The Protective Effects Of Long Non-coding RNA-ANCR On Arterial Calcification

Arterial calcification is common in diabetes,end-stage renal disease patients and the elderly,and is an important cause of cardiovascular disease development and death.Studies showed that arterial calcification is an active regulation process similar to bone formation.The differentiation of vascular smooth muscle cells(VSMCs)into osteoblast-like cells is the cytological basis and key link of arterial calcification.However,its specific pathogenesis remains unclear.Long non-coding RNA(lncRNA)is considered to be a key regulator of gene transcription and cell differentiation.Previous studies have confirmed that lncRNA affects proliferation and apoptosis of VSMCs.Anti-differentiation non-coding RNA(ANCR)is a type of lncRNA.Recent studies have shown that ANCR can inhibit human embryonic osteoblast differentiation.Our previous studies have shown that ANCR can inhibit the differentiation of mouse VSMCs induced by ?-glycerophosphate(?-GP).However,it is unclear whether ANCR has a protective effect on arterial calcification in vivo.The purpose of this study was to investigate the protective effect of lncRNA-ANCR on arterial calcification.C57BL/6 male mice were divided randomly into 4 groups,control group,vitamin D3 group,vitamin D3+ lentivirus ANCR group,vitamin D3+ control lentivirus group,except for the control group,the other 3 groups were injected in high dose of 500,000 IU / kg / day of vitamin D3,in control group,mice were injected with the equal dose of olive oil for 7 days.The ANCR overexpressed lentiviral vector was constructed at a concentration of 5×108 TU/ml.The lentivirus ANCR and the control lentivirus were injected into the vitamin D3+ lentivirus ANCR group and the vitamin D3+ control lentivirus group respectively,via tail vein.All mice were sacrificed four week later,and the mouse thoracic aorta was isolated to make frozen tissue sections.The histological structure and calcification of the mouse thoracic aorta were visualized by Alizarin red S and Von kossa staining,and Runt-related transcription factor 2(Runx2)and Bone morphology protein-2(BMP-2)were evaluated by immunofluorescence staining(IF)staining.The expression of Runx2 and BMP-2 were measured by Image J software and data were analyzed by Graphpad Prism7.0.Alizarin red staining showed that 3 groups of mice aorta after subcutaneous injection of vitamin D3 showed different degrees of orange-red calcified nodules in the middle layer of the thoracic aorta,and the elastic fibers in the arterial wall were disorderly arranged.Compared with the control group,the intact one arterial wall structure was lost.Von kossa staining showed that different degrees of black calcified nodules in the middle layer of the thoracic aorta in the three groups,and no calcified nodules in the control group.The data analysis showed that there was significant arterial calcification in vitamin D3 group and vitamin D3 + control lentivirus group compared with control group,and the difference was statistically significant(p<0.001).Compared with vitamin D3 group and vitamin D3 + control lentivirus group,the arterial calcification of vitamin D3+ lentivirus ANCR group was significantly weaker,and the difference was statistically significant(p<0.001).Immunofluorescence staining showed that the expression of Runx2 and BMP-2 were different in the thoracic aorta of the four groups of mice.The results of data analysis showed that the expression of Runx2 and BMP-2 in vitamin D3 group and vitamin D3 + control lentivirus group was significantly increased compared with control group,and the difference was statistically significant(p<0.05).Compared with vitamin D3 group and vitamin D3 + control lentivirus group,the expression of Runx2 and BMP-2 in vitamin D3 + lentivirus ANCR group was significantly weaker,and the difference was statistically significant(p<0.05).Therefore,we could draw conclusions that ANCR may play a protective role in arterial calcification and provide a new direction for the prevention and treatment of arterial calcification.ANCR may attenuate arterial calcification by inhibiting osteogenic differentiation of VSMCs.