Effect And Mechanism Of MiR-92b In The Zebrafish Heart Regeneration

The ideal repair after myocardial injury is to activate proliferation of endogenouscardiomyocytes or supply ectogenic ones, but actually mature mammalianmyocardial cells have little potentiality of differentiation and proliferation.Accordingly, myocardial injury will eventually lead to the loss of heart function.Recent studies have shown that mammals existed proliferative capacity ofcardiomyocytes, but this potentiality is extremely limited in adult cardiac repair.Therefore, only after fully grasping the regulation mechanism of cardiac regenerationprocess, we can not make it possible to induce myocardial proliferation for repairingdamaged heart.In2002, Poss et al. first found that adult zebrafish had the ability to regenerate theimpaired heart. During the experiment, zebrafish fully regenerated hearts of20%ventricular resection within2months. Regeneration occurs through robustproliferation of cardiomyocytes localized at the leading epicardial edge of the newmyocardium. After that, cardiovascular research on zebrafish heart regenerationbecame the hot spots in this field. At present, although there have been many studiesto clarify the mechanism of this regeneration course, the main regulatory mechanismremains unclear.Nowadays, with the development of gene chip technology, scholars have foundthat gene differentially expressed in the surrounding area of regeneration heart atdifferent time, which included multiple genes of signaling pathways related to cellmoving, inflammation and adhesion. Among them, PDGF-B was upregulated in theregeneration heart, which would stimulate DNA synthesis of zebrafish myocardialcells in primary culture, while PDGF-B receptor inhibitors could slowdown DNAsynthesis of zebrafish cardiomyocytes in vitro and in vivo. Moreover, reportedevidences showed that PDGF-B signaling pathway is required for cardiomyocytesproliferation in heart regeneration process.Recent studies have shown that microRNA (miRNA) plays a key role in the genesregulation of apoptosis, differentiation and proliferation. In the zebrafish finregeneration study, evidence showed that Wnt signaling also subject was to miRNA regulation through miR-203control of Lef1in addition to regulation of Fgf signalingduring regeneration.Therefore, to find the key miRNA regulating PDGF-B and prove its role inzebrafish heart regeneration, more works should be done. It will further reveal theregulation mechanism of the zebrafish heart regeneration, enrich heart regenerationtheory, and provide a new target for activating endogenous myocardial cellsproliferation in the near future.Part I Establishment of zebrafish heart regeneration modelObjective: To establish a stable, reliable research platform on zebrafish heartregeneration.Method:1. Anesthetized adult zebrafish (more than6months of age) was placedupside down on the mold, heart was exposed under the microscope, part of theventricular apical ventricular was removed. After surgery, fishes were returned towater and stimulated to breathe by vigorously squirting water over the gills with apipette;2. Histological observation of the repair process of7dpa,14dpa and30dpaheart was performed;3. Mortality changes after partial amputation in differentresection area were analyzed;4. According to the zebrafish age, fishes were dividedinto three groups:0.5-1years,1-2years and more than2years old fishes. At thesame condition and resection area (20%), survival and cardiac repair capacity werecompared in these there groups.Results:1. Zebrafish had a high survival rate of over70%in excised heart arealess than20%, while in resection area greater than25%the rate was decreased to40%±7.1%.2Through histological observation, it was found that local fibroustissue was gradually reduced and myocardial cells were accumulated at the damagedarea of zebrafish heart during the regeneration process. At the end of30dpa, theheart almostly regenerated to be intact;3. Compared with group of0.5-1aged fishes,the other two groups had a lower level of survival rate (P<0.01), but it was found thatthe heart regeneration ability had no significant difference between the three groupsof different aged zebrafish (P>0.05).Conclusion: The establishment of the zebrafish heart regeneration model has ahigh success rate and stability. Adult zebrafish mortality increased with the resectionarea of heart. The ideal resection area is between15%-20%. Old zebrafish still hasthe ability of heart regeneration, but its ability to tolerate surgery declined. Part II Expression profilie of miRNAs targeted PDGF-B inthe zebrafish heart regenerationObjective: To screen the specific miRNA which has evident change in zebrafishheart regeneration process and one of its main targets is PDGF-B.Methods:1. Following the previous steps of zebrafish heart resection, protein andRNA were extracted after surgery according to the conventional methods, andconcentration determination and quality of identification were performed;2. Theexpression of PDGF-B during zebrafish heart regeneration was determined byWestern Blot and quantitative PCR;3. The3’untranslated region sequence ofPDGF-B gene was analyzed by bioinformatics software;4. miRNAs clone library ofzebrafish heart was established;5. The expression level of miRNAs highly expressedin zebrafish heart was detected by quantitative PCR during heart regenerationprocess.Results:1. Compared to sham group, PDGF-B gene expression levels in3dpa,7dpa and14dpa were significantly increased (P<0.01) by qRT-PCR and Western Blotdetection;2. Many miRNAs have high expression level in zebrafish heart clonelibrary, such as miR-1, miR-133a and let-7f;3. Conservative analysis found thatmost of miRNAs high expressed in zebrafish heart were highly conserved betweenzebrafish, mouse and human species, such as miR-92b, miR-1and miR-133a;4. Bysequence analysis of the3’non-translated region of PDGF-B, it was found that manyof miRNA binding sites existed, and these sites were highly conserved in evolution;5. By quantitative PCR, miR-199, miR-92b, miR-143, etc., were found that they hadsignificant changes in the zebrafish heart regeneration process;6. Compared withsham-operated, miR-92b expression levels in the heart after3dpa,7dpa,14dpadecreased significantly (P<0.05). Its expression decreased to the lowest expressionlevels in7dpa, and then began to rise. In the30dpa time, it returned to normal level.Conclusion: During zebrafish heart regeneration, miR-92b may play a key role inmyocardial regeneration by regulating of PDGF-B.Part III Role and mechanism of miR-92b in zebrafishcardiomyocytes proliferationObjective: To determine the impact of miR-92b on zebrafish cardiomyocytesproliferation and the underlying regulation mechanism of zebrafish heart regeneration.Method:1. Cardiomyocytes of zebrafish were separated and cultured;2,MicroRNAs mature sequences were obtained on Mirbas, its antisensecomplementary RNA were designed, and the sequence was synthesized chemicallyand modified on the base2’methoxy. Cardiomyocytes transfected with the antisensesequence (40nM) were incubated for48hours, and silencing efficiency of the targetmicroRNA was analyzed by quantitative PCR;3. Dual luciferase reporter genesystem predicted the target gene of miR-92b;4. The affinity of the four binding sitesof miR-92b and PDGF-B was analysized by bioinformatics software in the PDGF-Bgene3’untranslated region sequences;5. By immunofluorescence detection ofMEF-2, the proliferative ability of myocardial cells transfected with miR-92binhibitor was observed;6. qRT-PCR and Western Blot detected the PDGF-Bexpression levels in myocardial cells transfected with miR-92b inhibitor.Results:1. In293T cells transfected with miR-92b mimic24h later, theexpression of miR-92b improved significantly to20times of the level of controlgroup. In cells transfected with miR-92b inhibitor, miR-92b expression significantlyreduced to41%of the control group;2. The seed sequence of miR-92b and PDGF-Bhad7-8base pairing sites with high affinity;3. PDGF-B was predicted as the targetgene of miR-92b by the dual luciferase reporter gene system;4. In separatedzebrafish myocardial cells transfected with miR-92b inhibitor48h later, the level ofmiR-92b decreased to48.1%of the control group, and the total number of cells wasabout2times that of the control group. The number of PCNA positive cells intransfected group significantly increased, which was1.6times those of the controlgroup;5. By Western Blot and qRT-PCR assay, it was found that the expressionlevel of PDGF-B in myocardial cells transfected with miR-92b inhibitor wassignificantly higher (P<0.05) than that of the control group.Conclusion: MiR-92b may regulate myocardial cell proliferation throughPDGF-B.Summary: Zebrafish heart regeneration model has a high success rate andstability. The ideal resection area is between15%-20%, and aged zebrafish still hasrobust heart regeneration ability. Bioinformatics predicted the miRNA of targetingthe expression of PDGF-B, and zebrafish heart highly expressed miRNAs (miR-1,miR-133, miR-199, miR-21, miR-92) were identified by method of small RNAlibraries, then the expression levels of these miRNAs in the heart regeneration were tested. By the above method, results showed that miR-92b may be a key regulator ofPDGF-B. Luciferase reporter genes proved PDGF-B was the target gene of miR-92b.On cultured myocardial cells, inhibiting miR-92b promoted zebrafishcardiomyocytes proliferation. Our study found and proved that MiR-92b regulatedzebrafish heart regeneration through its target gene PDGF-B, which enriched themolecular mechanism of cardiac regeneration.