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Cloning and Functional Study of a Novel microRNA Regulating Osteoblast Differentiation

Author: LiHui
Tutor: LuoXiangHang
School: Central South University
Course: Internal Medicine
Keywords: Osteoblasts Small RNA Clone cDNA library Bone marrow stromal cells Differentiation Overexpression Transfection Target genes Histone deacetylase 5 Reporter gene Carrier
CLC: R329
Type: PhD thesis
Year: 2010
Downloads: 493
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Abstract

Objective: Find a new mouse osteoblast-specific expression preferentially expressed microRNA (miRNA), and their expression patterns in multiple tissues, organs and cells. Method: First extract the mouse osteoblasts small molecule RNA, Poly (A); plus tail and connect the 5 'end of the connector after the reverse transcription into cDNA, followed by PCR amplification, and recovery, connected to the pcDNA3.1 TOPO vector construct cDNA library of small RNA molecules, the last colony PCR, the positive clones were sequenced to select the size of the 19-26nt RNA bioinformatics analysis to identify new miRNA. Northern blot detection of miRNA in mouse osteoblasts, osteoclasts cells, bone, liver, heart, lung, kidney, brain, fat, spleen, and skeletal muscle expression. BMP-2-induced the ST2 cell to osteoblast differentiation, Northern blot hybridization detection of miRNA expression patterns in this process. Results: A total of 162 small molecule RNA cloned 68 miRNAs, including two new miRNAs, including one named after submitting the miRBase database of miR-2861. miR-2861 is located in mouse chromosome 2, there are conserved in the human genome. miR-2861 into bone cells and bone is highly expressed in the mouse lower expression in the liver did not express in other organizations and osteoclasts. miR-2861 ST2 cells do not express BMP-2-induced ST2 osteoblast differentiation 12h can detect the expression of miR-2861, and with the induction of differentiation time prolonged expression gradually increased. Conclusion: for the first time to build a the mouse osteoblasts small RNA cDNA library. For the first time found a new osteoblasts preferentially expressed miRNA-miR-2861. MiR-2861 are conserved between mouse and human, and its expression gradually increased with the progress of osteoblast differentiation, suggesting that it may be involved in the regulation of osteoblast differentiation process. Objective: To investigate the miR-2861 in mouse stromal cells ST2 and primary bone marrow stromal cells (BMSCs) to osteoblast differentiation. Methods: The primers were designed according to the precursor sequence of miR-2861, after annealing connected to pSilencer 4.1-CMV puro vector was constructed miR-2861 expression vector Pre-miR-2861. Pre-miR-2861 transfected ST2 cells and bone marrow stromal cells (bone marrow stromal cells, BMSCs), caused by over-expressing cells miR-2861 model, then I 300ng/ml BMP-2-induced differentiation, osteoblast differentiation was observed indicators changes. Alkaline phosphatase (alkaline phosphatase, ALP) activity spectrophotometer nitrophenol release obtained osteocalcin (Osteocalcin, OC) was measured by radioimmunoassay, the cell calcium deposition ratio of the amount Cresolphthalein complex copper color assay. Western blotting and quantitative real-time PCR were used to detect changes in Runx2 protein and mRNA expression. 2'-O-methyl-modified antisense oligonucleotide anti-miR-2861 instantaneous transfected ST2 cells and BMSCs same method to detect the activity of ALP, OC secretion, as well as changes in the amount of calcium deposits in the cells, factor Runx2 of MSCs protein and mRNA expression, respectively by Western blot and real-time quantitative PCR. The results: (1) transfected with miR-2861 the pSilencer 4.1CMV puro build the expression vector pre-miR-2861 cells stably high expression of miR-2861. (2) miR-2861 overexpression promote ST2 and BMSCs into increased ALP activity in osteoblast differentiation process and OC secretion, improve Runx2 protein expression, without affecting the level of Runx2 mRNA. ST2 cells transfected with pre-miR-2861 can increase calcium deposition in the cell (3) inhibition of miR-2861 to reduce the ST2 and BMSCs into ALP activity in bone cell differentiation process, reducing OC secretion caused by BMP-2-induced Runx2 protein expression increased, but does not affect the level of Runx2 mRNA transfected ST2 cells after 5 days, and the content of calcium deposition is lower than the control group. Conclusions: miR-2861 expression vector was successfully constructed, over-expression of miR-2861 ST2 and BMSCs can promote osteoblast differentiation, inhibition of miR-2861 is able to delay the ST2 and BMSCs to the osteoblast differentiation. Objective: To predict and verify the role of target genes of miR-2861 to clarify miR-2861 to promote osteoblast differentiation mechanism. Methods: multiple target gene prediction software to analyze the role of miR-2861 target genes. PCR amplification contain the target sites, including the target gene coding sequence (coding sequence, CDS), the product was ligated into the pGL3 vector to construct a wild-type target gene CDS luciferase reporter gene vector. As a template, using the QuickChange site-directed mutagenesis kit introduced in the target site mutation of two bases constructed mutant target gene CDS luciferase reporter gene vector. These two vectors were co-transfected with the pre-miR-2861 transfection ST2 cells detects the changes of luciferase activity to confirm whether the target gene is the role of a target gene of the miR-2861. ST2 cells individually transfected with pre-miR-2861, observed miR-2861 target gene expression, Western blot and real-time quantitative PCR were used to detect changes in target gene protein and mRNA levels, clear the role of miR-2861 target genes. PCR amplification of the target the gene CDS full-length cDNA, connected to the pcDNA3.1 () vector construct a wild-type target gene expression vector as template, using the QuickChange site-directed mutagenesis kit to introduce two base mutation at the target site constructed mutant target gene expression vector, two vectors were transfected with pre-miR-2861 or miR-C ST2 cells, plus BMP-2-induced differentiation, ALP activity was observed and Target Gene Expression, ALP activity using spectrophotometric photometer detection of nitrophenol release target gene protein expression using Western blot analysis. Results: (1) software predicted the HDAC5 gene targets for miR-2861. (2) Compared with the control group of the transfected with miR-C the HDAC5 CDS miR-2861 over-expression group luciferase activity was significantly decreased, while the the HDAC5 CDS transfected mutant luciferase activity did not change significantly. (3) separate transfected pre-miR-2861 can reduce HDAC5 protein levels had no effect on HDAC5 mRNA. (4) pre-miR-2861 with the wild-type HDAC5 CDS expression vectors were co-transfected ALP activity can increase, reduce HDAC5 protein expression, and pre-miR-2861 mutant HDAC5 CDS expression vectors were cotransfected not produce this role. That the role of miR-2861 in osteoblast differentiation completely or mainly through the role with HDAC5 completed. Conclusion: (1) successfully constructed the wild-type and mutant HDAC5 CDS luciferase reporter gene vector, wild-type and mutant HDAC5 expression vector. (2) software to predict and luciferase reporter gene assay confirmed HDAC5 target genes of miR-2861. (3) miR-2861 to inhibit HDAC5 expression through post-transcriptional level, and promote osteoblast differentiation. (4) HDAC5 is a target gene of the miR-2861 is the most important in osteoblast differentiation.

Cloning and Functional Study of a Novel microRNA Regulating Osteoblast Differentiation

Abstract

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