Interferon downregulation of miR-1225-3p as an antiviral mechanism through modulating Grb2

27 Pages·2017·2.11 MB·English

JBC Papers in Press. Published on March 1, 2018 as Manuscript RA117.000738
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA117.000738
Interferon downregulation of miR-1225-3p as an antiviral mechanism through modulating
Grb2-associated binding protein 3 expression

Min Cheng§, Yuqiang Niu§, Jingjing Fan§, Xiaojing Chi, Xiuying Liu, Wei Yang*

From the MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese
Academy of Medical Sciences & Peking Union Medical College, Beijing, China

Running title: miR-1225-3p inhibits viral infection through GAB3

§these authors contributed equally to this work

*To whom correspondence should be addressed: Wei Yang: Institute of Pathogen Biology, Chinese
Academy of Medical Sciences & Peking Union Medical College, Beijing, China 100176;
[email protected]; Telephone and fax: [phone]. D
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Keywords: interferon, microRNA (miRNA), virus, infection, host-pathogen interaction, miR-1225-3p, fro
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Growth factor receptor-bound protein 2-associated binding protein 3, GAB3 http
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ABSTRACT growth factor receptor-bound protein 2-associated .o
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Induction of interferons (IFNs) is a central binding protein 3 (GAB3) as a direct target of b/
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event of antiviral innate immunity. As crucial miR-1225-3p. GAB3 expression was upregulated ue
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post-transcriptional regulators, microRNAs by IFN, and overexpression of GAB3 t on
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(miRNAs) are important for IFN–mediated host demonstrated potent antiviral effects through pril 7
defense. Although screening has indicated a enhancing IFN response and virus–triggered , 2
0
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substantial number of miRNAs to be differentially innate immune activation. Taken together, our 9
expressed after IFN stimulation, the detailed findings reveal the biological function of
mechanisms of these miRNAs in the antiviral miR-1225-3p for the first time and propose a novel
response is underexplored and of great antiviral regulation pathway in which miRNA and
significance. Here, we show that hsa-miR-1225-3p GAB3 participate. This study contributes to the
is specifically downregulated by type I IFN understanding of host miRNA participation in
through IFN/JAK/STAT signaling pathway. antiviral processes of IFN.
Silencing endogenous miR-1225-3p inhibited
infection by multiple IFN-susceptive viruses,
including hepatitis C virus, Sendai virus and Chronic and emerging viral infection is a
Newcastle disease virus. In contrast, serious threat to public health. The host innate
overexpression of miR-1225-3p impaired the immune system is the first line of defense against
antiviral effect of IFNs and facilitated viral viral invasion or replication before the more
infection. Regarding the mechanism, we identified specific adaptive immune system is established.
1
miR-1225-3p inhibits viral infection through GAB3
Host pattern recognition receptors (PRRs), antiviral state (7, 10).
including Toll-like receptors (TLRs), retinoic MiRNAs that regulate host antiviral
acid-inducible gene I (RIG-I)-like receptors mechanisms are significant and promising
(RLRs), and nucleotide-binding oligomerization candidates for a broad-spectrum antiviral strategy.
domain (NOD)-like receptors (NLRs), recognize For example, IFN-β–induced miR-155 targeted
pathogenic components and activate intracellular suppressor of cytokine signaling 1, which is a
signaling. Signaling through members of the TLRs negative regulator of JAK/STAT signaling, and
and RLRs family activates immune response via enhanced the innate immune response (11, 12).
the cascades that involves NF-κB, IFN regulatory Moreover, IFN–regulated miRNAs can act as
factor 3 (IRF3) and IRF7 to induce repressors of antiviral pathways, which introduces
proinflammatory cytokines and type I IFNs an additional layer of self-regulation for IFN
production (1, 2). IFNs play a key role in the signaling. IFN-γ–induced miR-29a inhibited the
control of initial virus spread and have been antiviral response by targeting the IFN alpha and
approved for treating a variety of diseases, such as beta receptor subunit 1 (IFNAR1) (13, 14).
hepatitis C and B virus infection (3, 4). IFNs IFN-downregulated miR-27a inhibited the D
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inhibit viral propagation through distinct antiviral innate response by upregulating two nlo
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mechanisms, though the most widely accepted negative regulators of IFN production, namely, de
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mechanism is the transcriptional regulation of sialic acid-binding Ig-like lectin 1 (Siglec1) and fro
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hundreds of IFN–stimulated genes (ISGs). These E3 ubiquitin ligase tripartite motif-containing http
ISGs control viral infection by either directly protein 27 (TRIM27) (15). Significantly, ://w
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targeting pathways required for viral cell entry, increasing evidence suggests that the important .jb
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replication, translation and egress or leading to cell impact of miRNA expression may occur through .o
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death under serious infection conditions. mutual regulation between the innate and adaptive b/
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Beyond conventional protein-coding ISGs, responses (16). Given the importance of host ue
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contemporary genome-wide and high-throughput non-coding RNAs in the antiviral response, we t on
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approaches have identified a large number of and other researchers profiled host miRNA pril 7
IFN–regulated non-coding RNAs that participate expression patterns in response to multiple types , 2
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in antiviral innate immunity through positive and of IFN stimuli through high-throughput screening 9
negative mechanisms (5–8). MicroRNAs (17, 18). Overlapping and distinct data were
(miRNAs) are an abundant class of short (18–25 obtained from different labs, but further functional
nt), highly conserved non-coding RNAs that investigations of these identified IFN–regulated
function as posttranscriptional regulators to miRNAs are obviously urgent and of great
suppress gene expression by binding to their target significance. Our previous screening showed that
mRNAs (9). By modifying miRNA transcription hsa-miR-1225-3p might be a novel IFN–regulated
or activity, IFNs may regulate host epigenetic miRNA (18). Hsa-mir-1225 was reported as a
mechanisms in the regulation of viral infection "mirtron", which is formed by a short intron of
through either the direct recognition of viral PDK1 with hairpin potential (19). However,
sequences, which affects only the infection process mir-1225 is processed through non-canonical
of the targeted virus, or the mechanism of mirtron biogenesis (20). MiR-1225-3p may be
targeting components at many steps of the antiviral related to microphthalmia-associated transcription
pathway, which induces a non-virus-specific factor dysfunction in melanocytes (21).
2
miR-1225-3p inhibits viral infection through GAB3
Growth factor receptor-bound protein 2 downregulated after viral infection. We found that
(Grb2)-associated binding (GAB) infection with three RNA viruses, hepatitis C virus
scaffolding/adapter proteins are a family of three (HCV), Newcastle disease virus (NDV) and
members including mammalian GAB1, GAB2, Sendai virus (SeV), also induced miR-1225-3p
and GAB3 (22). GABs participate in multiple downregulation in Huh7 or 293T cells (Fig.
signaling pathways by recruiting enzymes into the 1C–1G). To evaluate the abundance of endogenous
signaling network or placing the enzymes close to miR-1225-3p, we used absolute quantification
substrates (22). In particular, GABs have been RT-PCR to amplify miR-1225-3p in four
found to play important roles in immune signaling. hepatoma cell lines, primary fetal liver cells
GAB1 is needed for T/B cell receptor signal (PFLC) and 293T cells, and found that
transduction and TLR- and RIG-I-triggered innate miR-1225-3p could be readily detected (Fig. 1H).
immune responses (22, 23). A study showed that These results verified the inhibition of
GAB3 had high sequence similarity to GAB1 and miR-1225-3p by IFN stimulation and virus
facilitated macrophage differentiation upon infection.
stimulation with macrophage colony-stimulating D
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factor (24). Up to now, the functions of GAB3 in IFN/JAK/STAT pathway is required for nlo
antiviral immunity signaling have not been miR-1225-3p downregulation ade
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reported. Binding to membrane receptors (IFNAR1/2) fro
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In this study, we confirmed the modulation of and activating the JAK/STAT pathway are required http
miR-1225-3p expression by IFNs and further for IFN-α2b to trigger downstream signaling and ://w
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antiviral effects. To investigate cell membrane w
elucidated the roles and mechanisms of .jb
miR-1225-3p in host antiviral regulation. Our receptor usage specificity by IFN-α2b to suppress c.o
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findings highlight the role of an miR-1225-3p expression, we performed a receptor b/
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IFN–downregulated miRNA in the positive knockdown assay. We transfected Huh7 cells with gue
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regulatory mechanism for innate antiviral siRNAs targeting IFNAR1 or IFNAR2 and then t on
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response. ternedaotegde nothues mceilRls- 12w2i5th-3 pI FlNev-αel2sb. Wbeef ofroeu ntde sttihnagt pril 7, 2
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Results IFN-α2b treatments of mock- or negative control 19
IFN downregulates miR-1225-3p expression (NC)-siRNA-transfected cells led to a significant
In our previous attempt to identify reduction in miR-1225-3p expression. However,
IFN–regulated non-coding RNAs in Huh7 transfection with siRNAs targeting IFNAR1 or
hepatoma cells using microarrays, miR-1225-3p IFNAR2 could rescue the miR-1225-3p expression
was one of the top-ranked hits among the from 40% inhibition to 8% inhibition in the
differentially expressed miRNAs (18). In this presence of IFN-α2b (Fig. 2A). The knockdown of
study, to further investigate the biological IFNAR1 or IFNAR2 by specific siRNAs was
significance of miR-1225-3p during viral infection, confirmed (Fig. 2B). The stimulation of OAS1 and
we first validated the downregulation of IFIT1 by IFN-α2b was partially abolished in the
miR-1225-3p by type I IFN (IFN-α2b) in Huh7 absence of IFNAR1 and IFNAR2 (Fig. 2C). Using
cells using qRT-PCR (Fig. 1A and 1B). Because small molecule inhibitor fludarabine, we found
virus infection induces IFN production, we that inhibition of STAT1 activation efficiently
determined whether miR-1225-3p was blocked IFN-α2b–induced miR-1225-3p
3
miR-1225-3p inhibits viral infection through GAB3
downregulation (Fig. 2D). Consistently, miR-1225-3p by IFN participates in the
phosphorylation of STAT1 and expressions of IFN-mediated inhibition of viral infection, we
OAS1 and IFIT1 were also suppressed by counteracted the IFN-elicited decrease in
fludarabine (Fig. 2E and 2F). Therefore, these miR-1225-3p expression by transfecting
results demonstrate that IFN downregulates miR-1225-3p mimic into cells and then analyzed
miR-1225-3p expression mainly through the antiviral effect of IFN. As shown in Fig. 4A
IFN/JAK/STAT pathway. and 4B, IFN-α2b treatment markedly suppressed
viral replication, but virus suppression was
Silencing miR-1225-3p inhibits viral infection
rescued by the overexpression of miR-1225-3p.
To determine whether the downregulation of
Furthermore, overexpression of miR-1225-3p
miR-1225-3p by IFN affects viral infection, we
attenuated the induction and phosphorylation of
transfected miR-1225-3p inhibitors and analyzed
STAT1, the activation of interferon-stimulated
the antiviral effects of silencing miR-1225-3p
response element (ISRE) and the stimulations of
using HCV, a positive single strand RNA (ssRNA)
IFIT1 and OAS1 by IFN (Fig. 4C–4E). These
virus susceptible to IFN treatment, as the indicator findings suggest that the downregulation of D
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virus. We observed that miR-1225-3p miR-1225-3p might contribute to the antiviral nlo
downregulation significantly inhibited HCV effect and JAK/STAT signaling mediated by ade
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infection (Fig. 3A and 3B), though the IFN-α2b. fro
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overexpression of miR-1225-3p per se had no h
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effect on HCV (data not shown). Significant GAB3 is one of the targets of miR-1225-3p ://w
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decreases in infection by NDV at both 18 hours An online search of the miRWalk 2.0 w
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post-infection (hpi) and 24 hpi (Fig. 3C–3E) were database revealed hundreds of predicted targets of c.o
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observed. SeV infection was inhibited in a miR-1225-3p. Several predicted targets ranked in b/
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dose-dependent manner after miR-1225-3p was the top were verified. We found that the 3'UTR of gu
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silenced (Fig. 3F). The transfection of miRNAs GAB3 mRNA contained two putative t o
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did not induce any cell viability changes (Fig. 3G). miR-1225-3p seed-matching sites, suggesting that p
The inhibition of viral infection by miRNAs might GAB3 may be a potential target for miR-1225-3p ril 7, 2
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occur by either directly targeting viral RNA or (Fig. 5A). To verify the bioinformatics predication, 1
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regulating host genes that mediate an antiviral the wild or mutant type of GAB3 3’UTR was
state. To eliminate the possibility that constructed and inserted into the pmirGLO
miR-1225-3p targets virus genome, sequence reporter vector (Fig. 5A). A luciferase reporter
alignments between miR-1225-3p and the assay was then performed. As shown in Fig. 5B,
genomic RNAs of HCV, NDV and SeV were transfection of cells with the miR-1225-3p mimic
performed, and no putative miR-1225-3p target significantly decreased the luciferase activity of
sites were found in viral RNA. These data the co-transfected wild-type GAB3 mRNA
elucidate that downregulation of miR-1225-3p reporter construct, whereas the stepwise addition
shows significant antiviral effects, likely through of binding-site mutations to the GAB3 mRNA
modulating host targets as the mechanism. 3’UTR progressively restored luciferase activity,
with a complete reversal of the inhibition of
Overexpression of miR-1225-3p attenuates the
luciferase activity achieved when all binding sites
antiviral effect of IFN
were mutated. To further establish the direct
To further determine whether the reduction of
4
miR-1225-3p inhibits viral infection through GAB3
interaction between miR-1225-3p and the GAB3 5 and 6) suggests the involvement of GAB3 in the
3’UTR, we created mutant miR-1225-3p (defined antiviral effect. To verify this hypothesis, we
as mut-1225-3p) in which seed match sites for the measured the antiviral ability of GAB3. As
3’UTR of GAB3 mRNA were abolished but a expected, the overexpressed GAB3 inhibited HCV
perfect match for the mutant GAB3 3’UTR was infection at the levels of viral RNA abundance and
obtained (Fig. 5A). In contrast to miR-1225-3p, viral protein expression (Fig. 7A). Aside from
mut-1225-3p mimic had no significant effect on HCV, we showed that infections with NDV at both
the luciferase activity of wild-type GAB3 mRNA 14 hpi and 24 hpi (Fig. 7B–7D) and with SeV at 6
reporter construct but significantly inhibited the hpi, 12 hpi and 24 hpi (Fig. 7E) were suppressed
reporter activity in the cells transfected with significantly by GAB3 overexpression. These
pmirGLO-GAB3-3’UTR-Mut, with a progressive results highlight the antiviral activity of GAB3.
enhancement of the inhibition of luciferase activity
GAB3 enhances the activation of IFN signaling
appearing with the stepwise addition of
The GAB family has been identified as
complementary mutations to the GAB3 mRNA
3’UTR (Fig. 5B). Furthermore, silencing scaffolding adapter molecules contributing to D
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endogenous miR-1225-3p resulted in an increase antiviral immune signal transduction (22, 25). nlo
of the luciferase activity from the wild-type According to the fact that miR-1225-3p affect the ade
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reporter construct but not the mutant construct activation of IFN signaling (Fig. 4), GAB3, as a fro
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(Fig. 5C). To experimentally confirm the target of miR-1225-3p, might regulate the IFN http
regulatory effect of miR-1225-3p on GAB3, we signal transduction. To test this hypothesis, we ://w
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measured the regulation of IFN/JAK/STAT w
transfected Huh7 cells with miR-1225-3p mimics .jb
and observed significant GAB3 decreases on both cascade by GAB3. As shown in Fig. 8, IFN-α2b c.o
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the mRNA and protein levels; in contrast, activated STAT1, ISRE and ISG expression (IFIT1 b/
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miR-1225-3p inhibitors increased the expression and OAS1) were further enhanced by GAB3 gue
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of GAB3 (Fig. 6A–6F). These findings suggest overexpression. t on
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that GAB3 is one of the target genes of Suppression of miR-1225-3p or overexpression of pril 7
miR-1225-3p. , 2
GAB3 enhance virus–induced innate immune 01
9
activation
GAB3 expression is downregulated following
To further investigate the biological
IFN treatment
significance of GAB3 in antiviral immunity, we
Based on our confirmation of GAB3 as a
tested whether GAB3 affects virus–triggered
miR-1225-3p target and on the observation that
innate immune activation. Considering the
miR-1225-3p could be downregulated by IFN-α2b,
importance of IFNs and ISGs in antiviral immunity,
we hypothesized that IFN-α2b might regulate
we evaluated the mRNA levels of IFN-β, IFN-λ3
GAB3 expression. As expected, IFN-α2b was able
and RANTES in SeV–infected cells. As shown in
to increase GAB3 expression (Fig. 6G).
Fig. 9A, the induction of IFN-β, IFN-λ3 and
Overexpressed miR-1225-3p attenuated the
RANTES was significantly enhanced by GAB3
upregulation of GAB3 by IFN-α2b (Fig. 6H).
overexpression at multiple time points after SeV
Overexpressed GAB3 shows antiviral activity infection. Using IFN-β and ISRE reporter
The fact that silencing miR-1225-3p inhibited plasmids, we found that SeV-activated reporter
viral infection (Fig. 3) but upregulated GAB3 (Fig. gene expressions were enhanced in
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miR-1225-3p inhibits viral infection through GAB3
GAB3–overexpressed cells, compared with those miR-1225-3p increases GAB3, which promotes an
in control vector transfected cells (Fig. 9B and 9C). antiviral immune response, thus constituting a
These results suggest that GAB3 enhances positive loop for IFN response (Fig. 10). Zheng et
virus–triggered IFN and ISG production. In al. reported that IFN-downregulated miR-27a can
accordance with the antiviral effect of upregulate Siglec1 and TRIM27 expression, which
miR-1225-3p suppression and its regulation of inhibits type I IFN production, thus constituting a
GAB3, we detected the effect of miR-1225-3p negative loop for innate antiviral response (15).
inhibitor on virus–triggered innate immune This study and our present report highlight the
activation. Similar to the role of GAB3, importance of epigenetic modification in immune
miR-1225-3p inhibition effectively enhanced response by IFN-downregulated miRNAs. As a
SeV–induced expression of IFNs and ISG and special class of ISGs, IFN-downregulated
activation of IFN-β and ISRE reporter genes (Fig. miRNAs, through their posttranscriptional
9D–9F). These data suggest that miR-1225-3p and regulation of the immune system, are worthy of
its targeting to GAB3 might be involved in host more attention and research (29). Furthermore,
antiviral innate immunity. overexpression of miR-1225-3p results in partial D
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Taken together, our findings describe a novel alleviation of the antiviral effects of IFN (Fig. 4A nlo
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antiviral regulation pathway. IFN-stimulated and 4B), suggesting that IFN-regulated gene de
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miR-1225-3p downregulation inhibits viral networks involved many factors are complex and fro
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infection by releasing the suppression of its target dynamic process (30). http
gene GAB3, allowing this scaffolding adapter MiRNAs are important elements of ://w
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protein to be upregulated and consequently to regulatory networks, providing positive or .jb
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enhance host antiviral response (Fig. 10). negative feedback between functional responses. .o
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Over the past decade, the number of miRNAs b/
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Discussion implicated in immune system function has gue
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Once IFN binds to its receptors, a series of increased dramatically (29, 30). Importantly, t on
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etrvaennstcsr ibaer eI SaGcst iv(2a6te, d2 7t)o. Itt raisn stdhue cnea tusirgen oalfs thaensde manutaltgipolnei smtiicR NfaAshsi ocnan, aacntd i na e istihnegrl ae cmooiRpeNraAti vme aoyr pril 7, 2
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genes and their magnitude, duration and cellular 1
regulate several targets in a gene network (31). We 9
context that will determine the outcome of the IFN
had tested other predicted targets of miR-1225-3p
response. In recent years, there has been great
such as IFNAR1, IFNLR1, IRF1, STAT1, STAT2,
progress in understanding the role of ISGs in the
JAK3, PTPA and SOCS7 (data not shown). No
innate immune system, which has evolved to
evidence showed that these genes were regulated
include non-coding RNA as well as ISGs (18, 28).
by miR-1225-3p. It remains to be determined
Several miRNAs have been reported to respond to
whether additional unidentified targets mediate the
IFN stimulation and contribute to IFN-dependent
antiviral effects of miR-1225-3p downregulation.
antiviral immunity (13-15, 18). miR-1225-3p
The GAB proteins occupy a potentially
attracts much attention from us because of its
pivotal position in receptor tyrosine kinase
downregulation, with inducibility by type I IFN,
(RTK)-mediated signaling pathways. GAB
and the significant anti-HCV ability that it shows
proteins contain binding sites for the Src
(Fig. 1A, 1B, Fig. 3A and 3B). Mechanistic
homology region 2 (SH2) and SH3 domains.
analysis revealed that IFN-downregulated
Through its tyrosine phosphorylation by RTKs,
6
miR-1225-3p inhibits viral infection through GAB3
the GAB family may generate an interacting relative miRNA expression, thus changing the
platform for proteins with SH2 and SH3 domains, expression pattern of GAB3 in originally
such as phosphatidylinositol 3-kinase (PI3K) and low-expression cells.
SH2 domain-containing phosphatase 2, and may Despite decades of developments in IFN
transfer these molecules to the plasma membrane, biology, we are still striving to understand
thereby contributing to their activation and IFN-regulated antiviral mechanisms. Numerous
RTK-mediated signal transduction (22, 25). Thus, IFN-regulated miRNAs and relevant epigenetic
it is not surprising to find GABs involved in innate events contribute to the antiviral state. Our
immune processes. Supporting this view, we find findings provide the first example of an
that GAB3 plays a positive role in antiviral IFN-downregulated miRNA, miR-1225-3p,
immune response (Fig. 7–9). The specific playing role in the positive regulation of antiviral
molecular mechanism of GAB3 in immune immune response (Fig. 10). This discovery raises
signaling, which is not discussed in the present the question of whether more miRNAs will have
study, needs to be researched in further detail. It roles in IFN responses. Furthermore, we need to
has been reported that GAB1 enhances TLR3/4- continue building on and identifying an integrated D
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and RIG-I-triggered innate responses by and comprehensive network architecture of how nlo
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promoting activation of the PI3K/Akt pathway multiple miRNAs interact with multiple mRNAs de
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through direct binding to p85 (23). The high in the immune system, which will allow us to fro
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conservation of functional domains among GAB1, identify nodes that may be viable therapeutic http
GAB2 and GAB3 prompts the speculation that targets. ://w
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GAB3 may utilize a similar molecular mechanism Experimental procedures .jbc
to regulate immune response (24). Given the .o
apparently central role played by GAB signaling Cells and reagents brg/
y
Human embryonic kidney 293T cells, PFLC g
via many receptors, delineating the precise ue
mechanism(s) of GAB-mediated signaling is (human primary fetal liver cell), HepG2, Huh7.5.1 st on
and Huh7 hepatoma cells have been described A
critical to understanding how cytokines mediate a previously (18, 32, 33). Hep3B were obtained pril 7
variety of biological activities. , 2
from ATCC and passaged in our laboratory. 01
Among the growing family of GAB proteins, 9
IFN-α2b was from Schering Plough (Kenilworth,
GAB3 has a unique and overlapping pattern of
NJ, USA). Fludarabine, an inhibitor of STAT1
expression compared with GAB1 and GAB2 (24).
activation, was from Selleck (Shanghai, China).
The expression of GAB3 is highest in the
Antibodies were obtained from Abcam
hematopoietic system and lymphocytes during the
(anti-GAB3, ab129553; anti-IFNAR1, ab45172;
steady state but is detectable at low levels within
anti-IFNAR2, ab56070; anti-STAT1, ab103813),
the kidney, lung, and other tissues. According to
Thermo Scientific (anti-HCV core, MA1-080),
our discovery that GAB3 is targeted by
MBL (anti-SeV, PD029), cell signaling
miR-1225-3p in hepatocytes (Fig. 5 and 6), we
technology (anti-pSTAT1, 7649; anti-STAT1,
deduce that posttranscriptional modification by
9172), Abmart (anti-GFP, P30010) and Sigma
miRNA is one of the mechanisms contributing to
(anti-Flag, F1804; anti-β-actin, A5316).
the low expression of GAB3 in non-hematopoietic
Horseradish peroxidase-conjugated secondary
tissues. Some stimuli such as cytokines relieve the
antibodies were from Jackson ImmunoResearch
restriction of GAB3 expression by regulating the
(West Grove, PA, USA).
7
miR-1225-3p inhibits viral infection through GAB3
Viruses miRNA–mRNA targets prediction
The generation of cell culture-derived The potential mRNA targets of miR-1225-3p
hepatitis C virus (strain JFH-1 HCVcc) and firefly were predicted using miRWalk 2.0 (34). The
luciferase reporter HCVcc (Jc1-Luc HCVcc) has predicted target mRNAs simultaneously identified
been previously described (32, 33). JFH-1 HCVcc by miRWalk, miRanda, miRDB, RNA22,
were concentrated using Amicon ultra centrifugal RNAhybrid, Targetscan algorithms were selected
filter (Millipore, 100kD). HCV infection was for next validation. The predicted targets which
evaluated by measuring virus RNA abundance and could be regulated by miR-1225-3p and inhibit
core protein level of JFH-1 HCVcc or luciferase viral infection by its overexpression were
activity (Promega, Madison, WI, USA) of Jc1-Luc considered as targets of miR-1225-3p.
HCVcc. SeV and NDV expressing green
Reporter constructs and luciferase reporter assay
fluorescent protein (NDV-GFP) were kindly
The pmirGLO vector (Promega) contains the
provided by Dr. Zhen-Dong Zhao (Chinese
firefly luciferase gene as the primary reporter to
Academy of Medical Sciences, Beijing, China).
monitor mRNA regulation and the Renilla
SeV replication was determined by western D
luciferase gene as a control reporter for ow
blotting using anti-SeV antibody. NDV-GFP normalization. There are two putative nlo
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infection was evaluated based on GFP expression. de
Cells infected with NDV-GFP were imaged with a miR-1225-3p seed match sites at positions 40–47 d fro
and 222–228 of the GAB3 3’UTR. A fragment m
Nikon Eclipse TE2000-U fluorescence microscope consisting of base pairs 1 to 1476 from the http
and NIS-Elements BR 2.30 software. The intensity ://w
wild-type 3’UTR of GAB3 was inserted into w
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of the GFP fluorescence was quantified using NIH pmirGLO to generate the .jb
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Image software and were normalized to the .o
pmirGLO-GAB3-3’UTR-Wild reporter construct. rg
intensity of the control fluorescence. b/
Site-directed mutagenesis was performed using the y g
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Plasmid constructs and transfection QuickChange site-directed mutagenesis system st o
n
After cDNA encoding human GAB3 (Stratagene, LaJolla, CA, USA) using KOD -Plus- A
p
(NM_001081573.2) was amplified from cDNA as the polymerase (Toyobo, Osaka, Japan) and the ril 7
made from Huh7 cells using the SuperScript III construct encoding the wild-type 3’UTR of GAB3 , 20
1
9
First-Strand Synthesis System (Invitrogen, as the template. As depicted in Fig. 4A, the
Carlsbad, CA, USA), it was cloned into the pmirGLO-GAB3-3’UTR-Mut-#1 construct
plasmid pMIR-cFlag to generate Flag-tagged contains a mutation at match site 1,
protein. The cloning primer sequences were as pmirGLO-GAB3-3’UTR-Mut-#2 contains a
follows: forward primer, mutation at site 2, and
5’-CTTGGATCCAGTGCGGGCGACGCAGTGT pmirGLO-GAB3-3’UTR-Mut-#1+#2 harbors
G-3’; reverse primer, mutations at both sites. All constructs were
5’-AAGGAAAAAAGCGGCCGCTACTTTGGAT verified by restriction enzyme digestion and
TGCCTTTCATC-3’ (BamH I and Not I double sequencing. IFN-β luciferase reporter plasmid,
digestion). DNA transfection of Huh7 or 293T ISRE luciferase reporter plasmid and
cells with Lipofectamine LTX (Invitrogen) was pRL-actin-Renilla-luciferase plasmid were gifts
performed according to the manufacturer’s from Dr. Zhen-dong Zhao. The luciferase reporter
instructions. assay was performed using a protocol that has
been described previously (18).
8
miR-1225-3p inhibits viral infection through GAB3
Quantitative real-time RT-PCR (qRT-PCR) the U6 gene was used as the endogenous control in
Total intracellular RNA was prepared using relative qRT-PCR. The miRNA standards of
TRIzol reagent (Invitrogen) according to the miR-1225-3p for absolute quantification was
manufacturer’s instructions. qRT-PCR analysis purchased from RiboBio (Guangzhou, China).
was performed with a QuantiFast SYBR Green
RT-PCR
RT-PCR kit (Qiagen, Düsseldorf, Germany) and a
100 ng of total RNA extracted with TRIzol
7900HT Fast Real-Time PCR System (Applied
reagent was reverse transcribed and PCR
Biosystems, Foster City, CA, USA). The
amplified in one reaction with a PrimeScript One
preparation of total intracellular RNA and the
Step RT-PCR Kit (Takara, Otsu, Japan). The
performance of qRT-PCR have been described
induction of IFN-β, IFN-λ3 and C-C motif
(18). The absolute quantification of HCV RNA
chemokine ligand 5 (RANTES) was analyzed by
has been described in detail previously (33). For
One Step RT-PCR with 21, 27 and 18 cycles,
the relative quantification of mRNA, primer pairs
respectively, of 94 ℃ for 30 seconds, 60 ℃ for
for GAB3 (forward,
30 seconds and 72 ℃ for 20 seconds. The
5’-AATGGAGGAGCACCGAACAG-3’; reverse, D
specific primers for these cytokines were as ow
5’-GAAGTTTCTGCTGCATGGGG-3’), follows: IFN-β (forward, nlo
a
interferon-induced protein with tetratricopeptide de
repeats 1 (IFIT1, forward, 5’-CCAACAAGTGTCTCCTCCAA-3’; reverse, d fro
5’-ATAGTCTCATTCCAGCCAGT-3’), IFN-λ3 m
5’-CCTCCTTGGGTTCGTCTACA-3’; reverse, (forward, 5’-ATGACCGGGGACTGCATGCC-3’; http
5’-AGTGGCTGATATCTGGGTGC-3’), IFNAR1 ://w
reverse, w
w
(forward, 5’-CGCTGGCAACACAATTCAGGTC-3’), .jb
c
5’-TCCAGTACATTGTATAAAGACCACAGT-3 .o
RANTES (forward, rg
’; reverse, b/
5’-CCTCGCTGTCATCCTCATTG-3’; reverse, y g
5’-GTTCTGATTTTGGACACTGACTTC-3’) and ue
IFNAR2 (forward, 5’-GGTAGGATAGTGAGGGGAAG-3’). The st on
expression level of β-actin served as the A
5’-TAGCCTCCCCAAAGTCTTGA-3’; reverse, endogenous control and was detected at 15 cycles pril 7
5’-AAATGACCTCCACCATATCCA-3’) were of PCR amplification. , 20
1
designed. Primers used for qRT-PCR 9
RNAs and RNA transfection
amplification of 2'-5'-oligoadenylate synthetase 1
The mimic and inhibitor for miRNA-1225-3p,
(OAS1) and β-actin were described previously (18,
the negative control mimic and inhibitor
33). The expression of β-actin served as the
(cel-miR-67-3p), the mimic for customized mutant
endogenous control.
miR-1225-3p (defined as mut-1225-3p) and all of
For the quantification of miRNA, a mirVana
the small interfering RNAs (siRNA) were obtained
miRNA isolation kit (Ambion, Austin, TX, USA)
from RiboBio. The sequences for siRNAs were as
was used to isolate total RNA. The reverse
follows: IFNAR1 siRNA,
transcription reaction and quantitative PCR were
5’-GCUUUCCUACUUCCUCCAGUCUUUA-3’;
then carried out using a TaqMan microRNA
IFNAR2 siRNA,
reverse transcription kit, TaqMan universal PCR
5’-UGAACCACCAGAGUUUGAGAUUGUU-3’.
master mix and TaqMan microRNA assays
Transfection of miRNA and siRNA was performed
(Applied Biosystems) according to the
using Lipofectamine RNAiMAX (Invitrogen)
manufacturer’s protocols. The expression level of
9
miR-1225-3p inhibits viral infection through GAB3
according to the manufacturer’s protocol. zolium bromide (MTT) assay as described
elsewhere (35).
Western blotting
These experiments were conducted according Statistics
to the standard procedures of our laboratory, as Statistical analyses of two sample comprison
described elsewhere (33). The densitometries of were performed by independent sample t-test.
the bands were quantified using Image J software One-way ANOVA and Dunnett's multiple
and were normalized to the density of the control comparisons test were made across multiple
band. samples. Two-way ANOVA and Sidak's multiple
comparisons test were made in grouped graphs. A
Cytotoxicity assay
P value <0.05 was considered statistically
The cytotoxicity of miRNA transfection was
significant.
evaluated via the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetra

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