Research Paper Crucial Role Of MiR-433 In Regulating .

Theranostics 2016, Vol. 6, Issue 12in this study. MI was generated by ligating the leftanterior descending coronary artery (LAD) using a7/0 silk thread while sham was created by the ed cardiomyopathy mouse modelwas induced by chronically treating mice with eitherdoxorubicin or phosphate-buffered saline (PBS) byfour intraperitoneal (i.p) injections (day 0, 2, 4 and 6)at a dose of 4 mg/kg. All mice were sacrificed after 4weeks.To determine if inhibition of miR-433 is sufficientto prevent cardiac fibrosis in vivo, mice were injectedvia tail vein with 80 mg/kg antagomir (a 2’OME 5’chol modified miR-433 inhibitor) or the scramblecontrol (Ribobio, Guangzhou, China) for 3consecutive days and subjected to LAD ligation. AAVrepresents an efficient and safe vector for in vivo genetransfer and serotype 9 is significantly cardiotropic[23-26]. Thus, besides miR-433 antagomir, thecardiotropic miR-433 sponge AAV9 was used todetermine further if cardiac inhibition of miR-433 issufficient to prevent fibrosis in vivo. In brief, micewere randomly chosen to receive a single-bolus tailvein injection of either miR-433 sponge AAV9 ormiR-scramble (Hanheng Biotechnology, Shanghai,China) at 1*1011 vg (viral genomes) per animal. After 1week, mice were subjected to LAD ligation and finallysacrificed at 3 weeks post-MI.miRNA Array and Gene-Chip AnalysisTotal RNA extracted from ventricular tissues 21days post-MI or sham control was used for miRNAarrays based on Affymetrix 4.0 (OE Biotech’s,Shanghai, China). Additionally, total RNA extractedfrom ventricular tissues 21 days post-MI injected withmiR-433 antagomir or scramble control was used forgene-chip analysis based on Agilent SurePrint G3Mouse GE (8*60K, Design ID: 028005) Microarray (OEBiotech’s, Shanghai, China). The MIAME compliantdata have been submitted to Gene ExpressionOmnibus (GEO, platform ID: GSE74135 for miRNAarray and GSE74206 for gene-chip analysis,respectively).Quantitative Real-time Polymerase ChainReactions (qRT-PCRs)Total RNAs were extracted from cardiacfibroblasts and heart samples by using miRNeasyMini Kit (Qiagen, Hilden, Germany) according tomanufacturer’s instructions. Total RNAs (400 ng)were reverse transcribed using Bio-Rad iScriptTMcDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) toobtain cDNAs. The expression levels of TGF-β,α-SMA, Col1a1, and Col3a1 were analyzed by usingBio-Rad SYBR qPCR (Bio-Rad, Hercules, CA, USA) on2070ABI-7900 Real-Time PCR Detection System (7900HT,Applied Biosystems, CA, USA). 18S RNA was used asan internal control for gene expressions. Primersequences used in the study are listed inSupplemental Table 1. For quantitative miRNAanalysis, the Bulge-LoopTM miRNA qPCR Primer Set(RiboBio) was used to determine the expression levelsof miRNAs with Takara SYBR Premix Ex TaqTM (TliRNaseH Plus) on ABI-7900 Real-Time PCR DetectionSystem (Applied Biosystems). U6 was used as aninternal control for miRNA template normalization.Pharmacokinetics of miRNAmiR-433 antagomir or the scramble control(Ribobio, Guangzhou, China) was prepared in PBSand administered via tail vein at a dose of 7.5 mg/kgfor each mice. Subsequently, mice were sacrificed andplasma and heart tissues were collected immediatelyat different time points of 5, 10, 15, 30, 60, 120, 240,480, 1320 and 1440 minutes after injection (n 5 pergroup for each time point) [27]. miR-433 expressionlevels in plasma and heart samples were determinedusing qRT-PCRs as described above.Immunofluorescence and EdU StainingCardiac fibroblasts were fixed in 4%paraformaldehyde (PFA) for 20 min at roomtemperature. Cells were then permeabilized with 0.2%Triton X-100 for 20 min and blocked with 10% goatserum in PBS-Tween for 1 h at room temperature.Subsequently, cardiac fibroblasts were incubated withα-SMA-Cy3 antibody (1:500, Sigma, St. Louis, MO,USA) diluted in 10% goat serum overnight at 4 C. Todetect proliferation, EdU assays were performedusing Click-iT Plus EdU Alexa Fluor 488 Imaging Kit(Invitrogen) according to manufacturer’s instructions.Cell nuclei were counterstained with DAPI and thenumber of EdU-positive nuclei was calculated. Fifteenfields/sample (200 x magnification) were viewedunder a confocal microscope (Carl Zeiss, Thuringia,Germany).Sections of heart samples were cut at a thicknessof 5-6 μm. Subsequently, the sections were fixed in 4%PFA for 20 min at room temperature, permeabilizedwith 0.2% Triton X-100 for 20 min, and then blockedwith 10% goat serum in PBS-Tween for 1 h at roomtemperature. Next, the sections were incubated withdiluted primary antibodies at 4 C overnight; thefollowing antibodies were used: α-SMA-Cy3 antibody(1:500, Sigma), Vimentin antibody (1:100, Abcam),Ki67 antibody (1:100, Abcam), and pHH3 antibody(1:100, Abcam). After three washes with PBS for 5 mineach, the sections were incubated with secondaryantibodies or other dyes at room temperature for 2 h.Fifteen fields/sample (400x magnification) werehttp://www.thno.org

Theranostics 2016, Vol. 6, Issue 122071viewed under a confocal microscope (Carl Zeiss).and averaged for each mouse.Western Blotting AnalysisTTC stainingCardiac fibroblasts and heart samples were lysedusing RIPA buffer (Beyotime Institute ofBiotechnology, Nantong, China), which contained aprotease inhibitor cocktail (Sigma). The concentrationof protein samples was evaluated by BicinchoninicAcid Protein Assay Kit (Thermo Fisher, Waltham,MA, USA). Equal amounts of protein were separatedin SDS-PAGE and blotted onto PVDF membranes.The primary antibodies used were from the followingsources: α-SMA (1:1000, Sigma), TGF-β (1:1000, CellSignaling Technology, Boston, MA, USA), p38 (1:1000,CST), p-p38 (1:1000, CST), ERK (1:1000, CST), p-ERK(1:1000, CST), p-Smad3 (1:1000, CST), Smad3 (1:1000,CST), JNK1 (1:1000, CST), AZIN1 (1:500, Proteintech,Wuhan, China), CTGF (1:500, Proteintech, Wuhan,China), Col1a1 (1:500, Proteintech), Col3a1 (1:500,Proteintech), MMP2 (1:500, Proteintech), MMP9(1:500, Proteintech) and GAPDH (1:10000, Kangchen,Shanghai, China). All proteins were visualized byECL Chemiluminescence Kit (Thermo Fisher) and thequantification of each band was performed usingImagelab Software (Bio-Rad) with GAPDH as aloading control.At 3 days’ post LAD ligation, mice wereanesthetized with intraperitoneal injection of 0.5mg/g tribromoethanol. Subsequently, 1 ml Evansblue (BioSharp, Anhui, China) was slowly injectedinto inferior vena and the heart was removedimmediately. After storage for 15 minutes at -20 C,the heart was cut into 5 transverse slices at 1 mmthickness across the long axis. The slices were thenstained with 1% triphenyltetrazolium chloride (TTC,Amresco, OHIO, USA) in PBS for 10 min at 37 Cfollowing which the slices were fixed with 4% PFAand analyzed. The final infarct size was calculated byImage J Software (National Institutes of Health).Luciferase Reporter AssayA fragment of the 3’UTRs of AZIN1 or JNK1containing the target site of miR-433 was obtained byPCR amplification and then cloned into thepGL3-Basic Vector (Promega, Madison, WI, USA) togenerate the AZIN1 or JNK1 wt-luc vector. TheAZIN1 or JNK1 mutant-luc vector was generated byusing the MutaBest kit (Takara, Tokyo, Japan).Forty-eight hours after transfection, luciferaseactivities were measured using a dual luciferasereporter assay system (Promega) following a standardprocedure.EchocardiographyThree weeks after the injection of miR-433antagomir, mice were anesthetized with 1.5–2%isoflurane and then evaluated by Vevo 2100echocardiography (VisualSonics Inc, Toronto,Ontario, Canada) with a 30 MHz central frequencyscan head to detect cardiac function. The followingparameters were measured from M-mode imagestaken from the parasternal short-axis view atpapillary muscle level: left ventricular fractionalshortening (FS) and left ventricular ejection fraction(EF). The left ventricle internal diameter (LVID),interventricular septum (IVS), and left ventricleposterior wall (LVPW) in diastole or systole were alsomeasured. At least three measurements were obtainedMasson’s Trichrome StainingHeart samples were fixed in 4% PFA and thenembedded in paraffin. Five μm-thick sections weresubjected to Masson’s trichrome staining following astandard procedure. Images of the left ventriculararea of each section were taken by Nikon model (200xmagnification) with Spot Insight camera. Image JSoftware (National Institutes of Health) was used toquantify fibrotic region in each section. Thepercentage of fibrosis was measured as fibrosisareas/total left ventricular areas x 100%.Collagen content assayA quantitative dye-binding method was used todetermine the collagen content. Analysis of hearttissues was performed using Sircol assay (Biocolor,Carrickfergus, UK) according to manufacturer'sinstructions. In this assay, each heart sample wasweighed and homogenized with pepsin. The BioTekSoftware (Hercules, CA, USA) was used to quantifycollagen content in each sample.Statistical AnalysisData were presented as mean SE. A Student’st-test, Chi-squares test or one-way ANOVA followedby Bonferroni’s post-hoc test was used to compare theone-way layout data when appropriate. P values lessthan 0.05 were considered to be statistically different.All analyses were performed using GraphPad Prism5.ResultsmiR-433 is Increased in Cardiac FibrosismiRNA arrays were used to determine aberrantexpressions of miRNAs, which might contribute tocardiac fibrosis in the post-MI ventricle at a time pointnotable for prominent fibrosis. A total of 26 miRNAswere found to be dysregulated (Fold change 2.0;http://www.thno.org

Theranostics 2016, Vol. 6, Issue 12P 0.05; Figure 1A and Supplemental Table 2).Interestingly, the top 3 dysregulated miRNAsincluding miR-34b-3p, 34c-5p, and 34c-3p belong tothe miR-34 family, whose inhibition has been shownto attenuate pathological cardiac remodeling [13].Since miR-433 (number fourth) has previously beenreported to participate in kidney and liver fibrosis [21,22] but has not so far been explored in themyocardium and during cardiac fibrosis, we exploredits function further.Based on the qRT-PCR analysis, we confirmedthat miR-433 was upregulated in heart samples withfibrosis from mice 3 weeks post-MI (Figure 1B). Toexclude the possibility that increased miR-433 isspecific to cardiac fibrosis post-MI, we alsodetermined its expression in doxorubicin-inducedcardiomyopathy rodent model and in human dilated2072cardiomyopathy (DCM) (Figure 1B). The clinicalinformation and echocardiography parameters forDCM patients are presented in Supplemental Table 3.The DCM sample size is small due to the difficulty ofacquiring human heart tissues. Interestingly, miR-433was consistently upregulated in all three models, i.e.,in heart tissues with fibrosis, in doxorubicin-inducedcardiomyopathy, and in patients with DCM (Figure1B). Thus, there appeared to be a strong correlationbetween the presence of cardiac fibrosis and anincrease in miR-433 expression in several differentcardiac diseases. Furthermore, miR-433 was alsoincreased in cultured neonatal rat cardiac fibrosismodels stimulated by TGF-β or Ang II (Figure 1C-D).Taken together, these data supported a potential rolefor miR-433 in cardiac fibrosis.Figure 1: miR-433 is increased in cardiac fibrosis. A, dysregulated miRNAs in hearts from 21 days post-myocardial infarction (MI) versus sham control mice(n 4); B, upregulated miR-433 in ventricle samples from 21 days post-MI mice (n 4), a rodent model of doxorubicin (Dox)-induced cardiomyopathy (n 6), andhuman dilated cardiomyopathy (n 4); C-D, increased miR-433 in two in vitro cardiac fibrosis model induced either by TGF-β or Angiotensin II (n 6); E, expressionof miR-433 in neonatal cardiac fibroblasts (NRCF) compared to cardiomyocytes (NRCM) (n 6); F, markers for pathological hypertrophy (ANP, BNP and Myh7) andextracellular matrix proteins (CTGF, TSP-1, Col1a1 and Col3a1) in cardiomyocytes with miR-433 overexpression (n 6). Scale bar: 50 μm. *, P 0.05, **, P 0.01, ***,P 0.001 versus respective controls.http://www.thno.org

Theranostics 2016, Vol. 6, Issue 122073Figure 2: Antagonizing miR-433 attenuates cardiac fibrosis and preserves ventricular function post-myocardial infarction. A, decreased miR-433in hearts from mice treated with miR-433 antagomir (n 6); B, preserved left ventricular fractional shortening (FS) and ejection fraction (EF); C, reduced cardiacfibrosis; D, decreased collagen content in myocardial infarction (MI) with miR-433 inhibition, as evidenced by echocardiography (n 6), Masson’s trichrome staining(n 4), and Sircol assay (n 4); E, no difference in the infarct size between mice treated with miR-433 antagomir or negative control 3 days post-MI (n 7). Scale bar:100 μm. *, P 0.05, **, P 0.01, ***, P 0.001 versus respective controls.In vivo Inhibition of miR-433 Preserves CardiacFunction and Prevents FibrosisNext, we determined the relative expressionlevel of miR-433 in isolated neonatal rat cardiacfibroblasts versus cardiomyocytes, and demonstratedhigher expression level in fibroblasts compared tocardiomyocytes (Figure 1E). Forced expression ofmiR-433 in cardiomyocytes did not lead to anelevation of markers for pathological hypertrophy(ANP, BNP, and Myh7) or extracellular matrixproteins (CTGF, TSP-1, Col1a1 and Col3a1) (Figure1F) supporting a more prominent role for miR-433 infibroblasts rather than cardiomyocytes.To evaluate the effect of miR-433 inhibition oncardiac fibrosis, we administrated miR-433 antagomirin mice via tail vein to downregulate miR-433 in vivo.First, the pharmacokinetic analysis for miR-433antagomir was performed by measuring miR-433expression level in both plasma and heart samples atdifferent time points after mice were administratedwith a single bolus of miR-433 antagomir at the doseof 7.5 mg/kg as previously reported [27]. Thepharmacokinetic analysis showed that miR-433 wassignificantly downregulated in plasma and heartsamples at 10 min post injection maintaining the lowexpression level thereafter (Supplemental Figure 1).Next, to explore whether antagonizing miR-433attenuates cardiac fibrosis and preserves ventricularfunction post-MI, we treated mice with miR-433antagomir or scrambled negative control via tail veininjection for 3 consecutive days and subjected them toMI or sham surgery. Then mice were sacrificed 3weeks after MI and the loss of miR-433 in the heartwas confirmed by qRT-PCRs (Figure 2A).Echocardiography showed that miR-433 antagomirpreserved cardiac function including FS and EF(Figure 2B), and also reversed MI-induced increase insystolic left ventricle internal diameter (LVID;s) anddiastolic left ventricle internal diameter (LVID;d) asshown in Supplemental Table 4. Importantly,inhibition of miR-433 also attenuated cardiac fibrosisas evidenced by reduced collagen deposition andcontent in MI heart tissues (Figure 2C-D). Inhttp://www.thno.org

Theranostics 2016, Vol. 6, Issue 12particular, we evaluated the effect of miR-433inhibition on cardiac infarction 3 days after MI; thepurpose was to determine whether miR-433 inhibitionpredominantly protects against cardiac fibrosis in theremodeling phase after MI or prevents cardiacinfarction in the acute phase after MI. Based on TTCstaining, there was no difference in the infarct sizebetween mice treated with miR-433 antagomir ornegative control, strongly suggesting that miR-433inhibition predominantly protects against cardiacfibrosis in the remodeling phase after MI (Figure 2E).To further confirm the effect of miR-433inhibition in preventing cardiac fibrosis, we used acardiotropic AAV9 delivery system to achieve cardiacinhibition of miR-433 in vivo. Mice received asingle-bolus tail vein injection of either miR-4332074sponge AAV9 or miR-scramble. After 1 week, micewere subjected to LAD ligation and sacrificed at 3weeks post-MI. Using qRT-PCR, we confirmed thatmiR-433 sponge AAV9 efficiently reduced miR-433expression level in heart tissues (Figure 3A).Furthermore, our data showed that AAV9-mediatedinhibition of miR-433 could significantly preserve leftventricular EF and FS (Figure 3B), and reduceincreased systolic LVID and diastolic LVID in mice 3weeks post-MI (Supplemental Table 5). Cardiacinhibition of miR-433 also reduced collagendeposition and collagen content in hearts post-MI(Figure 3C-D). These data provide strong evidencethat inhibition of miR-433 has cardioprotective effectagainst fibrosis.Figure 3: Cardiac inhibition of miR-433 via AAV9 attenuates cardiac fibrosis and preserves ventricular function post-myocardial infarction. A,decreased miR-433 in hearts from mice treated with miR-433 sponge AAV9 (n 6); B, preserved left ventricular fractional shortening (FS) and ejection fraction (EF);C, reduced cardiac fibrosis; D, decreased collagen content in myocardial infarction (MI) interfered with miR-433 sponge AAV9, as evidenced by echocardiography(n 6), Masson’s trichrome staining (n 4), and Sircol assay (n 4). Scale bar: 100 μm. *, P 0.05, **, P 0.01, ***, P 0.001 versus respective controls.http://www.thno.org

Theranostics 2016, Vol. 6, Issue 122075Figure 4: Antagonizing miR-433 attenuates cardiac fibroblasts proliferation and their differentiation into myofibroblasts in vivo. A-B, decreasedcardiac fibroblasts proliferation; C, reduced differentiation into myofibroblasts in myocardial infarction (MI) with miR-433 inhibition, as determined byimmunofluorescent staining for Vimentin and Ki-67 or pHH3 or α-SMA (n 4); D, decreased α-SMA, Col1a1, and Col3a1 in MI mice with miR-433 inhibition (n 4);E, Agilent gene arrays and KEGG pathway analysis identified extracellular matrix (ECM) receptor interaction as the most affected pathway in MI hearts with miR-433inhibition (n 4); F, decreased TGF-β, CTGF, Col1a1, Col3a1 and α-SMA and increased MMP2 and MMP9 after treatment with miR-433 antagomir in MI mice (n 4).Scale bar: 20 μm. *, P 0.05, **, P 0.01, ***, P 0.001 versus respective controls.Inhibition of miR-433 Attenuates CardiacFibroblast Proliferation and MyofibroblastDifferentiation In Vivo and In sts is a critical event in the genesis ofcardiac fibrosis [28, 29]. We determined the effects ofmiR-433 inhibition on cardiac fibroblasts proliferationand their differentiation into myofibroblasts in bothpost-MI mice and cultured cardiac fibroblasts. Basedon the heart samples from in vivo antagonizing miR-433 decreased cardiac imentinorphospho-HistoneH3(pHH3)/Vimentin double positive cells (Figure 4A-B).Furthermore, miR-433 inhibition also attenuated blasts as shown by decreased number ofcells double-positive for α-SMA and Vimentin (Figure4C). Consistent with this, the expression levels ofα-SMA, Col1a1, and Col3a1 in the ventricle followingMI were also attenuated by miR-433 inhibition (Figure4D). Agilent gene arrays were used to compare thedifference of gene expressions between ventriclesamples from miR-433 antagomir or scramblednegative control post-MI (Supplemental Tables 6-7).The KEGG pathway analysis based on dysregulatedgenes showed that extracellular matrix (ECM)receptor interaction was the most affected pathway(Figure 4E). Also, the protein levels of pro-fibroticgenes (TGF-β, α-SMA, CTGF, Col1a1, and Col3a1)were decreased, while genes responsible for collagendegradation (MMP2 and MMP9) were furtherincreased by miR-433 inhibition in post-MI hearts(Figure 4F). Similar results were obtained forfibrosis-associated genes in miR-433 spongeAAV9-treated MI mice (Supplemental Figure 2).To gain mechanistic insight into the role ofmiR-433 in regulating fibrosis, we investigated theeffect of miR-433 overexpression in cardiac fibroblastsin .org

Theranostics 2016, Vol. 6, Issue 12proliferation and differentiation of cardiac fibroblasts,as evidenced by an increase in EdU and α-SMAstaining and increased expression levels of α-SMA,Col1a1, Col3a1, CTGF, and TSP-1 (Figure 5).However, up-regulation of miR-433 failed to furtherenhance cardiac fibroblasts proliferation anddifferentiation in the presence of either TGF-β or AngII stimulation (Figure 5). Contrary to the effects ofmiR-433 overexpression, inhibition of miR-433decreased cardiac fibroblasts proliferation anddifferentiation (Figure 6). Collectively, these dataindicat

5’chol modified miR-433 inhibitor) or the scramble control (Ribobio, Guangzhou, China) for 3 consecutive days and subjected to LAD ligation. AAV represents an efficient and safe vector for in vivo gene transfer and serotype 9 is significantly cardiotropic [23-26]. Thus, besides miR-433 antagomir, the cardiotropic miR-433 sponge AAV9 was used to