| Objectives：Myocardial infarction is myocardial necrosis caused through coronaryartery persistent ischemia and hypoxia, and serious harm to human health.After myocardial infarction, ventricular remodeling, myocardial hypertrophyand myocardial fibrosis occurred. Finally it leads to heart failure. Over theyears, though there are many treatment methods including intervention, drugtreatment, surgery and stem cell transplantation, but they cannot interrupt theprogress of heart failure. Therefore seeking more effective and more advancedtreatment has become to be a difficult task. In recent years, gene therapy hasbeen accepted widely. Many studies showed that microRNAs (miRNAs)involved in the regulation of cell proliferation, differentiation, apoptosis, andmetabolic processes. Recent studies about miRNAs and cardiovascular diseaseincrease gradually, which microRNA-214(miR-214) is a newly discoveredfactor. Recent studies have reported that miR-214has myocardial protectiveeffect, but the mechanism of cardiac fibroblasts is not clear. When myocardialcontractility decreased, cardiac output reduced and blood flow of kidneydiminish. Then renin-angiotensin-aldosterone system (RAAS) is activatedand increased angiotensin (AngII) stimulated fibroblasts secrete collagen,leading to myocardial interstitial fibrosis and ventricular remodeling. So ourstudy adopts the AngII to stimulate cardiac fibroblasts (CFS) and establishmyocardial fibroblasts fibrosis model. After transfecting miR-214to cell, theeffect of miR-214on CFS fibrosis by AngII stimulation was observed. Weaim to find out the effective methods of delaying ventricular remodeling andinhibiting myocardial fibrosis.Methods：1Primary culture of neonatal rat cardiac fibroblasts and identification The hearts of1-3-day-old Sprague–Dawley rats (Hebei MedicalUniversity Laboratory Animal Center) were removed afterhypothermia-induced anesthesia. The ventricles were minced with scissors.The minced tissue was dispersed，filtrated, centrifuged and resuspended thecells were collected by Tyrisin digestion method. Based on the different ratesof attachment among the various cell types fibroblasts attached to cultureplates. Non-adherent cells were removed. The expression of cardiac myocytesspecific Vimentin was detected by immunocytochemical stain to identify thecardiac fibroblasts.2Effects on cell viability in CFS after stimulation with AngIICardiac fibroblasts in the2~3passage were cultured in serum-freemedium for24h before experiment. Then cardiac fibroblasts were treated witheither normal control or AngII (10-8，10-7，10-6，10-5mol/L) for24h. Cell vitalitywas measured by MTT. Then Cardiac fibroblasts were treated by10-6mol/LAngII with either normal control or AngII (6，12，24h),cell vitality wasmeasured by MTT too.3Hydroxyproline content in CFS AngII-inducedCardiac fibroblasts in the2~3passage were placed in serum-freemedium for24h before experiment. Cardiac fibroblasts were treated witheither normal control or AngII (10-8，10-7，10-6，10-5mol/L) for24h. Afterwards,Hydroxyproline content was measured by Hydroxyproline checkerboard. ThenCardiac fibroblasts were treated by10-6mol/L AngII with either normal controlor AngII (6，12，24h) Hydroxyproline content was measured byHydroxyproline checkerboard too.4miR-214expression in CFS AngII–induced with different concentration byQ-PCR analysisCardiac fibroblasts in the2~3passages were placed in serum-freemedium for24h before experiment. Cardiac fibroblasts were treated witheither normal control or AngII (10-8,10-7,10-6,10-5mol/L) for24h. Afterwards,miR-214expression in each group were analyzed by real time quantitativePCR. 5miR-214expression in CFS after transfection by Q-PCR analysisThe experiment is divided into five groups: normal control, miR-214precursor (pre-miR-214), pre-miR-214(pre-scramble), miR-214inhibitor(anti-miR-214), anti-miR-214(anti-scramble). They were added to the culturemedia at a final oligonucleotide concentration of30nM. Cardiac fibroblastswere placed in serum-free medium for24h and then treated with AngII(10-6mol/L) for24h. Afterwards, miR-214expression in each group wereanalyzed by real time quantitative PCR.6COLI、COLIII、TGFβ1、TIMP-1、MMP-1、MMP-2mRNA expression inCFS with AngII after transfection by Q-PCR analysisCardiac fibroblasts were incubated in good condition for24h beforemiR-214transfection. Cells were placed in serum-free medium for24h andthen treated with AngII (10-6mol/L) for24h. RNA was then isolated from thecultured cells using an RNA isolation kit. The experiment is divided into sixgroups: normal control, AngII, AngII+pre-miR-214,AngII+pre-scramble,AngII+anti-miR-214, AngII+anti-scramble. Afterwards, mRNA expression ofCOLI、COLIII、TGFβ1、TIMP-1、MMP-1and MMP-2in each group wereanalyzed by real time quantitative PCR.7COLI、COLIII、TGFβ1、MMP-1、MMP-2expression in CFS with AngIIafter transfection by Western-blot analysisCardiac fibroblasts were incubated in good condition before transfectionfor24h before miR-214transfection. Cells were placed in serum-free mediumfor24h and then treated with AngII (10-6mol/L) for24h. Proteins was thenisolated from the cultured cells using a RIPA cracking liquid. The experimentis divided into six groups: normal control, AngII, AngII+pre-miR-214,AngII+pre-scramble, AngII+anti-miR-214, AngII+anti-scramble. Afterwards,miR-214Proteins expression of COLI、COLIII、TGFβ1、TIMP-1、MMP-1andMMP-2in each group were analyzed by western blotting.Results:1Primary culture of neonatal rat cardiac fibroblasts and identificationCardiac fibroblasts showed a trend of ’S’ type growth over all, they grow in the logarithmic phase in2~4days and become to be steady after4days.The cardiac fibroblasts showed to be triangular, shuttle or polygonal undermicroscope; Immunohistochemical results for Vimentin showed the purity of95%.2Effects on cell viability in CFS AngII-inducedMTT assay results suggested that the proliferation (OD value) of cardiacfibroblast increased gradually with the increase of concentration of AngII andtime and the effect was almost dose and time dependent. Therefore,concentration of about50%cell proliferation induced by AngII (10-6mol/L)for24h was selected to establish cardiac fibroblasts fibrosis model.3Effects of AngII on collagen secretion in CFS HydroxyprolineCheckerboard testing results suggested, compared with normal controlgroup, secreting collagen was increased with concentration and time of AngII.4miR-214expression of CFS AngII-induced with different concentrationQ-PCR detection showed that miR-214expression of AngII groupdecreased significantly compared with normal control group and graduallydecreases with increase of AngII concentration.5miR-214expression of CFS after miRNA transfectionQ-PCR detection showed that the miR-214expression in pre-miR-214group was obvious higher than pre-scramble group, and miR-214expressionin anti-miR-214group was obvious lower than anti-scramble group.Compared with and anti-scramble group there is no statistical differenceamong normal control group, pre-scramble group and anti-scramble group. Sotransfection is success.6COLI、COLIII、TGFβ1、MMP-1、MMP-2、TIMP-1mRNA expressionof CFS with AngII after transfectionQ-PCR detection showed that COLI、COLIII、TGF β1、TIMP-1mRNAexpression of CFS in AngII group are significant higher than the normalcontrol group (P<0.01); COLI、COLIII、TGFβ1、TIMP-1mRNA expressionof AngII+pre-miR-214group in CFS are lower than that in AngII group(P<0.01) and there is no statistical differences compared with normal control group (P>0.05). COLI、COLIII、TGFβ1、TIMP-1mRNA expression of AngII+anti-miR-214group are significant higher than the AngII group (P<0.05).There is no statistical difference among pre-scramble, anti-scramble group andnormal control group (P>0.05). MMP-1、MMP-2mRNA expression of CFS inAngII group are significant lower than the normal control group (P<0.05);MMP-1、MMP-2mRNA expression of AngII+pre-miR-214group in CFS arehigher than that in AngII group (P <0.01), there is no statistical differencescompared with normal control group (P>0.05). MMP-1、MMP-2mRNAexpression of AngII+anti-miR-214group are significant lower than the AngIIgroup (P<0.01).There is no statistical difference among pre-scramble,anti-scramble group and normal control group (P>0.05).7COLI、COLIII、TGFβ1、MMP-1、MMP-2protein expression of CFS withAngII after transfectionWestern-blot detection showed that COLI、COLIII、TGF β1proteinexpression of CFS in AngII group in CFS are significant higher than thenormal control group (P<0.05); COLI、COLIII、TGFβ1protein expression ofAngII+pre-miR-214group in CFS are lower than that in AngII group(P<0.05), COLI、COLIII protein expression no statistical differencescompared with normal control group (P>0.05), while TGFβ1proteinexpression slightly higher than the AngII group(P<0.05). COLI、COLIII、TGFβ1protein expression of AngII+anti-miR-214group are significantlyhigher than the AngII group (P<0.05).There is no statistical difference amongpre-scramble, anti-scramble group and normal control group (P>0.05).MMP-1、MMP-2protein expression of AngII group in CFS are significantlylower than the normal control group (P<0.01); MMP-1、MMP-2proteinexpression of CFS in AngII+pre-miR-214group higher than that in AngIIgroup (P<0.05), and there is no statistical differences compared with normalcontrol group (P>0.05). MMP-1、MMP-2protein expression of AngII+anti-miR-214group are significantly lower than the AngII group (P<0.05).MMP-1protein expression of pre-scramble and anti-scramble group slightlylower than the AngII group (P<0.05). MMP-2protein expression no statistical difference among pre-scramble, anti-scramble group and normal control group(P>0.05).Conclusions1Cardiac fibroblasts can produce more collagen by AngII-induced andthe effect was almost dose and time dependent. About50%cell proliferationrate was obtained by inducing with AngII (10-6mol/L) for24h.2In cardiac fibroblasts, the expression of miR-214is decreased withincreasing concentrations of AngII. miR-214overexpression can inhibitcollagen I、III synthesis. It showed miR-214is associated with cardiac fibrosis.3miR-214can inhibit the expression of fibrotic factor TGF-β1and thesynthesis of TIMP1, thereby promote the expression of MMP-1and MMP-2,and inhibit the production of collagen.In a word miR-214can inhibit cardiacfibrosis. |
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