Hepatitis B virus inhibits intrinsic RIG-I and RIG-G immune signaling via inducing miR146a

Abstract

Previous studies showed that hepatitis B virus (HBV), as a latency invader, attenuated host anti-viral immune responses. miRNAs were shown to be involved in HBV infection and HBV-related diseases, however, the precise role of miRNAs in HBV-mediated immunosuppression remains unclear. Here, we observed that down-regulated RIG-I like receptors might be one critical mechanism of HBV-induced suppression of type I IFN transcription in both HBV(+) hepatoma cell lines and liver cancer tissues. Then, miR146a was demonstrated to negatively regulate the expression of RIG-I-like receptors by directly targeting both RIG-I and RIG-G. Further investigation showed that antagonizing miR146a by anti-sense inhibitors or sponge approach accelerated HBV clearance and reduced HBV load both in vitro and in a HBV-carrying mouse model. Therefore, our findings indicated that HBV-induced miR146a attenuates cell-intrinsic anti-viral innate immunity through targeting RIG-I and RIG-G, and silencing miR146a might be an effective target to reverse HBV-induced immune suppression.

Figure 1. HBV infection inhibited expression of RIG-I like receptors.

(a) Western blot analysis of RIG-I, MDA-5, IFIT1 and RIG-G expression levels in HepG2 and HepG2.2.15 cells. (b) Western blot assay of RIG-I, MDA-5 and RIG-G protein levels in liver paracancerous tissues from 4 HBV⁺, 3 HBV⁻ HCC and 1 intrahepatic cholangiocarcinoma (ICC) patients. (c) HepG2 cells were co-transfected with 0.05 μg RIG-I CARD, IFN-β–luc and pRL-TK reporter vectors, as well as RIG-G constructs or 100 nM RIG-I/RIG-G siRNA. After 36 hours, luciferase activity was measured. (d) RIG-I CARD/RIG-G constructs (0.5 μg/ml) and pAAV/HBV1.2 plasmid were co-transfected into HepG2 cells, together with the IFN-β–luc and pRL-TK reporter vectors, and luciferase activity was measured 36 hours later. (e) RIG-I CARD/RIG-G constructs (0.5 μg/ml) and pAAV/HBV1.2 plasmid (0.5 μg/ml), as well as 100 nM RIG-I siRNA were co-transfected into HepG2 cells, together with the IFN-β–luc and pRL-TK reporter vectors, and ELISA method was used to measure IFN-β production 36 hours later. Data are expressed as the mean ± SD from at least 3 independent experiments. *p < 0.05: versus the control vector–transfected group.

Figure 2. miR146a level correlated with HBV infection both in vitro and in vivo.

(a) The primary transcript (left) and precursor (right) of miR146a in HepG2 and HepG2.2.15 cells were assessed by qRT-PCR. (b) HBV genome-transfected HepG2 cells were established by transfecting HepG2 cells with the LMP-HBV1.2 plasmid, followed by puromycin selection. RNA from these transfected and control cells was isolated, and miR146a expression levels were evaluated by qRT-PCR. miR146a levels in HBV⁺ cells were expressed as the fold of the level in control cells. GAPDH was used as the internal control. (c) HBV-carrying BALB/c mice were established by hydrodynamic injection of pAAV/HBV1.2 at a dose of 6 μg per mouse. After 2 weeks, serum samples were harvested, and HBsAg levels were measured by ELISA. Simultaneously, miR146a levels in primary hepatocytes isolated by collagenase perfusion were measured as above. Data are expressed as the mean ± SD from at least 3 independent experiments. *p < 0.05: versus HepG2-vector cells.

Figure 3. miR146a suppressed RIG-I like receptor- mediated innate immune response.

(a) RIG-I, MDA-5, IFIT1 and RIG-G protein levels in HepG2 cells treated with miR146a mimics (left), and HepG2.2.15 cells treated with miR146a inhibitors (right) for 48 hours were analyzed by Western blot. One representative of at least 3 independent experiments. (b) HepG2 cells were transfected with 0.5 μg/ml RIG-I CARD construct and 100 nM miR146a mimics or inhibitors, together with the IFN-β–luc and pRL-TK reporter vector, and then luciferase activity was measured 36 hours later. (c) HepG2.2.15 cells were co-transfected with 0.5 μg/ml of the RIG-I CARD constructs, IFN-β–luc and pRL-TK reporter vectors, as well as RIG-G constructs or 100 nM of miR146a inhibitors, and then luciferase activity was measured 36 hours later. (d) HepG2.2.15 cells were transfected with pSIREN-PGK-miR146a-sponge plasmid (left) or pIRESpuro3-RIG-G plasmid (right) at a final dose of 1.5 μg/mL. After 24 hours, these cells were transfected with 3p-RNA at a final concentration of 1 μg/mL. After another 6 hours, the mRNA levels of IFN-α, IFN-β and TNF-α were measured by qRT-PCR. Data are expressed as the mean ± SD from at least 3 independent experiments. Empty, control vector pIRESpuro3; “-” represents miR146a mimic or inhibitor negative control. *p < 0.05: versus negative control RNA or control-vector–transfected group.

Figure 4. miR146a might directly target RIG-I and RIG-G.

(a) Sequence alignment between miR146a and the 3′-UTRs of RIG-I. The 3′-UTR of human RIG-I contained 2 potential miR146a binding sites. (b) Sequence alignment between miR146a and the 3′-UTRs of RIG-G and ORF region of RIG-G. (c) HEK293 cells were transfected with pmiR-reporter-RIG-I 3′-UTR or mutation constructs and miR146a mimics, (m1, contains a mutation in potential binding site 1; m2, contains a mutation in potential binding site 2; m1+m2, contains mutations in both potential binding sites 1 and 2). (d) HEK293 cells were co-transfected with pmiR-reporter-RIG-G 3′-UTR or mutation constructs and miR146a mimics or negative control (mNC). Luciferase activity was measured after 36 hours. Data are representative of 3 independent experiments and are expressed as the mean ± SD. *p < 0.05, **p < 0.01.

Figure 5. Silencing miR146a reversed HBV-induced immune suppression in vivo.

HBV-carrying BALB/c mice were injected with pSIREN-PGK-miR146a-sponge plasmid or control vector by hydrodynamic tail-vein injection. Four weeks later, HBV clearance was valuated. (a) Serum HBV DNA levels were measured by qRT-PCR. (b) Total HBV DNA and RNA levels in mouse liver tissues were analyzed by Southern blotting (upper) and Northern blotting (lower), respectively, with the same HBV probes. Mouse GAPDH was used as the loading control for Northern blotting. (c) Serum HBsAg (left) and HBeAg levels (right) were measured by ELISA. (d) HBsAg and HBcAg expression in liver tissue were measured by immunohistochemical staining. (e) mRNA levels of RIG-I and RIG-G in primary hepatocytes were evaluated by qRT-PCR (left). The concentrations of IFN-α and IFN-β in liver homogenates were evaluated by ELISA (right). C, control vector; S, pSIREN-PGK-miR146a-sponge plasmid. Data represent 2 independent experiments with 6 mice per group. *p < 0.05: versus control-vector–injected group.

Figure 6. Working model.

(1) HBV infection promoted endogenous miR146a transcription in hepatocytes. (2) miR146a directly targeted RIG-I and RIG-G mRNA, and then impaired RIG-I pathway axis-induced type I IFN production. (3) miR146a also inhibited STAT1 in a sequence-dependent manner, attenuates IFN-induced anti-viral genes expression and ultimately weaken anti-HBV response.