Cloning And Functional Study Of A Novel MicroRNA Regulating Osteoblast Differentiation

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