The Study On The Effect And Mechanism Of MicroRNA-205-3p On Rheumatic Heart Disease And Ischemia Reperfusion Injury Through IL-1b

Objective:Cardiovascular and cerebrovascular diseases are among the most lethal diseases in the world.Rheumatic heart disease and ischemic heart disease are the two most common diseases in heart related diseases.In recent years,the study of small RNA is increasing.It has become a consensus and research hotspot that microRNAs play a role in regulating metabolism and pathogenesis.However,the expression profiles,modes of expression and how microRNAs participate in or regulate processes in two heart diseases remain unclear.In this study,high-throughput sequencing of microRNAs,histopathological examination,and molecular biological methods were used to evaluate the role of microRNAs in the regulation of two types of heart diseases.Methods:1.Serum samples from normal subjects and patients with valvular heart disease were collected and microRNAs were extracted for high-throughput sequencing(HiSeq deep sequencing)to compare with those from congenital valvular heart disease and normal subjects.2.Using miRDeep2 and Piano to select small RNA and select individual specific miRNA for Q-PCR verification.3.Using RNA hybrid,microanda and TargetScan to predict target genes,the targeting relationship between microNA and predicted targeting mRNA was validated by double fluorescein reporting system.The expression levels of IL1beta and IL1R1 in mitral valve were detected and evaluated by immunohistochemistry and Weatern blot.4.Functional enrichment was carried out by GO and KEGG.5.Blood samples from patients with PCI were collected before and after operation.6.Langendorff isolated heart perfusion system was used to carry out ischemia-reperfusion in isolated hearts.30min ischemia is first experienced,followed by 20 to 60 minutes of perfusion.7.The myocardial cells were cultured in vitro and treated with hypoxia and hypoglycemia to simulate the process of ischemia and reperfusion.8.Increase or decrease the content of miR-205-3p through cardiac muscle cells through lentivirus.9.Add safflower yellow to treat animal models and cell models.10.To observe and measure the changes of miR-205-3p,IL-1b and other inflammatory factors in patients,animal models and cell models.Results:1.Serum microRNA sequencing showed that the expression of 13 microRNAs in the valves of four patients with rheumatic heart valvular disease was up-regulated and 91 microRNAs were down-regulated.2.The expression trend of Q-PCR verification was consistent with the sequencing results.3.Target prediction using Targetscan showed that miR-205-3p targeted the 3’UTR region of IL-1b and hsa-miR-3909 targeted the 3 ’UTR region of L-1R1.Dual luciferase reporter analysis confirmed the targeting relationship between miR-205-3p and hsa-miR-3909 to IL-1b and IL-1R1.4.Mitral valve tissues from patients with rheumatic heart disease were detected by immunohistochemistry and Western blot.The results showed that the levels of IL-lbeta and IL-1R1 in mitral valve tissues from patients with rheumatic heart disease were higher than those from patients with congenital heart disease.5.Mi-205-3p and 1L-1b increase simultaneously when myocardial ischemia-reperfusion injury occurs.1L-1b content decreases when the expression of microRNAs-205-3p is inhibited by lentiviral transduction into anti-microRNA205-3p.6.Safflower yellow can inhibit the damage of pre-miR-205-3p to cells and decrease the level of IL-1b.7.Safflower yellow pretreatment could maintain LVDP well,and the elevated value of LVEDP was significantly lower than that of the control group,while reducing the size of myocardial infarction.8.Safflower yellow can prevent the increase of NLRP3 and ROS levels in ischemia-reperfusion injury and decrease the secretion of caspas-1p10.Conclusion:1.The expression profiles of serum microRNAs in patients with rheumatic heart valvular disease were significantly different from those in normal subjects.The down-regulation of serum hsa-microRNA-205-3p and hsa-microRNA-3909 in patients with rheumatic heart valvular disease is conducive to the up-regulation of IL-lb and IL-1R1 expression in valvular tissue,while the up-regulation of IL-lb and IL-1R1 expression in patients with rheumatic heart valvular disease reveals that chronic inflammation still exists after the acute phase of rheumatic heart valvular disease and even several years.2.MiR-205-3p plays a regulatory role in IL-lb.3.Safflower yellow can alleviate ischemia/reperfusion,and its mechanism may be related to inhibiting miR-205-3p down regulating IL-1b.4.Safflower yellow also has strong anti-inflammatory and antioxidant effects,maintains LVDP,delays the elevation of LVEDP,reduces infarct size,and has high medicinal value.