Differential Expression Analysis Of CircRNAs In Plasma Of Patients With Coronary Artery Calcification And In Vascular Calcification Of Mice

Aim1.To explore the differential expression of circRNAs in peripheral blood in patients with coronary artery calcification and its diagnostic value;2.Explore the expression of circRNAs:mmu-PEX13-0003 and mmu-GALK2-0007 in the formation of arterial calcification in mice.Methods1.Differential expression profiles of circRNAs in calcified and non-calcified human vascular smooth muscle cells were analyzed by high-throughput microarray microarray;2.Since the main purpose of this study was to screen biomarkers for the diagnosis of coronary artery calcification（CAC）,we focused on circRNAs with up-regulated expression in the calcification group and selected those with significantly up-regulated expression from the calcification group as candidate circRNAs;3.Further validated in the CAC patients plasma high flux circRNAs data processing results of gene chip,we through the qRT-PCR technique to detect from 31 cases of coronary artery calcification（CACs>100）patients and 40 cases of coronary artery calcification（CACs=0）controls the expression levels of plasma candidate circRNAs（relative expression level）expression;At the same time analysis the plasma differentially expressed circRNAs and the correlation between coronary artery calcification score;4.Receiver operating characteristic curve（ROC）was used to evaluate the diagnostic efficacy of plasma differential expression of circRNAs in coronary artery calcification;5.Aortic vascular calcification in mice model,8 weeks of C57BL/6 mice ordinary adaptive feed 2 weeks,the experimental application of nicotine in mice after 25 mg/kg dissolved in peanut oil immersion of mice by gastric,nine hours after the same doses of nicotine compound lavage treatment,vitamin D3（300000 iu/kg）soluble in anhydrous ethanol after the left hind leg ministry muscle injection,and at the same time give foods with high fat feed vascular calcification in mice model was established;The mice in normal control group were given the same dose of peanut oil intragastric administration,and the mice in normal control group were given the same dose of peanut oil intragastric administration 9 hours later,and the mice were injected with anhydrous ethanol into the left hind limb and femoral region.Free drinking water,food and drink,ambient temperature controlled at 22-25℃,humidity<60%,natural lighting,cage feeding.Vascular calcification was induced at 0,2,4 and 6 weeks,respectively.Aortic tissue was extracted at 8 weeks and Von Kossa staining was used to observe the vascular calcification at the aortic arch of mice in each group.The expression of BMP2,BMP4,Runx2 and other calcification markers in mouse aorta was detected by immunohistochemistry.Western blot was used to detect the expression of BMP2,BMP4 and other calcification markers in mouse aorta tissues;6.Based on the results of differential expression of circRNAs in coronary artery calcification verified by plasma,primers were designed for homologous circRNAs in mice,and qRT-PCR was used to determine the expression level（relative expression level）of circRNAs in the aorta of mice in each group.Results1.The differential expression profile of circRNAs in the calcified and non-calcified human vascular smooth muscle cells of the three groups was analyzed by high-fluxmicroarray chip.The results showed that there were 46 differentially expressed circRNAs in the calcified and non-calcified of human vascular smooth muscle cells,of which 15 were up-regulated and 31 were down-regulated in the calcified group;2.We further selected the significantly upregulated circRNAs from the 15 circRNAs up-regulated in the calcification group as candidate circRNAs,including hsa-circ-0008534,hsa-circ-0002622,hsa-circ-0008488,hsa-circ-0001478,and hsa-circ-0019225;3.After repeated trials,the plasma expression levels（relative expression level）of hsa-circ-0008534,hsa-circ-0002622,and hsa-circ-0008488 were statistically significant different between the coronary artery calcification group and the control group.However,hsa-circ-0001478 and hsa-circ-0019225 were not detected at the plasma level.Spearman correlation analysis of hsa-circ-0008534,hsa-circ-0002622,hsa-circ-0008488 and coronary artery calcification score（CACs）in CAC patients showed that hsa-circ-0008534 was positively correlated with CACs（r=0.557;P=0.001）;There was a positive correlation between hsa-circ-0008488 and CACs（r=0.487;P=0.005）;hsa-circ-0002622 was not associated with coronary artery calcification severity（r=0.271;P=0.140）;4.Receiver operating characteristic curve（ROC curve）showed that the relative expression level of plasma hsa-circ-0008534,hsa-circ-0002622,and hsa-circ-0008488 had the diagnostic efficacy of CAC（AUC:0.790;95%CI:0.676-0.905;P<0.001）,（AUC:0.733;95%CI:0.614-0.831;P=0.001）,（AUC:0.801;95%CI:0.689-0.886;P<0.001）;5.Since the establishment of the model,the body weight of C57BL/6 mice in the experimental group gradually decreased,and gradually recovered two weeks later;Von Kossa calcium deposition staining showed that after 8 weeks of induced aortic calcification,the vascular ring at the aortic arch had obvious calcification.The results of immunohi stochemi stry and Western blot showed that BMP2,BMP4 and other calcification markers increased significantly 8 weeks after establishment of the model.All the above experimental results proved that the model of vascular calcification in C57BL/6 mice was established successfully;6.According to the homology of human and mouse genes,the circRNAs of mice homologous with human hsa-circ-0008534 and hsa-circ-0008488 were identified as mmu-Pex 13-0003 and mmu-Galk2-0007,respectively,and their primer sequences were designed.The expression levels of mmu-Pex13-0003 and mmu-Galk2₀007 in the aorta of mice in each group（relative expression）were measured by qRT-PCR.The results showed that the relative expression levels of mmu-Pex 13-0003 in the aorta of mice were higher than those in the control group（P=0.002）and mmu-Galk2-0007 were higher than those in the control group（P=0.023）after 8 weeks of induced vascular calcification.Conclusion1.The relative expression levels of plasmahsa-circ-0008534,hsa-circ-0002622,and hsa.circ-0008488 are good diagnostic markers for coronary artery calcification,and the relative expression levels of plasma hsa-circ-0008534 and hsa-circ-0008488 can well predict the severity of coronary artery calcification in CAC patients;2.Nicotine gavage,vitamin D3 intramuscular injection combined with high-fat diet induced vascular calcification in C57BL/6 mice is a good animal model for the study of vascular calcification;3.The relative expression levels of mmu-Pex 13-0003 and mmu-Galk2-0007 in calcified mouse aorta tissues were higher than those in the control group,and mmu-Pex 13-0003 and mmu-Galk2-0007were involved in the progression of vascular calcification.