Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas, and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

<div><p>Hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) has a poor prognosis with high in-hospital mortality. Hepatic and circulating inflammatory cytokines, such as fibrinogen like protein 2 (fgl2), FasL/Fas, and TNFα/TNFR1, play a significant role in the pathophysiology of ACLF. This study aimed to investigate the therapeutic effect of recombinant adenoviral vectors carrying constructed DNA code for non-native microRNA (miRNA) targeting mouse fgl2 (mfgl2) or both mFas and mTNFR1 on murine hepatitis virus (MHV)-3-induced fulminant hepatitis in BALB/cJ mice. Artificial miRNA eukaryotic expression plasmids against mfgl2, mFas, and mTNFR1 were constructed, and their inhibitory effects on the target genes were confirmed <i>in vitro</i>. pcDNA6.2-mFas-mTNFR1- miRNA，which expresses miRNA against both mFas and mTNFR1 simultaneously，was constructed. To construct a miRNA adenovirus expression vector against mfgl2, pcDNA6.2-mfgl2-miRNA was cloned using Gateway technology. Ad-mFas-mTNFR1- miRNA was also constructed by the same procedure. Adenovirus vectors were delivered by tail-vein injection into MHV-3-infected BALB/cJ mice to evaluate the therapeutic effect. 8 of 18 (44.4%) mice recovered from fulminant viral hepatitis in the combined interference group treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA. But only 4 of 18 (22.2%) mice receiving Ad-mfgl2-miRNA and 3 of 18 (16.7%) mice receiving Ad-mFas-mTNFR1- miRNA survived. These adenovirus vectors significantly ameliorated inflammatory infiltration, fibrin deposition, hepatocyte necrosis and apoptosis, and prolonged survival time. Our data illustrated that combined interference using adenovirus-mediated artificial miRNAs targeting mfgl2, mFas, and mTNFR1 might have significant therapeutic potential for the treatment of fulminant hepatitis.</p> </div

Combined interference increased the survival rate and improved liver function and histopathology in mice.

<p>(<b>A</b>): Combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA had a higher survival rate than that of interference with either construct alone in MHV-3–infected BALB/cJ mice. Ad-mfgl2-miRNA, Ad-mFas-mTNFR1-miRNA, or irrelevant miRNA control adenovirus were introduced into BALB/cJ mice by tail vein injection. The mice then received 20 PFU of MHV-3 intraperitoneally 24 hours later to promote the development of fulminant viral hepatitis. Survival data are presented. Serial serum ALT levels (<b>B</b>) and histopathology (<b>C</b>) (H&E staining; original magnification, ×400) at 24, 48, and 72 h post MHV-3 infection were evaluated in the five groups of mice. (<b>B</b>) Effect of Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA on serum ALT levels. Values represent means and standard error of three independent experiments done in triplicate. *<i>P</i> <0.05, #P <0.01 compared with the negative miRNA control adenovirus group. (<b>C</b>) Effect of Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA on liver histopathology in MHV-3–infected BALB/cJ mice. Livers were collected from Ad-mfgl2-miRNA–treated (<b>g</b>, <b>h</b>, and <b>i</b>), Ad-mFas-mTNFR1-miRNA–treated (<b>j</b>, <b>k</b>, and <b>l</b>), combined interference-treated (<b>m</b>, <b>n</b>, and <b>o</b>), irrelevant miRNA adenovirus-treated (<b>d</b>, <b>e</b>, and <b>f</b>), or PBS-treated (<b>a</b>, <b>b</b>, and <b>c</b>) BALB/cJ mice at 24 h (a, d, g, j, and m), 48 h (b, e, h, k, and n), and 72 h (c, f, i, l, and o) after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic areas with the inflammation. </p

The constructed miR adenovirus did not affect the expression of certain apoptosis-related proteins, but significantly decreased cleavage of caspase-3.

<p>(<b>A</b>) There was significantly decreased cleavage of caspase-3 in Ad-mFas-mTNFR1-miRNA treated mice and combined interference group at 72 h after MHV-3 infection. (<b>B</b>) Both Ad-mFas-mTNFR1-miR and combined interference with the two adenoviral miRNAs did not affect the expression of pro-apoptotic proteins, including Bax and Bad, and anti-apoptotic proteins, including Bcl-2 and c-IAP2 at 72 h after MHV-3 infection. The average protein expression from Negative miRNA control group was designated as 1. *<i>P</i> <0.05.</p

Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA inhibited target genes at both mRNA and protein levels <i>in</i><i>vivo</i>.

<p>The treatment process was the same as that described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082330#pone-0082330-g002" target="_blank">Figure 2</a>, and livers were collected from treated BALB/cJ mice 0, 24, 48, and 72 h after MHV-3 infection. (<b>a</b>) qRT-PCR showed both Ad-mfgl2-miRNA and combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mfgl2 mRNA expression 48 h and 72 h after MHV-3 infection. Both Ad-mFas-mTNFR1-miRNA and combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mFas (<b>b</b>) and mTNFR1 (<b>c</b>) mRNA expression 48 h and 72 h after MHV-3 infection also. Values represent means and SE of three separate experiments done in triplicate.*<i>P</i> <0.05 compared with Negative miRNA control adenovirus group. (<b>d</b>-<b>f</b>) Western blot analysis showed combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mfgl2 (<b>d</b>), mFas (<b>e</b>), and mTNFR1 (<b>f</b>) protein expression at 72 h after MHV-3 infection. The average protein expression from Negative miRNA control group was designated as 1. *<i>P</i> <0.05.</p