The Molecular Mechanism Of PiRd9 In Regulating Endothelial Cell Function And Atherosclerosis

Objective:By studying the role and mechanism of Piwi-interacting RNA（piRNA）in regulating human aortic endothelial cells（HAEC）apoptosis and normal physiological function,and assessing the regulatory role of piRNA on atherosclerosis（AS）,this study may provide a new idea for the clinical targeted therapy of AS.Methods:（1）Atherosclerosis model was established by feeding APOE^-/-mice with high-fat diet:after successful modeling.The expression of potential piRNA in the C57 and APOE^-/-mice tissues,HAECs and vascular smooth muscle cells（VSMC）was detected by Real-time quantitative polymerase chain reaction（RT-q PCR）.The tissue and cell specificity of piRNA expression was determined.（2）Tumor Necrosis Factor（TNF-α）treated HAEC to induce AS model in vitro,and the success of induction was verified by WB（western blot）experiments.The expression of potential piRNA in vitro AS model was detected by RT-q PCR to determine the target piRNA.Fluorescence in situ hybridization（FISH）was used to detect the distribution of piRNA in cells.（3）RNA was extracted from serum samples of healthy people and AS patients（stenosis degree>70%in coronary angiography results,excluding patients with severe inflammation,tumor and type 2 diabetes）,and the expression level of piRNA was detected by RT-q PCR.（4）Knockdown and overexpression of piRNA to verify the function of regulating HAEC:WB was used to detect the effects of piRNA on HAEC apoptosis and the expression of adhesion molecules.Annexin V-FIIC/PI experiment was used to further detect the effects of piRNA on HAEC apoptosis.（5）Mass spectrometry predict the target protein in the downstream of piRNA,pull-down and RNA binding protein immunoprecipitation（RIP）to verify their direct binding,and detect its expression changes by RT-q PCR and WB.The expression of downstream target proteins in vitro AS models was detected by RT-q PCR.（6）Endothelial cells were treated with TNF-αto establish the AS model in vitro,and RT-q PCR were used to detect the regulatory effect of downstream targeted proteins on AS.（7）The mice were divided into NC group,AS group and piRNA group,and piRNA or DEPC water was injected through the tail vein.Finally,the aorta and aortic root of the mice were dissected,and the effect of piRNA on atherosclerosis was determined by observing the plaque size by oil red staining.Results:（1）Through the aorta oil red staining confirmed the formation of plaque together with blood lipid analysis show that the atherosclerotic model of mice was successfully established,and the target piRNA was highly expressed in blood vessels and endothelial cells.（2）RT-q PCR experiment verified that piRd9 expression was decreased in APOE^-/-mice and in vitro AS model.HAEC was selected for the next experiment,and FISH experiment proved that piRd9 was mainly distributed in the nucleus of HAEC.（3）Thirty AS patients were selected according to the exclusion and inclusion criteria.RT-q PCR results showed that the expression of piRd9 in blood samples of clinical AS patients was significantly lower than that of healthy people,and the results of mouse AS model were consistent.（4）After 48 hours of TNF-αtreatment,the experiments showed that the expression of apoptosis-related proteins IL6,Active-Caspase3,Bax/Bcl2 and adhesion molecules ICAM-1,VCAM-1 increased.（5）HAEC was transfected with synthetic piRd9mimic and piRd9 inhibitor to over-express or knock down piRd9,and the transfection effect was verified by RT-q PCR（P<0.05）.The results of WB experiment showed that overexpression of piRd9 could inhibit the apoptosis and expression of adhesion molecules of HAEC induced by TNF-α（P<0.05）,and knockdown of piRd9 could promote the apoptosis and expression of adhesion molecules of HAEC induced by TNF-α（P<0.05）.Annexin V-FIIC/PI experiment further showed that overexpression of piRd9 inhibited the apoptosis of HAEC induced by TNF-α（P<0.05）,and knockdown of piRd9 promoted the apoptosis of HAEC induced by TNF-α（P<0.05）.（6）The results of mass spectrometry showed that piRd9 binds to the heterogeneous ribonucleoprotein A2B1（hnRNP A2B1）,RNA pull-down and RIP experiments showed that piRd9 directly bound to hnRNP A2B1.Western blot and RT-q PCR experiments showed that there was no significant change in the expression level of hnRNP A2B1 RNA and protein among NC group,piRd9 mimic group and piRd9 inhibitor group.RT-q PCR showed that the expression of hnRNP A2B1RNA decreased after TNF-αtreatment of HAEC（P<0.05）.（7）Compared with NC group,there were obvious plaques in aortic arch and aortic root of mice in AS group and the plaques in aorta and aortic root arch of mice in piRd9 group were reduced and the area was significantly smaller than that in AS group.Conclusion:In this study,we identified a piRNA:piRd9,which regulates endothelial cell apoptosis and normal physiological function and thus has a regulatory role in AS.Moreover,we further demonstrated that piRd9 may regulate HAEC apoptosis and the expression of adhesion molecules by targeting hnRNP A2B1,thereby affecting the occurrence and development of AS.This might provide a new target and idea for clinical diagnosis and treatment of AS.