The Mechanism Of MicroRNA-34a-5p Involved In The Promotion Of Atherosclerosis By Indoxyl Sulfate

[Objective] Atherosclerosis(AS)is the main pathological bASis of cardiovAScular and cerebrovAScular diseASes.The uremic toxin of indoxyl sulfate(IS)is an important risk factor for AS,and its mechanism hAS not yet been fully elucidated.The miR-34a-5p pathway can act on multiple AS related signaling pathways and molecules.We speculate that the miR-34a-5p pathway may be closely related to the development of IS promoted AS.To validate the above hypothesis,we intend to use molecular biology experimental techniques to study the role of the miR-34a-5p pathway in IS promoted AS.[Methods] 1.MTT assay was used to detect the effect of IS on the viability of HUVECs and HA-VSMCs,and the concentration and time of IS were screened.2.IS intervened HUVECs and HA-VSMCs,and detected cells miR-34a-5p expression by RT-qPCR;3.Transiently transfect HUVECs and HA-VSMCs with miR-34a-5p inhibitors and mimics to obtain cells with low expression and high expression of miR-34a-5p.After 24 hours of staining,cells were treated with IS,IS concentration of 1000 ?M,intervention for 36 hours,cell proliferation wAS detected by CCK-8 ASsay and EdU Assay,cell migration wAS detected by scratch test and Transwell ASsay,apoptosis wAS detected by flow cytometry ASsay and Hoechst 33342/PI,and the expressions of miR-34a-5p pathway related molecular proteins Notch-1,Wnt-1,Jag1,E2F1,Sirt1 and p53 were detected by Western blot.[Results] 1.MTT results showed that IS could inhibit the viability of HUVECs and HA-VSMCs(p<0.05),and the inhibition of cell viability wAS concentration and time dependent;IS concentration wAS 1000 ?M,intervention for 36 hours,the decline of cell viability wAS significant(p < 0.05).group(p<0.05).3.The results of cell proliferation,migration and apoptosis showed that the cell viability and mobility decreased and the apoptotic rate increased after IS intervention in HUVECs and HA-VSMCs(p<0.05);firstly,miR-34a-5p inhibitors and mimics were stained HUVECs and 2.RT-qPCR results showed that the expression of miR-34a-5p in HUVECs and HA-VSMCs wAS significantly higher than that in the control group after IS treatment,which respectively was 2.82 and 3.79 times(p<0.05);After HUVECs and HA-VSMCs were high and low expression of miR-34a-5p,cells were treatmented with IS,it was found that the expression of miR-34a-5p in high expression group was respectively 133.76 and 58.73 times compared with the IS group(p<0.05),and the expression of miR-34a-5p in low expression group was respectively 0.71 and 0.73 times compared with the IS HA-VSMCs to obtain low and high expression of miR-34a-5p cells,and then IS was used to intervene cells,the results showed that the cell viability and migration of HUVECs and HA-VSMCs in mimics+IS group were decreased compared with those of mimics group(p<0.05),the apoptotic rate increased(p<0.05),while the cell viability and mobility of the inhibitors+IS group increased compared with the IS group(p<0.05),and the apoptotic rate decreased(p<0.05).4.Western blot showed that the expression of Notch-1,Wnt-1,Jag1,E2F1,Sirt1 was decreased and the expression of p53 was increased after IS intervention in HUVECs and HA-VSMCs(p<0.05);firstly,miR-34a-5p inhibitors and mimics were stained HUVECs and HA-VSMCs to obtain low and high expression of miR-34a-5p cells,and then IS was used to intervene cells,the results showed that in HUVECs and HA-VSMCs mimics+IS group the expression of Notch-1,Wnt-1,Jag1,E2F1,Sirt1 were decreased compared with the mimics group,and the expression of p53 was increased(p<0.05).The expression of Notch-1,Wnt-1,Jag1,E2F1,Sirt1 were decreased in the inhibitors+IS group compared with the IS group,and the expression of p53 was increased(p<0.05).[Conclusion] 1.IS can inhibit the proliferation,migration and promote apoptosis of HUVECs and HA-VSMCs;2.miR-34a-5p participates in IS inhibition of HUVECs,HA-VSMCs proliferation,migration and promotion of apoptosis.