Effects Of MiR-32-deficiency On Vascular Calcification In Mice

Objective In this study,we aimed to discuss the role of miR-32 in the process of vascular calcification in mice and to provide experimental evidence for the pathogenesis and prevention strategies of vascular calcification.Methods1.In order to observe the effects of miR-32 in the vascular calcification in mice,the study induced the vascular calcification of8-10-week-old male wild type mice and miR-32 knockout mice by subcutaneous injection with VitD₃ in the concentrations of 5×10⁵IU?kg^-1d^-1for 7 days.The mice were executed on 8th day and the aortas were taken out.The degree of the vascular calcification of the two groups of mice was detected by HE Staining,Alizarin red s Staining and Von kossa Staining in both paraffin sections and integral aortas.2.Establishment of the miR-32/OPG double gene knockout mice:Establishing the miR-32/OPG double gene knockout mice through the sexual hybridization between the heterozygote-progenies of miR-32^-/-mice and OPG^-/-mice,which is a classical model of vascular calcification.3.In order to observe the effects of miR-32 in the vascular calcification of OPG^-/-mice,the aortas were taken out from the20-week-old OPG^-/-mice and miR-32/OPG double gene knockout mice.The HE staining,Alizarin red s staining and Von kossa staining were used to detect the degree of vascular calcification.Then the arteries and bone structure of miR-32/OPG double gene knockout mice and OPG^-/-mice were detected by dual energy CT and MRI technology.Results1.MiR-32 deficiency can inhibit the vascular calcification induced by VitD₃ in mice:The results of HE staining showed that both the number of deep blue calcium salt nodules and their range of miR-32^-/-mice decreased significantly when compared with WT mice.The results of Alizarin red s staining showed that the range of orange red calcium salt chelates of miR-32^-/-mice decreased obviously when compared with WT mice.The results of Von kossa staining showed that the range of substitutions of silver ions in black of miR-32^-/-mice decreased when compared with WT mice.In addition,the three kinds of staining showed that the vascular wall structure of the aortas were relatively intact in miR-32^-/-mice,whlie the the vascular elastic lamina and structure of vessel walls were destructed obviously in WT mice.The results indicating that the degree of vascular calcification of miR-32^-/-mice aortas were significantly relieved than WT mice.Mean while the results of Alizarin red s staining of aortic tissues showed the staining-color of miR-32^-/-mice were lighter than that of WT mice,which suggested that the degree of calcification of the aortas in miR-32^-/-mice were lighter when compared with WT mice.2.Establish the miR-32/OPG double gene knockout mice successfully:Amplifying the mice tail DNA of progenies of the heterozygote-progenies of the miR-32^-/-mice and the OPG^-/-mice by PCR and agarose gel electrophoresis.After screening,the offspring of miR-32/OPG double gene knockout mice were obtained and the ethnic group of miR-32/OPG double gene knockout mice were established successfully.3.MiR-32 deficiency can inhibit the vascular calcification in OPG^-/-mice:The results of HE staining,Alizarin red s staining and Von kossa staining showed that vascular calcification of miR-32/OPG double gene knockout mice of the aortas were significantly inhibited compared with OPG^-/-mice.MRI and CT examinations showed no obvious vascular calcification in the aortas of miR-32/OPG double gene knockout mice,but varying degrees of calcification in OPG^-/-mice.Conclusions1.MiR-32 deficiency can inhibit the vascular calcification induced by Vit D₃ injection in mice.2.MiR-32 deficiency can inhibit the vascular calcification in OPG^-/-mice.