Functional Analyses Of The Regulatory Feedback Loop Between MiR-X/miR-2861 Cluster And Transcription Factor Runx2 In Osteoblast Differentiation

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