Inhibiting MiR-21Expression Attenuates Experimental Hepatic Fibrosis

【Background and objective】Hepatic fibrosis characterized by excessive accumulation of extacellular matrix (ECM)is a common proess of chronic hepatic injury. It has been well established that activationand proliferation of resident hepatic stellate cell (HSC) was the central event in thedevelopment of hepatic fibrosis. Moreover, recent evidences suggest that, hepatocytes andbiliary epithelial cells could transform to the activated fibroblast cells throughepithelial-mesenchymal transition (EMT), which was considered as the importantcontributor to promoting liver fibrogenesis.It has been proved that, multiple signal pathways are concerned in the activation of HSC,including extracellular signal-regulated kinase1(ERK1) signal pathway, which is one ofthe major mitogen-activated protein kinase (MAPK) pathways to promote HSC activation.We have previously revealed that suppressing the expression of ERK1, one of the centralcomponents of the pathway, could significantly inhibit HSC activation and even blockEMT process of HSC, hepatocyte and biliary epithelial cell. In addition, our previous studyhas also substantially demonstrated that, as a pathogenetic principle involved inexacerbating liver fibrosis, EMT of hepatocyte could be inhibited significantly by forcedexpression of hepatocyte nuclear factor4α (HNF4α), which resulted in remarkablealleviation of experimental hepatic fibrosis and improvement of liver functions.microRNAs (miRNAs) are small non-protein-coding RNA about22nucleotide (nt)which regulate gene expression at the posttranscriptional level through incomplete orcomplete complementary to target the3′-untranslated region (3′-UTR) of target genemessenger RNA (mRNA), effecting mRNA stability and translation. Recently,dysregulation of miRNAs has been considered as the important contributor to thedevelopment of a variety of diseases, including hepatic fibrosis. In our previous study, werevealed that miR-21level enhanced in activated HSC and fibrotic liver tissues of rats. Weindicated that miR-21could directly interact with the3′-UTR of mRNAs of sprouty2 (SPRY2) and HNF4α, the latter have been demonstrated to inhibit ERK1pathway andblock EMT process in HSC and hepatocyte in vitro, respectively.TuD RNA (ToughDecoy RNA) is a more effective microRNA inhibitor, with doublechain structure to binding double molecule miRNA and the stem loop structure to resistdegradation by intracellular nuclease. TuD-RNA against miR-21(TuD-21) has miR-21binding sequence (MBS) in each chain structure, which could efficiently inhibite miR-21expression by tightly binding to miR-21.In the present study, we used adenoviral TuD-21expression vectors (Ad-TuD-21) toinhibit the miR-21expression in vivo, further explored the regulatory effect of miR-21onthe activation of ERK1pathway in HSC and EMT process in hepatocyte during hepaticfibrogenesis, which might confer synergistically therapeutic strategy for hepatic fibrosis.【Methods】1. Construction of recombinant replication-deficient adenovirus Ad-TuD-21TuD-21cDNA fragment containing two miR-21binding sequence was synthesised bychemical method. TuD-21cDNA was cloned into the shuttle plasmid pAVsiRNA1.1digested with Age I and EcoR I to generate expression plasmid pAVsiRNA1.1-TuD-21.The pAVsiRNA1.1-TuD-21was transformed into HEK293cells with backbone vectorpBHG lox ΔE1,3Cre to establish the recombinant adenoviral Ad-TuD-21by AdMaxTMadenoviral recombinant system. Using the same method, the control adenovirusAd-TuD-NC was obtained, which only expresed green fluorescent protein (GFP). Virustiter was detected as plaque forming units (pfu). The expression of miR-21in HSC-T6celllines was detected by real time RT-PCR after Ad-TuD-21infection for48h.2. Inhibiting miR-21expression attenuated experimental hepatic fibrosisThe model of experimental hepatic fibrosis was induced with40%carbontetrachloride(CCl4)/Olive Oil by subcutaneous injection into male Sprague–Dawley (SD) rats(Shanghai Laboratory Animal Center of Chinese Academy of Sciences, weighing180–200g). Rats in normal control group (4rats) served as that with saline subcutaneous injection (2ml/kg weight) for2times per week up to12weeks. Rats in CCl4-induced hepaticfibrosis group (24rats) were subcutaneous injected with40%CCl4/Olive Oil (2ml/kgweight) for2times per week up to12weeks. After CCl4was given for8weeks, rats inCCl4-induced hepatic fibrosis group were randomly divided into three groups andmaintained as follows: Model group (8rats) injected with1ml normal saline via the tailvein for2times per week up to4weeks; Ad-TuD-NC group (8rats) and Ad-TuD-21group(8rats) injected via the tail vein with5×109pfu Ad-TuD-NC or Ad-TuD-21dissolved in1ml normal saline for2times per week up to4weeks, respectively. At the end of12weeks,all of the animals were sacrificed.The expression of miR-21, fibrosis related genes α-smooth muscle actin (α-SMA),collagen typeⅠ(COLⅠ), matrix metalloproteinase2(MMP2), MMP9and tissue inhibitorof metalloproteinase-1(TIMP1) was detected by real time RT-PCR. Protein levelexpression of α-SMA, COLⅠand MMP2was evaluated by western blot analysis.All liver tissues were paraffin-embedded, cutted with4μm, stained withhematoxylin-eosin (H&E), Masson’s trichrome, Van Gieson (VG) and Sirius red stainingfor the evaluation of the degree of hepatic fibrosis. Semiquantitative analysis of the fibrosisarea was detected with Image-Pro plus (IPP)6.0software.3. Exploring the mechanism of downregulating miR-21expression on hepaticfibrosisThe levels of HSC ERK1pathway related genes SPRY2, ERK1, RSK2, α-SMA andhepatocyte EMT progress related genes HNF4α, albumin, E-cadherin, vimentin weredetected by real time RT-PCR in liver tissues from each group.All liver tissues were paraffin-embedded, cutted with4μm and blocked with5%horseserum. The actived HSC marked by α-SMA was co-localizated with ERK1pathway relatedgenes (SPRY2, ERK1and RSK2), and hepatocyte marked by albumin was co-localizatedwith EMT progress related genes (HNF4α, E-cadherin and vimentin) using doubleimmunofluorescence histochemistry method. Sections were co-incubated overnight withdouble first antibodies (α-SMA co-incubated with SPRY2, ERK1or RSK2, albumin co-incubated with HNF4α, E-cadherin or vimentin). In the next day, the sections wereincubated with the fluorescence-conjugated secondary antibodies together with DAPI fornuclear staining. Luorescence pictures were viewed by fluorescence microscopy.4. Statistical analysisStatistical analyses were performed with SPSS software (18.0version). The analysis ofvariance (ANOVA) and Student’s t test were used for comparison among the groups andbetween paired data, respectively. Data not normally distributed were compared using theMann-Whitney tests. A P value <0.05considered significant.【Results】1. Successful construction of recombinant replication-deficient adenovirusAd-TuD-21to inhibit miR-21expressionThe sequence of chemical synthesis TuD-21cDNA was tested, and GFP expressioncould be observed at the second day after plasmids pAVsiRNA1.1-TuD-21orpAVsiRNA1.1-TuD-NC was co-transformed into HEK293cells with backbone vectorpBHG lox ΔE1,3Cre. At last, about2×1010pfu/ml of Ad-TuD-21or Ad-TuD-NC wascollected after amplification and purifying.Real time RT-PCR analysis showed that miR-21expression level in HSC-T6infected byAd-TuD-21decreased more than90%compared with that in the Ad-TuD-NC group(P<0.01), which means successful construction of recombinant replication-deficientadenovirus Ad-TuD-21.2. Inhibiting miR-21expression attenuates experimental hepatic fibrosisAs expected, H&E staining showed hepatocyte necrosis, fibrosis area and the hepaticparenchyma with loss of the lobular architecture in liver tissues in the CCl4induced hepaticfibrosis. VG, Masson’s trichrome and Sirius red staining also showed a lot of fibrosis scar.It suggested that the experimental hepatic fibrosis was successfully established.The fibrosis area of the Ad-TuD-NC group is3.0times than that in the Ad-TuD-21group by VG staining (p<0.01). Fibrosis area in the Ad-TuD-21group reduced by57%and 63%(p<0.01) compared with that in the Ad-TuD-NC group by Masson or Sirius redstaining, respectively.Real time RT-PCR results revealed that the expression of miR-21decreased significantlyin the Ad-TuD-21group compared with that in the Ad-TuD-NC group (P<0.05). Theexpression of α-SMA, COLⅠ and TIMP1in the Ad-TuD-21group reduced by73%,53%and52%respectively compared with that in the Ad-TuD-NC group (P<0.01). Theexpression of MMP2and MMP9in the Ad-TuD-21group increased by30%and42%compared with that in the Ad-TuD-NC group (P<0.05).According to the western blot analysis，the expression of COLⅠand TIMP1decreasedsignificantly in the Ad-TuD-21group, along with the enhancement of MMP2expression.3. Inhibiting miR-21expression repressed ERK1pathway activity by targetingSPRY2in HSC, and blocked hepatocyte EMT by promoting HNF4α expression invivoReal time RT-PCR results revealed that the expression of miR-21decreased significantlyin the Ad-TuD-21group compared with that in the Ad-TuD-NC group (P<0.05). Theexpression of α-SMA in the Ad-TuD-21group decreased by73%(P<0.01) compared withthat in Ad-TuD-NC group. The expression of ERK1and RSK2in the Ad-TuD-21groupdecreased by73%(P<0.01) and47%(P<0.05) compared with that in Ad-TuD-NC group.The expression of HNF4α, albumin or E-cadherin in the Ad-TuD-21group increased by1.73fold (P<0.05),1.88fold (P<0.01) or1.56fold (P<0.01) compared with that in theAd-TuD-NC group. The expression of vimentin decreased by49%(P<0.01) in theAd-TuD-21group.α-SMA was co-localizated with SPRY2and ERK1pathway related genes (ERK1andRSK2) by double immunofluorescence histochemistry, and the results showed theincreased expression of SPRY2, accompanied with lower levels of ERK1, RSK2andα-SMA in the Ad-TuD-21group. In addition, albumin was co-localizated with HNF4α andEMT related genes (E-cadherin and vimentin) by double immunofluorescencehistochemistry, and the results revealed the expression of HNF4α and E-cadherin increased accompanied with upregulation of albumin in the Ad-TuD-21group, while vimentinexpression decreased significantly.【Conclusions】1. Recombinant replication-deficient adenovirus Ad-TuD-21, which couldeffectively inhibit miR-21expression, was successfully constructed by AdMaxTMsystem.2. Inhibiting miR-21expression in vivo could ameliorate hepatic fibrosis and mightbe an effective strategy for treatment of hepatic fibrosis.3. Down-regulating miR-21could repress the activity of ERK1pathway by targetingSPRY2in HSC, and block hepatocyte EMT process by promoting HNF4αexpression in vivo.