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Combined adenovirus-mediated artificial microRNAs targeting mfgl2, mFas, and mTNFR1 protect against fulminant hepatic failure in mice

PLoS ONE (2013) 8(11)
DOI: 10.1371/journal.pone.0082330
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Abstract

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 in vitro. 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 tailvein 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. © 2013 Xi et al.

Figures

  • Figure 1. The constructed miRNA expression plasmids significantly inhibited target gene expression in CHO cells. (a-c) qRT-PCR showed that the miRNA eukaryotic expression plasmids targeting mfgl2, mFas, and mTNFR1, pcDNA6.2-mfgl2-miRNA (a), both pcDNA6.2-mFas-miRNA and pcDNA6.2-mFas-mTNFR1-miRNA (b), and both pcDNA6.2-mTNFR1-miRNA and pcDNA6.2mFas-mTNFR1-miRNA (c), respectively, inhibited mfgl2, mFas, and mTNFR1 mRNA expression. Negative miRNA control: irrelevant miRNA plasmid, Blank control: CHO cells not treated. *P <0.05, #P <0.01 compared with the negative miRNA control group. (d-f) Western blot analysis showing that pcDNA6.2-mfgl2-miRNA (d), both pcDNA6.2-mFas-miRNA and pcDNA6.2-mFasmTNFR1-miRNA (e), and both pcDNA6.2-mTNFR1-miRNA and pcDNA6.2-mFas-mTNFR1-miRNA (f) inhibited mfgl2, mFas, and mTNFR1 protein expression, respectively. The average protein expression from Negative miRNA control group was designated as 1. *P <0.05.
  • Figure 2. Combined interference increased the survival rate and improved liver function and histopathology in mice. (A): 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) and histopathology (C) (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) 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. *P <0.05, #P <0.01 compared with the negative miRNA control adenovirus group. (C) 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 (g, h, and i), Ad-mFas-mTNFR1-miRNA– treated (j, k, and l), combined interference-treated (m, n, and o), irrelevant miRNA adenovirus-treated (d, e, and f), or PBS-treated (a, b, and c) 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.
  • Figure 3. Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA inhibited target genes at both mRNA and protein levels in vivo. The treatment process was the same as that described in Figure 2, and livers were collected from treated BALB/cJ mice 0, 24, 48, and 72 h after MHV-3 infection. (a) qRT-PCR showed both Ad-mfgl2-miRNA and combined interference with Ad-mfgl2miRNA and Ad-mFas-mTNFR1-miRNA inhibited mfgl2 mRNA expression 48 h and 72 h after MHV-3 infection. Both Ad-mFasmTNFR1-miRNA and combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mFas (b) and mTNFR1 (c) mRNA expression 48 h and 72 h after MHV-3 infection also. Values represent means and SE of three separate experiments done in triplicate.*P <0.05 compared with Negative miRNA control adenovirus group. (d-f) Western blot analysis showed combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mfgl2 (d), mFas (e), and mTNFR1 (f) protein expression at 72 h after MHV-3 infection. The average protein expression from Negative miRNA control group was designated as 1. *P <0.05.
  • Figure 4. Combined interference inhibited target gene expression and decreased fibrin deposition and intrahepatic apoptosis in mice. Livers from BALB/cJ mice treated with either Ad-mfgl2-miRNA or Ad-mFas-mTNFR1-miRNA, from combination interference mice treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA, and from control-treated mice were collected 72 h after MHV-3 infection. mfgl2 expression (A–D), fibrin deposition (E–H), mFas expression (I–L), mTNFR1 expression (M–P) were determined by immunohistochemistry analysis, and hepatocyte apoptosis (O–T) was determined by TUNEL. Arrows represent positive staining as shown in brown. Original magnification, ×400.
  • Figure 5. The constructed miR adenovirus did not affect the expression of certain apoptosis-related proteins, but significantly decreased cleavage of caspase-3. (A) There was significantly decreased cleavage of caspase-3 in Ad-mFasmTNFR1-miRNA treated mice and combined interference group at 72 h after MHV-3 infection. (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. *P <0.05.

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Xi, D., Wang, M., Ye, H., Luo, X., & Ning, Q. (2013). Combined adenovirus-mediated artificial microRNAs targeting mfgl2, mFas, and mTNFR1 protect against fulminant hepatic failure in mice. PLoS ONE, 8(11). https://doi.org/10.1371/journal.pone.0082330

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