The Mechanism Of MicroRNA-449c-5p Regulation In The Degenerative Calcific Aortic Valve Disease

Background and ObjectiveWith the decline in rheumatic fever incidence and the increasing aging population, Degenerative calcific aortic valve disease (DCAVD) becomes the most common heart valve disease in the elderly. At the present time, DCAVD has become one of the important reasons of heart failure, syncope or even sudden death in Chinese old people. There is no effective medicine to prevent or delay DCAVD. The operation is still the most effective treatment. These brings huge economic burden to patients and society. It will have great economic and social values to explore the pathogenesis of DCAVD.DCAVD was previously recognized as an irreversible passive process of calcium and phosphorus deposition; however, accumulating evidence has shown that its pathogenesis is an active process which is regulated by many factors. It may involve chronic inflammation, abnormal lipid metabolism, calcium deposition and so on. To date, the specific pathogenesis of DCAVD is still unknown and need for further research.Mohler et al. had cultured VICs and confirmed that it might acquire an osteoblastic phenotype and form calcium nodule under certain conditions. Previous studies have shown that the expression of many markers of the osteoblast and bone tissue is up-regulated, such as alkaline phosphatase, osteopontin and osteocalcin, which indicates calcification-related factors may participate in the pathogenesis of DCAVD. As important cellular non-coding regulatory molecules, microRNAs could regulate various physiological and pathological processes. VICs combined with miRNA is a new perspective to study the pathogenesis of DCAVD, which could provide the potential valve for early diagnosis and treatment.This research screens out related miRNAs of DCAVD through miRNA chip, then we further verify the functions of the selected miRNA-449c-5p, and finally we explore the mechanism of miRNA-449c-5p regulation through RT-PCT and western blot, in order to illuminate the pathogenesis of DCAVD.Infective endocarditis (IE) is a microbial infection involving the endocardium, sometimes with the formation of excrescence. In recent years, the incidence of IE is on the rise. Up to now, it is still controversial on the choice for surgery opportunity of IE. In the tradition, the medical treatment is firstly selected. The operation is usually performed after effective anti-infective treatment for about 4 to 6 weeks, however, cardiac insufficiency, embolism or even death may break out during this time. This study aims to analyze the distribution and drug sensitivity of pathogens in blood culture and evaluate the impact of early surgical treatment in active IE.MethodsPart 11. Screening of differentially expressed miRNAs and determination of experimental miRNA:Total RNA was extracted from calcific aortic valves and relatively normal valves. The miRNA expression was examined and differentially expressed miRNAs were screened out by gene chip. Then miRNA-449c-5p was chosen as research object.2. Isolation, culture and identification of VICs:VICs were isolated and amplified from human normal aortic valve in vitro by modified collagenase digestion method. The cell phenotype was identified by the immunofluorescent staining.3. Functional verification of miRNA-449c-5p:miRNA-449c-5p mimic and inhibitor were individually transfected into VICs, which stimulate overexpression and low expression of miRNA-449c-5p. Transfection efficiency was measured in a preliminary test. The osteoblastic activity of VICs was detected by RT-PCR, western blotting, ALP activity and Alizarin red staining in 7 days and 14 days after osteoblastic induction to further verify the function of miRNA-449c-5p on osteoblastic differentiation of VICs.4. Mechanism of miRNA-449c-5p regulation on osteoblastic differentiation of VICs: Preliminary target gene of miRNA-449c-5p was firstly predicted by the target gene professional forecasts site. Then we matched structure validation of target gene and miRNA-449c-5p through the dual luciferase experiment. At last, the target gene was confirmed by the siRNA interference technology.5. IL-6 regulation on miRNA-449c-5p expression during osteoblastic differentiation of VICs:Firstly, culture media were collected for detection of IL-6 24h and 48h after induction. Secondly, VICs were induced with growth medium containing IL-6 in the experimental group. In the control group, VICs were incubated in growth medium alone. Cellular RNA were extracted for miRNA-449c-5p detection 24h and 48h after induction.6. Animal experiment:miRNA-449c-5p agomirs and antagomirs were individually injected into Balb/c mice through tail vein, which induced overexpression and low expression of miRNA-449c-5p. Then the mice in each group were given subcutaneous injection of vitamin D3 to induce soft tissue calcification. Six week after the first injection, echocardiography was performed.Part 2Blood culture results and clinical data of 62 patients with active infective endocarditis in our hospital from May 2010 to July 2014 were retrospectively reviewed. All patients underwent early surgical treatment and the long-term follow-up was conducted.Results1. DCAVD-related differentially expressed miRNAs were successfully screened out. MiRNA-449c-5p was chosen as the research object from these differential miRNAs.2. VICs were successfully isolated. The cell phenotype was identified by the immunofuorescent staining, which proved to meet the experimental requirements.3. The functional research of miRNA-449c-5p was successfully conducted. miRNA-449c-5p regulates osteoblastic differentiation of VICs in animal level, cell level, protein level and mRNA level.4. Smad4 was predicted as the target gene of miRNA-449c-5p through the TargetScan site. Smad4 was finally verified as the target gene through the dual luciferase test. At last we found that miRNA-449c-5p regulates VICs osteoblastic differentiation through the TGF-β/ Smad pathway.5. We found that secreted IL-6 levels progressively increased during VICs osteoblastic differentiation. At the same time, miRNA-449c-5p expression significantly decreased in VICs after IL-6 stimulation. These results demonstrate that IL-6 may regulate miRNA-449c-5p expression in VICs.6. Streptococcus, Enterococcus and Staphylococcus aureus are the main pathogens in active IE. Two patients died during the perioperative period. Other 60 patients recovered smoothly. The number of patients recovered with I and II grade heart function were 42 and 15 respectively during the follow-up period. There was no relapse of endocarditis occurred during the follow-up period.Conclusion1. The DCAVD specific miRNA expression profile was identified.2. MiRNA-449c-5p regulates osteoblastic differentiation of VICs by targeting Smad4 and through the TGF-β/Smad pathway in vivo and in vitro.3. The pathogen of patients with active IE is mainly Gram-positive bacteria. Clinicians should choose the rational antibacterial drug according to the situation of resistance of pathogenic bacteria. Early surgical treatment could achieve satisfactory effectiveness for active IE.