MiR-221 accentuates IFN's anti-HCV effect by downregulatingSOCS1 and SOCS3

Gang Xu a,1, Fang Yang b,1, Cui-Ling Ding a,1, Jing Wang c, Ping Zhao a,Wen Wang a,n, Hao Ren a,nn

a Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai 200433, Chinab Department of Hepatobiliary Surgery, Fuzhou General Hospital of Nanjing Military Area Command, Fuzhou 350025, Chinac Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai 200433, China

a r t i c l e i n f o

Article history:Received 16 May 2014Returned to author for revisions11 June 2014Accepted 17 June 2014Available online 12 July 2014

Keywords:HCVMir-221SOCS1SOCS3IFN-α

a b s t r a c t

MiR-221 was reported to be upregulated and play roles in tumorigenesis of hepatitis C virus (HCV) associatedhepatocellular carcinoma (HCC). However, the role of miR-221 in HCV infection remains unknown. In thisstudy, it was found that miR-221 was upregulated in serum of HCV chronic hepatitis patients and Huh7.5.1cells infected with HCVcc. Further studies indicated that miR-221 mimic could accentuate anti-HCV effect ofIFN-α in HCVcc model, miR-221mimic could further repressed 10% HCV RNA expression and 35–42% HCV coreor NS5A protein expression in HCVcc infected Huh7.5.1 cells treated with 100 IU/mL IFN-α, and miR-221inhibitor resulted in the reverse effects. Furthermore, two members of suppressor of cytokine signaling (SOCS)family, SOCS1 and SOCS3, which are well established inhibitory factors on IFN/JAK/STAT pathway, wereidentified as the targets of miR-221 and were involved in the effect of miR-221. In conclusion, miR-221 couldaccentuate IFN's anti-HCV effect by targeting SOCS1 and SOCS3.

& 2014 Elsevier Inc. All rights reserved.

Introduction

Hepatitis C virus (HCV) is a blood-transmitted human virus andit is a global health problemwith estimated of 170 million infectedindividuals worldwide (Chisari, 2005). Seventy percent of acuteinfections are rapidly established as chronic infections, which is aleading cause of liver cirrhosis and hepatocellular carcinoma (HCC)(Bowen and Walker, 2005).

In the past decades, studies have focused on investigating thegenes and proteins underlying HCV infection, the host defenses andthe progression of HCC (Gale and Foy, 2005; Garaigorta and Chisari,2009; Liu and Gale, 2010). Recently, an increasing number of reportsdescribed a new class of small regulatory RNA molecules termedmicroRNAs (miRNAs) that are implicated in the progression ofhepatitis virus associated HCC (Negrini et al., 2011; Zhang et al.,2010; Braconi et al., 2010; Diaz et al., 2013) and in response to virusinfection (Berkhout and Jeang, 2007; Watanabe et al., 2007; Marquezet al., 2010). Specially, miR-122 has been shown to intensify thereplication of HCV by targeting the viral 50 non-coding region (Joplinget al., 2005). Moreover, the locked nucleic acid (LNA)—modifiedoligonucleotide (SPC3649) complementary to miR-122 leads to long-

lasting suppression of HCV viremia, with no evidence of viralresistance or side effects in HCV chronically infected chimpanzees(Lanford et al., 2010). The use of an antisense oligonucleotide thatsequesters mature miR-122 in a highly stable heteroduplex, miravir-sen, in patients with chronic HCV genotype 1 infection showedprolonged dose-dependent reductions in HCV RNA levels withoutevidence of viral resistance (Janssen et al., 2013). These findingssuggest miRNAs are involved in liver tumorigenesis at early stage ofHCV infection and have the potential to become novel drug targets invirally induced infectious or malignant diseases.

MiR-221 has been reported in a variety of cancers including HCC(Garofalo et al., 2012). MiR-221 is involved in liver fibrosis and HCCangiogenesis and tumorigenesis by regulating its target proteins (Parket al., 2011; Gramantieri et al., 2009; Pineau et al., 2010; Fornari et al.,2008; Ogawa et al., 2012; Santhekadur et al., 2012; Callegari et al.,2012). MiR-221 regulates CDKN1C/p57 and CDKN1B/p27 expressions(Park et al., 2011); miR-221 targets Bmf and correlates with tumormultifocality (Gramantieri et al., 2009); overexpression of miR-221contributes to tumorigenesis (Pineau et al., 2010); the knockdown ofmiR-221 blocks hepatocellular carcinoma and promotes survival(Fornari et al., 2008); miR-221 activates hepatic stellate cells andcontributes to liver fibrosis (Ogawa et al., 2012); miR-221 is involved inthe promotion of tumor angiogenesis by staphylococcal nucleasedomain containing 1 (SND1) (Santhekadur et al., 2012); miR-221 isfound to promote liver tumorigenicity in transgenic mouse model(Callegari et al., 2012). Although miR-221 has been reported to play

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Virology

http://dx.doi.org/10.1016/j.virol.2014.06.0240042-6822/& 2014 Elsevier Inc. All rights reserved.

n Corresponding author. Tel./fax: þ86 21 81870990.nn Corresponding author. Tel./fax: þ86 21 81870996.E-mail addresses: wonwin29@126.com (W. Wang), hmren@yahoo.com (H. Ren).1 These three authors contributed equally to this work.

Virology 462-463 (2014) 343–350

important roles in HCV associated HCC tumorigenesis, the mechanisticdetails of direct role of miR-221 in the regulation of HCV replicationhowever remain unclear. In the present study, miR-221 was found toaccentuate IFN's effect of repressing HCV replication by targetingsuppressor of cytokine signaling 1(SOCS1) and suppressor of cytokinesignaling 3(SOCS3).

Results

MiR-221 is upregulated by HCV infection

In serum of HCV infected patients with chronic hepatitis,miR-221 was upregulated around 4.5 folds in serum of patientswith CHC (n¼34) compared with healthy blood donors (n¼22)

(Fig. 1A). To verify whether the upregulation of miR-221 isassociated with HCV infection, the relative expression of miR-221 in HCVcc infected Huh7.5.1 cells was evaluated. Seventy twohours after HCVcc infection, miR-221 was upregulated around5.3 folds in Huh7.5.1 cells (Fig. 1B). Huh7.5.1 cells infected withHCVcc were harvested at different time points, and the relativeexpressions of HCV RNA and miR-221 were determined by real-time PCR. To confirm the upregulation of miR-221 by HCVccinfection, time and dose dependency assay were performed.Intracellular HCV RNA increased rapidly and reached to a relativeplateau at day 3 post-infection, and miR-221 expression increasedto a relative stable level at day 3 post-infection (Fig. 1C). Incontrast, there were no significant changes in expressions ofmiR-221 in uninfected group throughout the experiment (datanot shown). In addition, Huh7.5.1 cells were infected with different

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Fig. 1. MiR-221 is upregulated by HCV infecton. (A) The expression of miR-221 in serum of HCV chronic hepatitis patients. (B) The expression of miR-221 in Huh7.5.1 cells72 h after HCVcc infection. (C) Expressions of HCV RNA and miR-221 in Huh7.5.1 cells at the indicated time points post-infection. (D) Expressions of HCV RNA and miR-221 inHuh7.5.1 cells after infection with different levels of HCVcc. Intracellular HCV RNA and miR-221 are analyzed by RT-PCR. GAPDH and U6 are used as an internal control.Column, mean of three independent experiments; bars, SD; n, Po0.01, nn, Po0.001.

G. Xu et al. / Virology 462-463 (2014) 343–350344

amounts of HCV for 3 days. The expression of HCV RNA andmiR-122 in each group (105 IU/ml group, 106 IU/ml group and107 IU/ml group) increased to different levels (Fig. 1D). Theseresults indicated that HCV infection could induce the upregulationof miR-221.

MiR-221 accentuates IFN's repression of HCV replication

To investigate the possible role of miR-221 in HCV infection, theeffect of miR-221 on HCV replication was evaluated in Huh7.5.1cells infected with HCVcc and transfected with or without, miR-221 mimic, miR-221 inhibitor, mm-NC or mi-NC. The expression ofmiR-221 was increased 23.6 folds in cells transfected with 20 nMmiR-221 mimic, but decreased 68.4% in cells transfected with20 nM miR-221 inhibitor (Fig. 2A). By RT-PCR assay, 100 IU/mlIFN-α could repress more than 80% HCV replication in HCVccinfected Huh7.5.1 cells. In addition, HCV RNA expression wasfurther deceased by 43.6% by miR-221 mimic, but increased36.5% by miR-221 inhibitor (Fig. 2B). However, miR-221 mimic orinhibitor showed no effect on HCV RNA expression in HCVccinfected Huh7.5.1 cells without IFN-α treatment (Fig. 2B). Similarresults were seen by Western blotting. The expressions of HCVcore and NS5A protein were further decreased 35% and 42%,respectively, in miR-221 mimic transfected cells; but increased105% and 123%, respectively, in miR-221 inhibitor transfected cellsonly with IFN-α treatment (Fig. 2C). These results indicate thatmiR-221 is most likely involved in the repression effect of IFN-αsignaling on HCV replication.

To verify the mechanism of miR-221, the effects of miR-221 onIFN-α downstream signaling pathway were evaluated. In HCVccinfected Huh7.5.1 cells, the induction of exogenous IFN-α upregu-lated the phosphorylation of JAK1, STAT1 and STAT3 as expected.

In addition, miR-221 mimic further increased but miR-221 inhi-bitor decreased the phosphorylation of JAK1, STAT1 and STAT3(Fig. 3A, left panel). In addition, without HCVcc infection, miR-221mimic also further increased and miR-221 inhibitor decreased thephosphorylation of JAK1, STAT1 and STAT3 induced by IFN-α(Fig. 3A, right panel). These results indicated that miR-221 couldupregulate IFN/JAK/STAT signaling pathway which is independenton HCV infection.

A number of proteins, including IRF7, ISG15, OAS1 and PKRinduced by the JAK/STAT pathway play a role in the antiviralresponses of IFN-α. We directly tested whether miR-221 mimicand miR-221 inhibitor could affect the expression of these ISGs. InIFN-a treated Huh7.5.1 cells, all of these 4 ISGs mRNA expressionswere significantly increased by RT-qPCR, miR-221 mimic couldfurther increase ISG15 and IRF7, but miR-21 inhibitor decreasedtheir mRNA levels, to 45.6-fold and 18.7-fold and 28.7-fold and16.2-fold, respectively (Fig. 3B). Taken together, these findingsindicate that miR-221 increases the expression of several impor-tant anti-HCV factors including ISG15 and IRF7 which correlateswith decreased HCV replication.

SOCS1 and SOCS3 are the targets of miR-221

It is generally accepted that miRNAs exert their functionthrough regulating the expression of their downstream targetgenes. SOCS1 and SOCS3 were predicted as potential targets ofmiR-221 by TargetScan and PicTar. The 30-UTR of SOCS1 and SOCS3mRNA contained a complementary site for the seed region of miR-221 (Fig. 4A). To validate whether SOCS1 and SOCS3 are the targetsof miR-221, 30UTR fragment of SOCS1 and SOCS3 containing miR-221 binding sequence were subcloned downstream of a luciferasereporter vector. In HEK293T cells cotransfected with the reporter

Fig. 2. MiR-221 represses HCV replication. (A) The expression of miR-221 in Huh7.5.1 cells with or without transfection of miR-221 mimic, miR-221 inhibitor or mm-NC,mi-NC. (B) HCV RNA expression is further repressed by miR-221 in Huh7.5.1 cells infected with HCVcc under 100 IU/ml IFN-α treatment by quantitative RT-PCR. (C) HCV Coreand NS5A protein expressions are further repressed by miR-221 in Huh7.5.1 cells infected with HCVcc under 100 IU/ml IFN-α treatment by Western blotting. U6 and GAPDHare used as an internal control. Column, mean of three independent experiments; bars, SD; *, Po0.01; **, Po0.001.

G. Xu et al. / Virology 462-463 (2014) 343–350 345

plasmids and miR-221 mimic, mutant miR-221 or mm-NC, theluciferase activity of the reporter that contained SOCS1 and SOCS330-UTR was significantly suppressed by miR-221 mimic, but theluciferase activity of the reporter that contained SOCS1 and SOCS3mutant 30-UTR remained no significant difference (Fig. 4B). Usingmutant miR-221, the luciferase activity of the reporter that con-tained mutant SOCS1 and SOCS3 30-UTR was significantly sup-pressed (Fig. 4B). The results indicated that miR-221 may suppressgene expression through miR-221 binding sequence at the 30-UTRof SOCS1 and SOCS3. Furthermore, transfection of miR-221 mimicdecreased SOCS1 and SOCS3 expressions and transfection of miR-221 inhibitor increased SOCS1 and SOCS3 expressions in Huh7.5.1cells at both mRNA (by qRT-PCR) and protein level (Westernblotting) (Fig. 4C and D), suggesting that SOCS1 and SOCS3expressions could be inhibited by miR-221. Together, these resultsshowed that miR-221 could regulate the expressions of humanSOCS1 and SOCS3 by directly targeting the 30-UTR of SOCS1 andSOCS3 mRNA.

MiR-221 is involved in IFN's repression on HCV replication bytargeting SOCS1 and SOCS3

To identify whether inhibition of SOCS1 and SOCS3, just likemiR-221 restoration, also resulted in HCV repression, the effects ofknockdown of SOCS1 and SOCS3 on HCV replication were exam-ined. Seventy two hours after transfection with SOCS1, SOCS3specific siRNAs or NC siRNA, the expressions of SOCS1 and SOCS3were decreased at 48.3% and 81.3% respectively (Fig. 5A). Theknockdown of SOCS1 or SOCS3 upregulated the phosphorylationof JAK1, STAT1 and STAT3 (Fig. 5A). Furthermore, knockdown ofboth SOCS1 and SOCS3 enhanced the repression of HCV replicationby IFN-α (Fig. 5B), and miR-221 showed no further effect in suchSOCS1 or SOCS3 knockdown cells (Fig. 5B). These results indicate

that SOCS1 and SOCS3 are most likely involved in the effect ofmiR-221 on IFN-α signaling.

Together with the above findings, it was concluded that miR-221 rescue JAK/STAT signaling by targeting SOCS1 and SOCS3, andrescued IFN/JAK/STAT signaling repress HCV replication moreefficiently, thus miR-221 accentuates anti-HCV activity of IFN-αby targeting SOCS1 and SOCS3 (Fig. 5C).

Discussion

Altered miRNA expression during viral infections includes bothmiRNAs that target viral sequences as well as miRNAs that targethost genes that influence the course of viral replication andpathogenesis (Kumar, 2011). A number of miRNAs have beenreported to be involved in HCV infection. MiR-491 was involvedin regulation of HCV replication via the PI3 kinase/Akt pathway(Ishida et al., 2011); miR-155 was found to be associated with HCVinfection (Bala et al., 2012); and let-7b can also regulate HCVreplication (Cheng et al., 2012).

In the present study, we demonstrated miR-221 could help torepress HCV replication, which might provide new insights intothe mechanisms underlying HCV infection, but also raise a doubton the exact functions of miR-221. As reported by previous studies,miR-221 is regarded as an oncogene which could enhance tumor-igenesis (Janssen et al., 2013; Garofalo et al., 2012; Park et al., 2011;Gramantieri et al., 2009; Pineau et al., 2010; Fornari et al., 2008;Ogawa et al., 2012). Our findings suggest that miRNAs might playdual roles in HCV infection. Research on miR-122 support such ahypothesis. On the one hand, miR-122 enhances the replication,translation and infectious virus production of HCV (Chang et al.,2008; Jangra et al., 2010; Roberts et al., 2011); overexpression ofmiR-122 even supports the entire HCV life cycle in HepG2 cells(Narbus et al., 2011). On the other hand, miR-122 plays an

Fig. 3. MiR-221 upregulates IFN-α induced JAK/STAT pathway. (A) MiR-221 rescued JAK/STAT signaling pathway. The key molecular of JAK/STAT pathway including p-JAK1,p-STAT1 and p-STAT3 are deregulated in IFN-α treated Huh7.5.1 cells transfected with miR-221 mimic or miR-221 inhibitor. (B) ISGs including ISG15 and IRF7 are furtherupregulated by miR-221 in IFN-α treated Huh7.5.1 cells. GAPDH is used as an internal control. Column, mean of three independent experiments; bars, SD; *Po0.01.

G. Xu et al. / Virology 462-463 (2014) 343–350346

inhibitory effect on HCC tumorigenesis since it was found thatmiR-122 suppresses cell proliferation and induces cell apoptosis inHCC by targeting Wnt/β-catenin pathway (Xu et al., 2012),inhibiting tumorigenicity (Bai et al., 2009; Hsu et al., 2012).Moreover, miR-122 was reported to repress HBV transcription ina p53-dependent manner (Wang et al., 2012; Fan et al., 2011),which could suggest a possible virus type dependent role ofmiRNAs. As Sendi (2012) discussed, miR-122 might play thedual role of in viral hepatitis, and therapeutic applications ofmiR-122 might differ based on the underlying disease. Thus,miRNAs could play different roles in viral infection and virus-associated tumorigenesis.

As the IFN system represents an integral part of the mamma-lian innate immunity, it is likely that miRNAs might either targetpattern recognition receptors and signaling components involvedin the IFN induction after pathogen recognition, or alter expressionof proteins involved in the IFN response such as the JAK/STATpathway. Several cellular miRNAs that are induced during viralinfection were reported to negatively modulate the IFN pathway.MiR-26a, miR-34a, miR-145, and let-7b were proved to be IFN-βinducible and directly regulated IFN-β expression during infectionas a negative feedback loop (Witwer et al., 2010). MiR-146 limitsinterleukin-1 receptor associated kinase 1 (IRAK1) and TNF recep-tor associated factor 6 (TRAF6) expression and directly attenuatedtranscriptional induction via the IFN-α/β receptor by limiting

expression of STAT1 (Taganov et al., 2006; Tang et al., 2009).Inducible expression of miR-155 was observed during both bacter-ial and viral infections, as well as after exposure of cells toproinflammatory cytokines such as IFN-β, IFN-γ, or tumor necrosisfactor alpha (TNF-α) (O’Connell et al., 2007).

It is generally accepted that miRNAs exert their functionthrough regulating the expression of their target genes. Nocandidate related to IFN/JAK/STAT pathway was predicted as thetarget of miR-221 by TargetScan and PicTar. Interesting, SOCS1 andSOCS3 were predicted as the targets of miR-221. SOCS family is amainly negative regulator of JAK/STAT signaling. So far, 8 membersof the SOCS family have been identified, termed SOCS1 to SOCS-7and CIS (Yoshimura, 2009). Both SOCS1 and SOCS3 can inhibit JAKtyrosine kinase activity directly through their kinase inhibitoryregions (KIR) (Yasukawa et al., 1999). For HCV infection, SOCS1suppression was associated with abnormal B cell activation(Moorman et al., 2009); HCV core protein exerts an inhibitoryeffect on SOCS1 expression (Miyoshi et al., 2005); and increasedexpression of SOCS3 was associated with non-response to antiviraltherapy (Walsh et al., 2006). Furthermore, SOCS1 has beenreported to be a target of miR-155 and miR-221 (Cardoso et al.,2012; Lu et al., 2011), while SOCS3 is the target of miR-203 ingingival epithelial cells (Moffatt and Lamont, 2011). We identifiedSOCS1 and SOCS3 as the targets of miR-221, which might providea further understanding on the regulation of SOCS1 and SOCS3.

Fig. 4. SOCS1 and SOCS3 are the targets of miR-221. (A) The putative miR-221 binding sequence in the 30-UTR of SOCS1 and SOCS3 mRNA. (B) Suppressed luciferase activityof wild type 30UTR of SOCS1 and SOCS3 by miR-221 mimic. HEK293T cells are cotransfected pMIR-SOCS1-30UTR or pMIR-SOCS3-30UTR, and miR-221 mimic or mm-NC, mi-NC. Firefly luciferase activity of each sample is measured 48 h after transfection and normalized to Renilla luciferase activity. (C and D) The expressions of SOCS1 and SOCS3mRNA (C) and protein (D) are regulated by miR-221. The expressions of SOCS1 and SOCS3 in Huh7.5.1 cells are analyzed 48 h after transfection with miR-221 mimic, miR-221inhibitor or mm-NC, mi-NC by quantitative RT-PCR (C) or Western blotting (D). GAPDH is used as an internal control. Column, mean of three independent experiments;bars, SD; *, Po0.01; **, Po0.001.

G. Xu et al. / Virology 462-463 (2014) 343–350 347

In summary, our findings suggest miR-221 could regulate SOCS1/SOCS3 and functions as an IFN enhancer in HCV infection. Herein,we provide characterization of potential miR-221 target genes andbroaden the understanding of IFN stimulated gene regulation.

Materials and methods

Serum specimens, cell lines and reagents

Serum samples used in this study were collected from 34 HCVinfected patients with chronic hepatitis (Qujing, Yunnan province,China). The diagnosis was determined by physical examination,blood chemistry, and magnetic resonance imaging. No patient hadreceived chemotherapy. Additional samples were collected from22 blood donors in Shanghai Changhai Hospital, in which standardmarkers for HBV or HCV infection tested negative. Clinical andpathological information was extracted from the patients' medicalcharts and pathology reports (Table 1). Written consent for tissuedonation (for research purposes) was obtained from the patientsbefore serum collection and the protocol was approved by theInstitutional Review Board of Second Military Medical University.The samples were frozen in liquid nitrogen and stored at �80 1Cuntil use.

Human embryonic kidney cells (HEK293T) and human HCCcells lines Huh7.5.1 were cultured in DMEM medium plus 10% FBS(Invitrogen, Carlsbad, CA, USA) at 37 1C in a humidified atmo-sphere containing 5% CO2.

SOCS1 siRNA, SOCS3 siRNA and nonrelative siRNAwere purchasedfrom Santa Cluz Biotechnology (Santa Cruz, CA, USA); Interferon-alpha (IFN-α) was purchased from Piscataway (Middlesex County, NJ,

USA); miR-221 mimic, mutant miR-221 mimic and nonrelative mimiccontrol RNA (mm-NC), miR-221 inhibitor and nonrelative inhibitorcontrol RNA (mi-NC) were purchased from Ribobio (Gangzhou,China).

Generation of HCV viral stocks and infection of Huh7.5.1 cells

Infectious HCV in cell culture (HCVcc) was generated as describedpreviously (Lindenbach et al., 2005). Briefly, plasmid pFLJ6/JFH1,containing the full-length chimeric genomic cDNA for HCV J6 andJFH-1was linearized and used as the template for transcription usingan in vitro MEGAscript kit (Promega). The in vitro-transcribed RNAwas delivered to Huh7.5.1 cells by electroporation. Viral stocks wereobtained by harvesting the culture supernatants on days 8 to 18 aftertransfection and aliquoted for storage at �80 1C. The titer of viral stockwas determined in Huh7.5.1 cells by immunostaining as described

Fig. 5. SOCS1 and SOCS3 are involved in the repression of HCV replication by miR-221. (A) MiR-221 as well as SOCS1 and SOCS3 knockdown could rescue JAK/STAT signalingpathway. The phosphorylation of key molecular of JAK/STAT pathway is deregulated in HCVcc infected Huh7.5.1 (with IFN-α) cells transfected with miR-221 mimic, miR-221inhibitor or SOCS1 siRNA, SOCS3 siRNA. GAPDH is used as an internal control. (B) SOCS1 and SOCS3 knockdown downregulated HCV protein expression in HCVcc infectedHuh7.5.1 cells as miR-221 do. U6 is used as an internal control. (C) Schematic representation of the main findings in this study. Column, mean of three independentexperiments; bars, SD; *, Po0.01; **, Po0.001.

Table 1Profiles of patients.

HCV patients (n¼34) Blood donors (n¼22) Ref. value

Age (years) 45711.7 46.5714.6Male/female 9/13 11/20HCV RNA(copies/ml)

3.9879.6�105 o500 o500

HBV DNA(copies/ml)

o500 o500 o500

ALT 68.9759.9 22.379.9 5–40 IU/LAST 77.7774.4 18.776.8 8–40 IU/LGGT 69.1744.6 16.575.1 7–32 IU/LALB 46.372.57 36.2572.22 35–55 g/L

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previously (Lindenbach et al., 2005). Briefly, HCVcc-infected cells werewashed, fixed with cold methanol, probed with anti-HCV-positivesera, washed, and probed with FITC-conjugated anti-human IgG(Jackson ImmunoResearch, UK). Finally, the cells were labeled withDAPI and images were acquired using a fluorescence microscope(IX81; Olympus, Tokyo, Japan). Stained foci were counted in quad-ruplicate wells, and the virus titer of focus-forming units (ffu)/mL wascalculated.

Huh7.5.1 cells were seeded in a 24-well plate, and following anovernight culture, a 50 uL HCVcc supernatant with 5�105 ffu/mLwas added to each well for 5 h, and then supplemented with freshculture medium after washing with culture medium 5 times. FormiRNA or IFN treatment, 20 nM miR-221 mimic or inhibitor wastransfected into the cells at day 1 after HCVcc infection; then,100 IU/mL IFN-α was added or not to the cells at day 2; finally,HCV protein was detected by western blotting with relevantantibodies and the HCV RNA was determined by quantitativeRT-PCR at day 3 or day 4.

Quantitative real time PCR

Total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad,CA, USA) according to the manufacturers' recommendations. AnA260 nm/A280 nm ratio of 1.9 and a 28S/18S ratio of 1.8 wereminimum requirements for the following PCR. Total RNA (2 μg)was reverse transcribed with the SuperScript III First-Strand Synth-esis System with specific reverse primers or Oligo (dT) (Invitrogen).The primers (Table S1) and SYBR RT-PCR kit (Takara, Shiga, Japan)were used for amplification. Reactions were incubated in a 96-welloptical plate at 95 1C for 5 min, followed by 40 cycles at 95 1C for 15 sand 60 1C for 1 min. PCR reactions were run on a StepOne Plus real-time PCR machine (Applied Biosystems, Foster City, CA, USA) and thedata were analyzed by SDS v2.3 software. U6 and GAPDH were usedas the control.

Plasmids construction

The fragments of 30-UTR of SOCS1 (790–1203nt, Genbankaccession NM_003745) and SOCS3 (2102–2621nt, Genbank accessionNM_003955) containing the two putative miR-221 binding sequences(1140–1147nt and 2381–2386nt) were amplified with the primersSOCS1-UTR and SOCS3-UTR (Table S1). The PCR product was clonedinto pMIR-REPORT™ miRNA Expression Reporter Vector (Ambion, TX,USA), termed as pMIR3-SOCS1-30UTR and pMIR-SOCS3-30UTR. Theplasmids which carried the mutated sequence in the complementarysites for the seed region of miR-221 (Fig. 2A) were generated based onpMIR3-SOCS1-30UTR and pMIR-SOCS3-30UTR plasmid by MutanBESTKit (Takara Bio, Shiga, JP), termed as pMIR3-SOCS1-mut-30UTR andpMIR-SOCS3-mut-30UTR. These plasmids were confirmed by sequen-cing analyses.

Transfection

The transfections were carried out using FuGene HD transfec-tion reagent (Roche, Indianapolis, IN, USA) following the manu-facturer's protocol. In brief, 2�104 Huh7.5.1 cells or 5�104

HEK293T cells in 24-well plates were transfected with indicatedplasmids, miRNA or siRNA, and collected 24–48 h after transfec-tion for assay.

Dual-luciferase reporter assay

HEK293T cells seeded in 24-well plates in triplicate were cotrans-fected with pMIR-SOCS1-30UTR, pMIR-SOCS3-30UTR, pMIR3-SOCS1-mut-30UTR or pMIR-SOCS3-mut-30UTR and miRNA-221 mimic,mutant miR-221 mimic or mm-NC by using FuGene HD transfection

reagent. The pRL-TK (Promega) was also transfected as a normalizationcontrol. Cells were collected 48 h after transfection, and luciferaseactivity was measured using a dual-luciferase reporter assay kit(Promega) and recorded by multi-plate reader (Synergy 2, BioTek).

Western blotting

Protein extracts from Huh7.5.1 cells were prepared by a modifiedRIPA buffer with 0.5% sodium dodecyl sulfate (SDS) in the presence ofproteinase inhibitor cocktail (Complete mini, Roche), 25 mg protein ofcells were electrophoresed in 10% SDS-PAGE mini gels and transferredonto PVDF membranes (Immobilon P-SQ, Millipore, Billerica, MA, USA).After blocking with 5% nonfat milk, the membranes were incubatedwith specific primary antibodies at 4 1C overnight, followed byincubation with HRP-conjugated goat anti-rabbit or goat anti-mouseantibody (1:10000 dilution, KPL, Gaithersburg, MA,USA) for 1 h atroom temperature. Finally, signals were developed with Super SignalWest Pico chemoluminescent substrate (Pierce), visualized by theGene Gnome HR Image Capture System (Syngene, Frederick, MD, USA)and analyzed by Gene tools (Syngene). The primary antibodies usedwere: HCV core and HCV NS5A (Thermo Fisher, Waltham, MA USA),JAK1, STAT1, STAT3, SOCS1, SOCS3, pJAK1, pSTAT1, pSTAT1 rabbitpolyconal antibody (SAB, College Park, MD,USA) and GAPDH mousemonoclonal antibody (ImmunoGen, shanghai, China).

Prediction of miRNAs targets

TargetScan (http://www.targetscan.org/) and PicTar (http://pictar.mdc-berlin.de/) programs were used to investigate the targetgenes of miRNAs and the conserved sites bound by the seed regionof miR-221.

Statistical analysis

Data are presented as mean7SD. Comparisons were made byusing a two-tailed t test or one-way ANOVA for experiments withmore than two subgroups. Po0.01 was considered statisticallysignificant.

Funding

This work was supported by Ministry of Science and Technol-ogy Key Program (2012ZX10002009-004), National 973 Project(2013CB531601) and Science Fund for Creative Research Groups,NSFC, China (81221061).

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.virol.2014.06.024.

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