A Functional Study On MicroRNA-133a Regulating Osteoblastic Differentiation Of Vascular Smooth Muscle Cells

Part One Study on the roles of miRNA-133a (miR-133a) in osteoblastic differentiation of mouse VMSCs.ObjectiveTo investigate the roles of miRNA-133a (miR-133a) in osteoblastic differentiation of mouse vascular smooth muscle cells.MethodsNorthern blot is used to check the expression of miR-133a in mouse heart, skeletal muscle, VSMCs and aorta.β-sodium glycerophosphate is used to induce osteoblastic differentiation of mouse vascular smooth muscle cell. Northern blot is used to detect the change of expressive level of miR-133a in this process.To observe the change of osteoblastic markers, the activity of ALP was detected by means of spectrophotometric measurement of P-nitrophenol release, OC was measured by means of immunoradiometric assay; cells were stained by Alizarin Red and quantification of Alizarin Red stain via extraction with cetylpyridinium chloride. Runx2protein and mRNA expression were detected by means of Western-blot and quantitative real-time PCR (qRT-PCR) respectively. According to the sequence of precursor of miR133a-1, Primers were designed, and were connected to pSilencer4.1-CMV puro vector to construct pre-miR-133a-1. Pre-miR-133a-1was transfected to VSMCs, and the model of overexpression of miR-133a was created successfully. And then10mMβ-GP was used to induce osteoblastic differentiation; the activity of ALP, the secrection of OC and Calcified tubercle were detected; Runx2protein was checked by means of Western-blot; mRNA expression was detected by means of qRT-PCR.Results(1) There were high expressive levels of miR-133a in heart and skeletal muscle, and there was little expression of miR-133a in VSMCs and aorta.(2) Compared with the control group, in the process of osteoblastic differentiation of VSMCs induced by β-GP, the activitiy of ALP and the secretion of OC increased obviously, the expressive level of Runx2protein increased and there were more Calcified tubercle.(3) Compared with non-transfected group, there was higher expressive level of miR-133a in VSMCs which were transfected with pre-miR-133a-1. The activity of ALP, the secretion of OC and the quantity of Calcified tubercle decreased with miR-133a overexpressing. The overexpression of miR-133a inhibited the expression of Runx2protein, while the expressive level of Runx2mRNA was not affected.ConclusionmiR-133a-1expressive vector was constructed successfully by using pSilencer4.1-CMV puro plasmid, and there was high expression of miR-133a in VSMCs transfected with miR-133a-1expressive vector. The overexpression of miR-133a inhibited the osteoblastic differentiation of VSMCs. Part Two Study on the mechanism of miR-133a regulating osteoblastic differentiation of VSMCsObjectiveTo predict and prove the target gene of miR-133a, and to provide theotical proof for the prevention and cure of arteriosclerosis.MethodsThe target gene of miR-133a was discovered by means of several miRNA target prediction software tools. To construct wild type (WT) target gene3'-UTR expression vector, a segment of the mouse target gene3'-UTR including the putative target site was PCR amplified from mouse genomic DNA. The product was then inserted into the pcDNA3.1(+) vector, resulting in WT target gene expression vector. The QuickChange site-directed mutagenesis kit was used to introduce two point mutations into the3'UTR of WT target gene, resulting in mutant (MUT) target gene3'UTR expression vector. These two expression vectors were co-transfected with pre-miR-133a-1or miR-C to VSMCs. β-GP is used to induce differentiation, and the protein of target gene is oberserved. Western blot is used to detect the exression of the protein of target gene.Results(1) Runx2was predicted as the target gene of miR-133a by means of multiple miRNA target prediction software tools.(2)Tranfection of MUT Runx23'UTR expression vector could eliminate the inhibition of pre-miR-133a-1to the expression of Runx2protein. It was suggested that in the osteoblastic differentiation Runx2was an important target of miR-133a.Conclusion(1) Runx2gene was the target gene of miR-133a.(2)If only there existed miR-133a and3'UTR of Runx2in VSMCs normally, miR-133a could be connected with the3'UTR of Runx2to regulate the expression of Runx2protein negatively.