179
[85]Liang Y, Ridzon D, Wong L, et al. Characterization of microRNA expression profiles in normal human tissues. BMC Genomics,2007,8:166
[86]Tang F, Hajkova P, Barton SC, et al. MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res,2006,34(2):e9
[87]Tang F, Hajkova P, Barton SC, et al.220-plex microRNA expression profile of a single cell. Nat Protoc,2006,1(3):1154-1159
[88]Lao K, Xu NL, Sun YA, et al. Real time PCR profiling of 330 human micro-RNAs. Biotechnol J,2007,2(1):33-35
[89]Ortega FJ, Moreno-Navarrete JM, Pardo G, et al. MiRNA expression profile of human subcutaneous adipose and during adipocyte differentiation. PLoS One,2010, 5(2):e9022
[90]Li W, Ruan K. MicroRNA detection by microarray. Anal Bioanal Chem, 2009.394(4):1117-1124
[91]Liu CG, Calin GA. Meloon B, et al. An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc Natl Acad Sci USA,2004,101(26):9740-9744
[92]Wilson KD, Venkatasubrahmanyam S, Jia F, et al. MicroRNA profiling of human-induced pluripotent stem cells. Stem Cells Dev,2009,18(5):749-758
[93]Bostjancic E, Zidar N, Glavac D. MicroRNA microarray expression profiling in human myocardial infarction. Dis Markers,2009,27(6):255-268
[94]Brennecke J, Stark A, Russell RB, et al. Principles of microRNA-target recognition. PLoS Biol,2005,3(3):e85
[95]Enright AJ, John B, Gaul U, et al. MicroRNA targets in Drosophila. Genome Biol,2003,5(1):R1
[96]Lewis BP, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalian microRNA targets. Cell,2003,115(7):787-798
[97]Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 2005,120(1):15-20
[98]Krek A, Grun D, Poy MN, et al. Combinatorial microRNA target predictions. Nat Genet,2005,37(5):495-500
[99]Grun D, Wang YL, Langenberger D, et al. microRNA target predictions across seven Drosophila species and comparison to mammalian targets. PLoS Comput Biol,2005, 1(1):e13
[100]Lall S, Grun D, Krek A, et al. A genome-wide map of conserved microRNA targets in C. elegans. Curr Biol,2006.16(5):460-471
[101]Miranda KC, Huynh T, Tay Y, et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell,2006,126(6):1203-1217
[102]Li Z, Hassan MQ, Jafferji M, et al. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem,2009, 284(23):15676-15684
[103]Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A, 2008.105(37):13906-13911
[104]Oskowitz AZ, Lu J, Penfornis P, et al. Human multipotent stromal cells from bone marrow and microRNA:regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci U S A,2008,105(47):18372-18377
[105]Winnard RG, Gerstenfeld LC, Toma CD, et al. Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts. J Bone Miner Res,1995,10(12):1969-1977
[106]Yang XB, Roach HI, Clarke NM, et al. Human osteoprogenitor growth and differentiation on synthetic biodegradable structures after surface modification. Bone, 2001,29(6):523-531
[107]Zhang Y, Feng XH, Derynck R. Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription. Nature,1998, 394(6696):909-913
[108]Jensen ED, Gopalakrishnan R, Westendorf JJ. Regulation of gene expression in osteoblasts. Biofactors,2010,36(1):25-32
[109]Kim YJ, Kim HN, Park EK, et al. The bone-related Zn finger transcription factor Osterix promotes proliferation of mesenchymal cells. Gene,2006, 366(1):145-151
[110]Bialek P, Kern B, Yang X, et al. A twist code determines the onset of osteoblast differentiation. Dev Cell,2004,6(3):423-435
[111]Xiao G, Jiang D, Ge C, et al. Cooperative interactions between activating transcription factor 4 and Runx2/Cbfal stimulate osteoblast-specific osteocalcin gene expression. J Biol Chem,2005,280(35):30689-30696
[112]Etheridge SL, Spencer GJ, Heath DJ, et al. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem Cells,2004,22(5):849-860
[113]Osyczka AM, Leboy PS. Bone morphogenetic protein regulation of early osteoblast genes in human marrow stromal cells is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling. Endocrinology, 2005.146(8):3428-3437
[114]Levy JB, Schindler C, Raz R, et al. Activation of the JAK-STAT signal transduction pathway by oncostatin-M cultured human and mouse osteoblastic cells. Endocrinology,1996,137(4):1159-1165
[115]Lee KS, Hong SH, Bae SC. Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene,2002,21(47):7156-7163
[116]Luzi E, Marini F, Sala SC, et al. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res,2008,23(2):287-295
[117]Huang J, Zhao L, Xing L, et al. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells,2010, 28(2):357-364
[118]Kapinas K, Kessler CB, Delany AM. miR-29 suppression of osteonectin in osteoblasts:regulation during differentiation and by canonical Wnt signaling. J Cell Biochem,2009,108(1):216-224
[119]Mizuno Y, Yagi K, Tokuzawa Y, et al. miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun,2008,368(2):267-272
[120]Itoh T, Nozawa Y, Akao Y. MicroRNA-141 and-200a are involved in bone morphogenetic protein-2-induced mouse pre-osteoblast differentiation by targeting distal-less homeobox 5. J Biol Chem,2009,284(29):19272-19279
[121]Mizuno Y, Tokuzawa Y, Ninomiya Y, et al. miR-210 promotes osteoblastic differentiation through inhibition of AcvRlb. FEBS Lett,2009,583(13):2263-2268
[122]Li H, Xie H, Liu W, et al. A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans. J Clin Invest,2009,119(12):3666-3677
[123]Kobayashi T, Lu J, Cobb BS, et al. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. Proc Natl Acad Sci U S A,2008, 105(6):1949-1954
[124]Lakshmipathy U, Love B, Goff LA, et al. MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells. Stem Cells Dev,2007, 16(6):1003-1016
[125]Suomi S, Taipaleenmaki H, Seppanen A, et al. MicroRNAs Regulate Osteogenesis and Chondrogenesis of Mouse Bone Marrow Stromal Cells. Gene Regul Syst Bio,2008,2:177-191
[126]Lin EA, Kong L, Bai XH, et al. miR-199a, a bone morphogenic protein 2-responsive MicroRNA, regulates chondrogenesis via direct targeting to Smadl. J Biol Chem,2009,284(17):11326-11335
[127]Sugatani T, Hruska KA. MicroRNA-223 is a key factor in osteoclast differentiation. J Cell Biochem,2007,101(4):996-999
[128]Sugatani T and Hruska KA. Impaired micro-RNA pathways diminish osteoclast differentiation and function. J Biol Chem,2009,284(7):4667-4678
[2]Zeng Y. Principles of microRNA production and maturation. Oncogene,2006, 25(46):6156-6162
[3]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993, 75(5):843-854
[4]Lim LP, Glasner ME, Yekta S, et al. Vertebrate microRNA genes. Science, 2003,299(5612):1540
[5]Lai EC, Tomancak P, Williams RW, et al. Computational identification of Drosophila microRNA genes. Genome Biol,2003,4(7):R42
[6]Lim LP, Lau NC, Weinstein EG, et al. The microRNAs of Caenorhabditis elegans. Genes Dev,2003,17(8):991-1008
[7]Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature,2000, 403(6772):901-906
[8]Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature,2005, 436(7048):214-220
[9]Kloosterman WP, Lagendijk AK, Ketting RF, et al. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol,2007,5(8):e203
[10]Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs. Science,2001,294(5543):853-858
[11]Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res,2004, 32(Database issue):D 109-111
[12]Griffiths-Jones S, Grocock RJ, van Dongen S, et al. miRBase:microRNA sequences, targets and gene nomenclature. Nucleic Acids Res,2006,34(Database issue):D140-144
[13]Griffiths-Jones S, Saini HK, van Dongen S, et al. miRBase:tools for microRNA genomics. Nucleic Acids Res,2008,36(Database issue):D154-158
[14]Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res,2003,31(13):3406-3415
[15]Ro S, Park C, Song R, et al. Cloning and expression profiling of testis-expressed piRNA-like RNAs. RNA,2007,13(10):1693-1702
[16]Ro S, Park C, Sanders KM, et al. Cloning and expression profiling of testis-expressed microRNAs. Dev Biol,2007,311(2):592-602
[17]Berezikov E, van Tetering G, Verheul M, et al. Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis. Genome Res,2006,16(10):1289-1298
[18]Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science,2001,294(5543):862-864
[19]Lagos-Quintana M, Rauhut R, Yalcin A, et al. Identification of tissue-specific microRNAs from mouse. Curr Biol,2002,12(9):735-739
[20]Lagos-Quintana M, Rauhut R, Meyer J, et al. New microRNAs from mouse and human. RNA,2003,9(2):175-179
[21]Houbaviy HB, Murray MF and Sharp PA. Embryonic stem cell-specific MicroRNAs. Dev Cell,2003,5(2):351-358
[22]Kim J, Krichevsky A, Grad Y, et al. Identification of many microRNAs that copurify with polyribosomes in mammalian neurons. Proc Natl Acad Sci U S A,2004, 101(1):360-365
[23]Fu H, Tie Y, Xu C, et al. Identification of human fetal liver miRNAs by a novel method. FEBS Lett,2005,579(17):3849-3854
[24]Ruby JG, Jan C, Player C, et al. Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell,2006, 127(6):1193-1207
[25]Long JE, Chen HX. Identification and characteristics of cattle microRNAs by homology searching and small RNA cloning. Biochem Genet,2009, 47(5-6):329-343
[26]Oskowitz AZ, Lu J, Penfornis P, et al. Human multipotent stromal cells from bone marrow and microRNA:regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci U S A,2008,105(47):18372-18377
[27]Li Z, Hassan MQ, Jafferji M, et al. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem,2009, 284(23):15676-15684
[28]Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A, 2008,105(37):13906-13911
[29]Chen H, Shalom-Feuerstein R, Riley J, et al. miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro. Biochem Biophys Res Commun,2010,394(4):921-927
[30]Chen JF, Mandel EM, Thomson JM, et al. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet,2006, 38(2):228-233
[31]Esau C, Davis S, Murray SF, et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab,2006,3(2):87-98
[32]Chen CZ, Li L, Lodish HF, et al. MicroRNAs modulate hematopoietic lineage differentiation. Science,2004,303(5654):83-86
[1]Hime GR, Somers WG. Micro-RNA mediated regulation of proliferation, self-renewal and differentiation of mammalian stem cells. Cell Adh Migr,2009, 3(4):425-432
[2]Friedman JM, Jones PA. MicroRNAs:critical mediators of differentiation, development and disease. Swiss Med Wkly,2009,139(33-34):466-472
[3]Shi Y, Jin Y. MicroRNA in cell differentiation and development. Sci China C Life Sci,2009,52(3):205-211
[4]Sheedy FJ, O'Neill LA. Adding fuel to fire:microRNAs as a new class of mediators of inflammation. Ann Rheum Dis,2008,67 Suppl 3:iii50-55
[5]Krutzfeldt J, Stoffel M. MicroRNAs:a new class of regulatory genes affecting metabolism. Cell Metab,2006,4(1):9-12
[6]Tsitsiou E, Lindsay MA. microRNAs and the immune response. Curr Opin Pharmacol,2009,9(4):514-520
[7]Shenouda SK, Alahari SK, MicroRNA function in cancer:oncogene or a tumor suppressor? Cancer Metastasis Rev,2009,28(3-4):369-378
[8]Chen JF, Mandel EM, Thomson JM, et al. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet,2006, 38(2):228-233
[9]Chen CZ, Li L, Lodish HF, et al. MicroRNAs modulate hematopoietic lineage differentiation. Science,2004,303(5654):83-86
[10]Tuddenham L, Wheeler G, Ntounia-Fousara S, et al. The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Lett,2006, 580(17):4214-4217
[11]Mizuno Y, Yagi K, Tokuzawa Y, et al. miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun,2008,368(2):267-272
[12]Luzi E, Marini F, Sala SC, et al. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res,2008,23(2):287-295
[13]Mizuno Y, Tokuzawa Y, Ninomiya Y, et al. miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b. FEBS Lett,2009,583(13):2263-2268
[14]Li Z, Hassan MQ, Jafferji M, et al. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem,2009, 284(23):15676-15684
[15]Banerjee C, Javed A, Choi JY, et al. Differential regulation of the two principal Runx2/Cbfal n-terminal isoforms in response to bone morphogenetic protein-2 during development of the osteoblast phenotype. Endocrinology,2001, 142(9):4026-4039
[16]Osyczka AM, Leboy PS. Bone morphogenetic protein regulation of early osteoblast genes in human marrow stromal cells is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling. Endocrinology, 2005,146(8):3428-3437
[17]Kim YJ, Kim HN, Park EK, et al. The bone-related Zn finger transcription factor Osterix promotes proliferation of mesenchymal cells. Gene,2006, 366(1):145-151
[18]Xiao G, Jiang D, Ge C, et al. Cooperative interactions between activating transcription factor 4 and Runx2/Cbfal stimulate osteoblast-specific osteocalcin gene expression. J Biol Chem,2005,280(35):30689-30696
[19]Bialek P, Kern B, Yang X, et al. A twist code determines the onset of osteoblast differentiation. Dev Cell,2004,6(3):423-435
[20]Lee KS, Hong SH, Bae SC. Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene,2002,21 (47):7156-7163
[21]Etheridge SL, Spencer GJ, Heath DJ, et al. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem Cells,2004,22(5):849-860
[22]Levy JB, Schindler C, Raz R, et al. Activation of the JAK-STAT signal transduction pathway by oncostatin-M cultured human and mouse osteoblastic cells. Endocrinology,1996,137(4):1159-1165
[23]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993, 75(5):843-854
[24]Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature,2000, 403(6772):901-906
[25]Krichevsky AM, Sonntag KC, Isacson O, et al. Specific microRNAs modulate embryonic stem cell-derived neurogenesis. Stem Cells,2006, 24(4):857-864
[26]Wang Q, Huang Z, Xue H, et al. MicroRNA miR-24 inhibits erythropoiesis by targeting activin type Ⅰ receptor ALK4. Blood,2008,111(2):588-595
[27]Fontana L, Pelosi E, Greco P, et al. MicroRNAs 17-5p-20a-106a control monocytopoiesis through AML1 targeting and M-CSF receptor upregulation. Nat Cell Biol,2007,9(7):775-787
[28]Anderson C, Catoe H, Werner R. MIR-206 regulates connexin43 expression during skeletal muscle development. Nucleic Acids Res,2006,34(20):5863-5871
[29]McCarthy JJ. MicroRNA-206:the skeletal muscle-specific myomiR. Biochim Biophys Acta,2008,1779(11):682-691
[30]Wong CF, Tellam RL. MicroRNA-26a targets the histone methyltransferase Enhancer of Zeste homolog 2 during myogenesis. J Biol Chem,2008, 283(15):9836-9843
[31]Lee SO, Masyuk T, Splinter P, et al. MicroRNA15a modulates expression of the cell-cycle regulator Cdc25A and affects hepatic cystogenesis in a rat model of polycystic kidney disease. J Clin Invest,2008,118(11):3714-3724
[32]张菊,关恒云,张鹏举,等.miRNA let-7al真核表达载体的构建及对肺癌细胞增殖的影响.中国病理生理杂志,2009,125(8)：1495-1500
[33]赵健,闫康,高杰,等.miR-21真核表达载体的构建及其活性鉴定.细胞与分子免疫学杂志,2009,25(11)：1047-1052
[34]Aubin JE, Liu F, Malaval L, et al. Osteoblast and chondroblast differentiation. Bone,1995,17(2 Suppl):77S-83S
[35]Levy MM, Joyner CJ, Virdi AS, et al. Osteoprogenitor cells of mature human skeletal muscle tissue:an in vitro study. Bone,2001,29(4):317-322
[36]Owen TA, Aronow M, Shalhoub V, et al. Progressive development of the rat osteoblast phenotype in vitro:reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol,1990,143(3):420-430
[37]Komori T. Regulation of bone development and maintenance by Runx2. Front Biosci,2008,13:898-903
[38]Otto F, Thornell AP, Crompton T, et al. Cbfal, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell,1997,89(5):765-771
[39]Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfal results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell, 1997,89(5):755-764
[40]Shui C, Spelsberg TC, Riggs BL, et al. Changes in Runx2/Cbfal expression and activity during osteoblastic differentiation of human bone marrow stromal cells. J Bone Miner Res,2003.18(2):213-221
[41]Huang J, Zhao L, Xing L, et al. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells,2010, 28(2):357-364
[42]Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A, 2008,105(37):13906-13911
[1]Carthew RW, Sontheimer EJ. Origins and Mechanisms of miRNAs and siRNAs. Cell,2009,136(4):642-655
[2]John B, Enright AJ, Aravin A, et al. Human MicroRNA targets. PLoS Biol, 2004,2(11):e363
[3]Lewis BP, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalian microRNA targets. Cell,2003,115(7):787-798
[4]Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 2005,120(1):15-20
[5]Krek A, Grun D, Poy MN, et al. Combinatorial microRNA target predictions. Nat Genet,2005,37(5):495-500
[6]Lytle JR, Yario TA, Steitz JA. Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5'UTR as in the 3'UTR. Proc Natl Acad Sci U S A, 2007,104(23):9667-9672
[7]Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res,2009, 19(7):1175-1183
[8]Tay Y, Zhang J, Thomson AM, et al. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature,2008, 455(7216):1124-1128
[9]Zhou X, Duan X, Qian J, et al. Abundant conserved microRNA target sites in the 5'-untranslated region and coding sequence. Genetica,2009,137(2):159-164
[10]Qin W, Shi Y, Zhao B, et al. miR-24 regulates apoptosis by targeting the open reading frame (ORF) region of FAF1 in cancer cells. PLoS One,2010, 5(2):e9429
[11]Miranda KC, Huynh T, Tay Y, et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell,2006,126(6):1203-1217
[12]Didiano D, Hobert O. Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions. Nat Struct Mol Biol,2006,13(9):849-851
[13]Javed A, Afzal F, Bae JS, et al. Specific residues of RUNX2 are obligatory for formation of BMP2-induced RUNX2-SMAD complex to promote osteoblast differentiation. Cells Tissues Organs,2009,189(1-4):133-137
14] Weissman AM. Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol,2001,2(3):169-178
[15]Hershko A. Ubiquitin:roles in protein modification and breakdown. Cell, 1983,34(1):11-12
[16]Ciechanover A, Orian A, Schwartz AL. The ubiquitin-mediated proteolytic pathway:mode of action and clinical implications. J Cell Biochem Suppl,2000, 34:40-51
[17]Zhao M, Qiao M, Harris SE, et al. Smurfl inhibits osteoblast differentiation and bone formation in vitro and in vivo. J Biol Chem,2004,279(13):12854-12859
[18]Zhao M, Qiao M, Oyajobi BO, et al. E3 ubiquitin ligase Smurfl mediates core-binding factor alphal/Runx2 degradation and plays a specific role in osteoblast differentiation. J Biol Chem,2003,278(30):27939-27944
[19]Kaneki H, Guo R, Chen D, et al. Tumor necrosis factor promotes Runx2 degradation through up-regulation of Smurfl and Smurf2 in osteoblasts. J Biol Chem, 2006,281(7):4326-4333
[20]Jeon EJ, Lee KY, Choi NS, et al. Bone morphogenetic protein-2 stimulates Runx2 acetylation. J Biol Chem,2006,281(24):16502-16511
[21]Schroeder TM, Kahler RA, Li X, et al. Histone deacetylase 3 interacts with runx2 to repress the osteocalcin promoter and regulate osteoblast differentiation. J Biol Chem,2004,279(40):41998-42007
[22]Schroeder TM, Westendorf JJ. Histone deacetylase inhibitors promote osteoblast maturation. J Bone Miner Res,2005,20(12):2254-2263
[23]Cho HH, Park HT, Kim YJ, et al. Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors. J Cell Biochem, 2005,96(3):533-542
[24]Luzi E, Marini F, Sala SC, et al. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res,2008,23(2):287-295
[25]Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A, 2008,105(37):13906-13911
[26]Huang J, Zhao L, Xing L, et al. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells,2010, 28(2):357-364
[27]Kapinas K, Kessler CB, Delany AM. miR-29 suppression of osteonectin in osteoblasts:regulation during differentiation and by canonical Wnt signaling. J Cell Biochem,2009,108(1):216-224
[28]Itoh T, Nozawa Y, Akao Y. MicroRNA-141 and-200a are involved in bone morphogenetic protein-2-induced mouse pre-osteoblast differentiation by targeting distal-less homeobox 5. J Biol Chem,2009,284(29):19272-19279
[29]Li Z, Hassan MQ, Jafferji M, et al. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem,2009, 284(23):15676-15684
[30]Mizuno Y, Tokuzawa Y, Ninomiya Y, et al. miR-210 promotes osteoblastic differentiation through inhibition of AcvRlb. FEBS Lett,2009,583(13):2263-2268
[1]Berezikov E, Guryev V, van de Belt J, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell,2005,120(1):21-24
[2]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993, 75(5):843-854
[3]Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature,2000, 403(6772):901-906
[4]Lau NC, Lim LP, Weinstein EG, et al. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science,2001, 294(5543):858-862
[5]Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science,2001,294(5543):862-864
[6]Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs. Science,2001,294(5543):853-858.
[7]Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res,2004, 32(Database issue):D109-111
[8]Griffiths-Jones S,Grocock RJ, van Dongen S, et al. miRBase:microRNA sequences, targets and gene nomenclature. Nucleic Acids Res,2006,34(Database issue):D 140-144
[9]Ambros V, Bartel B, Bartel DP, et al. A uniform system for microRNA annotation. RNA,2003,9(3):277-279
[10]Zhang BH, Pan XP, Cox SB, et al. Evidence that miRNAs are different from other RNAs. Cell Mol Life Sci,2006,63(2):246-254
[11]Pan X, Zhang B, San Francisco M, et al. Characterizing viral microRNAs and its application on identifying new microRNAs in viruses. J Cell Physiol,2007, 211(1):10-18
[12]Kim VN, Nam JW. Genomics of microRNA. Trends Genet,2006, 22(3):165-173
[13]Cullen BR. Transcription and processing of human microRNA precursors. Mol Cell,2004,16(6):861-865
[14]Cai X, Hagedorn CH, Cullen BR. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA,2004, 10(12):1957-1966
[15]Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase Ⅱ. EMBO J,2004,23(20):4051-4060
[16]Borchert GM, Lanier W, Davidson BL. RNA polymerase Ⅲ transcribes human microRNAs. Nat Struct Mol Biol,2006,13(12):1097-1101
[17]Han J, Lee Y, Yeom KH, et al. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell,2006,125(5):887-901
[18]Han J, Pedersen JS, Kwon SC, et al. Posttranscriptional crossregulation between Drosha and DGCR8. Cell,2009,136(1):75-84
[19]Denli AM, Tops BB, Plasterk RH, et al. Processing of primary microRNAs by the Microprocessor complex. Nature,2004,432(7014):231-235
[20]Gregory RI, Yan KP, Amuthan G, et al. The Microprocessor complex mediates the genesis of microRNAs. Nature,2004,432(7014):235-240
[21]Han J, Lee Y, Yeom KH, et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev,2004,18(24):3016-3027
[22]Du Z, Lee JK, Tjhen R, et al. Structural and biochemical insights into the dicing mechanism of mouse Dicer:a conserved lysine is critical for dsRNA cleavage. Proc Natl Acad Sci U S A,2008,105(7):2391-2396
[23]Zeng Y, Cullen BR. Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. Nucleic Acids Res,2004,32(16):4776-4785
[24]Yi R, Qin Y, Macara IG, et al. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev,2003,17(24):3011-3016
[25]Lund E, Guttinger S, Calado A, et al. Nuclear export of microRNA precursors. Science,2004,303(5654):95-98
[26]Bohnsack MT, Czaplinski K and Gorlich D. Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA,2004,10(2):185-191
[27]Shibata S, Sasaki M, Miki T, et al. Exportin-5 orthologues are functionally divergent among species. Nucleic Acids Res,2006,34(17):4711-4721
[28]Forstemann K, Tomari Y, Du T, et al. Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol,2005,3(7):e236
[29]Hutvagner G, McLachlan J, Pasquinelli AE, et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science,2001,293(5531):834-838
[30]Kurihara Y, Watanabe Y. Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci U S A,2004, 101(34):12753-12758
[31]Park MY, Wu G, Gonzalez-Sulser A, et al. Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci U S A,2005,102(10):3691-3696
[32]Lee Y, Hur I, Park SY, et al. The role of PACT in the RNA silencing pathway. EMBO J,2006,25(3):522-532
[33]Tay Y, Zhang J, Thomson AM, et al. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature,2008, 455(7216):1124-1128
[34]Lytle JR, Yario TA, Steitz JA. Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5'UTR as in the 3'UTR. Proc Natl Acad Sci U S A, 2007,104(23):9667-9672
[35]Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res,2009, 19(7):1175-1183
[36]Ruby JG, Jan CH, Bartel DP. Intronic microRNA precursors that bypass Drosha processing. Nature,2007,448(7149):83-86
[37]Okamura K, Hagen JW, Duan H, et al. The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila. Cell,2007,130(1):89-100
[38]Berezikov E, Chung WJ, Willis J, et al. Mammalian mirtron genes. Mol Cell, 2007,28(2):328-336
[39]Lau PW, MacRae IJ. The molecular machines that mediate microRNA maturation. J Cell Mol Med,2009,13(1):54-60
[40]Carthew RW, Sontheimer EJ. Origins and Mechanisms of miRNAs and siRNAs. Cell,2009,136(4):642-655
[41]Hornstein E, Mansfield JH, Yekta S, et al. The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development. Nature,2005,438(7068):671-674
[42]Bruno I, Wilkinson MF. P-bodies react to stress and nonsense. Cell,2006, 125(6):1036-1038
[43]Miyoshi K, Okada TN, Siomi H, et al. Characterization of the miRNA-RISC loading complex and miRNA-RISC formed in the Drosophila miRNA pathway. RNA, 2009,15(7):1282-1291
[44]Eulalio A, Behm-Ansmant I, Schweizer D, et al. P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol Cell Biol,2007, 27(11):3970-3981
[45]Schwarz DS, Hutvagner G, Du T, et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell,2003,115(2):199-208
[46]Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell,2003,115(2):209-216
[47]Hutvagner G. Small RNA asymmetry in RNAi:function in RISC assembly and gene regulation. FEBS Lett,2005,579(26):5850-5857
[48]MacRae IJ, Ma E, Zhou M, et al. In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci U S A,2008,105(2):512-517
[49]Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs:how many mechanisms? Trends Cell Biol,2007,17(3):118-126
[50]Kozak M. Faulty old ideas about translational regulation paved the way for current confusion about how microRNAs function. Gene,2008,423(2):108-115
[51]Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate translation. Science,2007,318(5858):1931-1934.
[52]Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res,2003,31(13):3406-3415
[53]Luciano DJ, Mirsky H, Vendetti NJ, et al. RNA editing of a miRNA precursor. RNA,2004,10(8):1174-1177
[54]Yang W, Chendrimada TP, Wang Q, et al. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases. Nat Struct Mol Biol,2006,13(1):13-21
[55]Yang Z, Ebright YW, Yu B, et al. HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2'OH of the 3'terminal nucleotide. Nucleic Acids Res,2006,34(2):667-675
[56]Wang JF, Zhou H, Chen YQ, et al. Identification of 20 microRNAs from Oryza sativa. Nucleic Acids Res,2004,32(5):1688-1695
[57]Berezikov E, Cuppen E, Plasterk RH. Approaches to microRNA discovery. Nat Genet,2006,38 Suppl:S2-7
[58]Lim LP, Lau NC, Weinstein EG, et al. The microRNAs of Caenorhabditis elegans. Genes Dev,2003,17(8):991-1008
[59]Wang X, Zhang J, Li F, et al. MicroRNA identification based on sequence and structure alignment. Bioinformatics,2005,21(18):3610-3614
[60]Lim LP, Glasner ME, Yekta S, et al. Vertebrate microRNA genes. Science, 2003,299(5612):1540
[61]Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet,2005,37(7):766-770
[62]Huang TH, Fan B, Rothschild MF, et al. MiRFinder:an improved approach and software implementation for genome-wide fast microRNA precursor scans. BMC Bioinformatics,2007,8:341
[63]Berninger P, Gaidatzis D, van Nimwegen E, et al. Computational analysis of small RNA cloning data. Methods,2008,44(1):13-21
[64]Creighton CJ, Reid JG, Gunaratne PH. Expression profiling of microRNAs by deep sequencing. Brief Bioinform,2009,10(5):490-497
[65]Hafner M, Landgraf P, Ludwig J, et al. Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods,2008, 44(1):3-12
[66]Addo-Quaye C, Miller W, Axtell MJ. CleaveLand:a pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics,2009, 25(1):130-131
[67]Hackenberg M, Sturm M, Langenberger D, et al. miRanalyzer:a microRNA detection and analysis tool for next-generation sequencing experiments. Nucleic Acids Res,2009,37(Web Server issue):W68-76
[68]Friedlander MR, Chen W, Adamidi C, et al. Discovering microRNAs from deep sequencing data using miRDeep. Nat Biotechnol,2008,26(4):407-415
[69]Bonnet E, Wuyts J, Rouze P, et al. Evidence that microRNA precursors, unlike other non-coding RNAs, have lower folding free energies than random sequences. Bioinformatics,2004,20(17):2911-2917
[70]Washietl S, Hofacker IL, Stadler PF. Fast and reliable prediction of noncoding RNAs. Proc Natl Acad Sci U S A,2005,102(7):2454-2459
[71]Washietl S, Hofacker IL, Lukasser M, et al. Mapping of conserved RNA secondary structures predicts thousands of functional noncoding RNAs in the human genome. Nat Biotechnol,2005,23(11):1383-1390
[72]Sewer A, Paul N, Landgraf P, et al. Identification of clustered microRNAs using an ab initio prediction method. BMC Bioinformatics,2005,6:267
[73]Xue C, Li F, He T, et al. Classification of real and pseudo microRNA precursors using local structure-sequence features and support vector machine. BMC Bioinformatics,2005,6:310
[74]Pfeffer S, Sewer A, Lagos-Quintana M, et al. Identification of microRNAs of the herpesvirus family. Nat Methods,2005,2(4):269-276
[75]Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression in zebrafish embryonic development. Science,2005,309(5732):310-311
[76]Kloosterman WP, Wienholds E, de Bruijn E, et al. In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods,2006,3(1):27-29
[77]Wheeler G, Ntounia-Fousara S, Granda B, et al. Identification of new central nervous system specific mouse microRNAs. FEBS Lett,2006,580(9):2195-2200
[78]Tuddenham L, Wheeler G, Ntounia-Fousara S, et al. The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Lett,2006, 580(17):4214-4217
[79]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods,2001, 25(4):402-408
[80]Tardif G, Hum D, Pelletier JP, et al. Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes. BMC Musculoskelet Disord,2009,10:148
[81]Benes V, Castoldi M. Expression profiling of microRNA using real-ime quantitative PCR, how to use it and what is available. Methods,2010,50(4):244-249
[82]Schmittgen TD, Jiang J, Liu Q, et al. A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res,2004,32(4):e43
[83]Jiang J, Lee EJ, Gusev Y, et al. Real-time expression profiling of microRNA precursors in human cancer cell lines. Nucleic Acids Res,2005,33(17):5394-5403
[84]Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res,2005,33(20):e179
[85]Liang Y, Ridzon D, Wong L, et al. Characterization of microRNA expression profiles in normal human tissues. BMC Genomics,2007,8:166
[86]Tang F, Hajkova P, Barton SC, et al. MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res,2006,34(2):e9
[87]Tang F, Hajkova P, Barton SC, et al.220-plex microRNA expression profile of a single cell. Nat Protoc,2006,1(3):1154-1159
[88]Lao K, Xu NL, Sun YA, et al. Real time PCR profiling of 330 human micro-RNAs. Biotechnol J,2007,2(1):33-35
[89]Ortega FJ, Moreno-Navarrete JM, Pardo G, et al. MiRNA expression profile of human subcutaneous adipose and during adipocyte differentiation. PLoS One,2010, 5(2):e9022
[90]Li W, Ruan K. MicroRNA detection by microarray. Anal Bioanal Chem, 2009.394(4):1117-1124
[91]Liu CG, Calin GA. Meloon B, et al. An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc Natl Acad Sci USA,2004,101(26):9740-9744
[92]Wilson KD, Venkatasubrahmanyam S, Jia F, et al. MicroRNA profiling of human-induced pluripotent stem cells. Stem Cells Dev,2009,18(5):749-758
[93]Bostjancic E, Zidar N, Glavac D. MicroRNA microarray expression profiling in human myocardial infarction. Dis Markers,2009,27(6):255-268
[94]Brennecke J, Stark A, Russell RB, et al. Principles of microRNA-target recognition. PLoS Biol,2005,3(3):e85
[95]Enright AJ, John B, Gaul U, et al. MicroRNA targets in Drosophila. Genome Biol,2003,5(1):R1
[96]Lewis BP, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalian microRNA targets. Cell,2003,115(7):787-798
[97]Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 2005,120(1):15-20
[98]Krek A, Grun D, Poy MN, et al. Combinatorial microRNA target predictions. Nat Genet,2005,37(5):495-500
[99]Grun D, Wang YL, Langenberger D, et al. microRNA target predictions across seven Drosophila species and comparison to mammalian targets. PLoS Comput Biol,2005, 1(1):e13
[100]Lall S, Grun D, Krek A, et al. A genome-wide map of conserved microRNA targets in C. elegans. Curr Biol,2006.16(5):460-471
[101]Miranda KC, Huynh T, Tay Y, et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell,2006,126(6):1203-1217
[102]Li Z, Hassan MQ, Jafferji M, et al. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem,2009, 284(23):15676-15684
[103]Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A, 2008.105(37):13906-13911
[104]Oskowitz AZ, Lu J, Penfornis P, et al. Human multipotent stromal cells from bone marrow and microRNA:regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci U S A,2008,105(47):18372-18377
[105]Winnard RG, Gerstenfeld LC, Toma CD, et al. Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts. J Bone Miner Res,1995,10(12):1969-1977
[106]Yang XB, Roach HI, Clarke NM, et al. Human osteoprogenitor growth and differentiation on synthetic biodegradable structures after surface modification. Bone, 2001,29(6):523-531
[107]Zhang Y, Feng XH, Derynck R. Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription. Nature,1998, 394(6696):909-913
[108]Jensen ED, Gopalakrishnan R, Westendorf JJ. Regulation of gene expression in osteoblasts. Biofactors,2010,36(1):25-32
[109]Kim YJ, Kim HN, Park EK, et al. The bone-related Zn finger transcription factor Osterix promotes proliferation of mesenchymal cells. Gene,2006, 366(1):145-151
[110]Bialek P, Kern B, Yang X, et al. A twist code determines the onset of osteoblast differentiation. Dev Cell,2004,6(3):423-435
[111]Xiao G, Jiang D, Ge C, et al. Cooperative interactions between activating transcription factor 4 and Runx2/Cbfal stimulate osteoblast-specific osteocalcin gene expression. J Biol Chem,2005,280(35):30689-30696
[112]Etheridge SL, Spencer GJ, Heath DJ, et al. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem Cells,2004,22(5):849-860
[113]Osyczka AM, Leboy PS. Bone morphogenetic protein regulation of early osteoblast genes in human marrow stromal cells is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling. Endocrinology, 2005.146(8):3428-3437
[114]Levy JB, Schindler C, Raz R, et al. Activation of the JAK-STAT signal transduction pathway by oncostatin-M cultured human and mouse osteoblastic cells. Endocrinology,1996,137(4):1159-1165
[115]Lee KS, Hong SH, Bae SC. Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene,2002,21(47):7156-7163
[116]Luzi E, Marini F, Sala SC, et al. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res,2008,23(2):287-295
[117]Huang J, Zhao L, Xing L, et al. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells,2010, 28(2):357-364
[118]Kapinas K, Kessler CB, Delany AM. miR-29 suppression of osteonectin in osteoblasts:regulation during differentiation and by canonical Wnt signaling. J Cell Biochem,2009,108(1):216-224
[119]Mizuno Y, Yagi K, Tokuzawa Y, et al. miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun,2008,368(2):267-272
[120]Itoh T, Nozawa Y, Akao Y. MicroRNA-141 and-200a are involved in bone morphogenetic protein-2-induced mouse pre-osteoblast differentiation by targeting distal-less homeobox 5. J Biol Chem,2009,284(29):19272-19279
[121]Mizuno Y, Tokuzawa Y, Ninomiya Y, et al. miR-210 promotes osteoblastic differentiation through inhibition of AcvRlb. FEBS Lett,2009,583(13):2263-2268
[122]Li H, Xie H, Liu W, et al. A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans. J Clin Invest,2009,119(12):3666-3677
[123]Kobayashi T, Lu J, Cobb BS, et al. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. Proc Natl Acad Sci U S A,2008, 105(6):1949-1954
[124]Lakshmipathy U, Love B, Goff LA, et al. MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells. Stem Cells Dev,2007, 16(6):1003-1016
[125]Suomi S, Taipaleenmaki H, Seppanen A, et al. MicroRNAs Regulate Osteogenesis and Chondrogenesis of Mouse Bone Marrow Stromal Cells. Gene Regul Syst Bio,2008,2:177-191
[126]Lin EA, Kong L, Bai XH, et al. miR-199a, a bone morphogenic protein 2-responsive MicroRNA, regulates chondrogenesis via direct targeting to Smadl. J Biol Chem,2009,284(17):11326-11335
[127]Sugatani T, Hruska KA. MicroRNA-223 is a key factor in osteoclast differentiation. J Cell Biochem,2007,101(4):996-999
[128]Sugatani T and Hruska KA. Impaired micro-RNA pathways diminish osteoclast differentiation and function. J Biol Chem,2009,284(7):4667-4678