| Objective：Interferon (IFN) is currently an important component duringthe antiviral treatment of viral hepatitis, either CHB or CHC. Interferontherapy has an advantage of finite duration and the convenience of withdrawalor change to other drug compared with nucleos(t)ide analogues during thetreatment of CHB; and the combination of pegylated interferon-α and ribavirinis the standard guideline for the treatment of CHC. Although dozens ofinterferon has been found until now, however, there is only interferon-α whichbelongs to type Ⅰ interferon applied to the treatment of viral hepatitis inchina. But interferon-α2a given for48weeks can induce the seroconversion ofhepatitis B e antigen (HBeAg) in only30%-40%of CHB patients, and about60%of patients will continue to suffer from chronic active hepatitis B despiteinterferon-α2a therapy. Meanwhile, about50%-60%of patients infected withgenotype1hepatitis C virus can’t achieve sustained virological response (SVR)despite interferon-α2a standard therapy. Besides that IFN-α treatment isassociated with adverse effects and some patients terminate the treatmentbecause of severe side effects. Further study identified that the negativeregulator of Janus activated kinase-signal transducer and activator oftranscription (JAK-STAT) pathway ubiquitin-specific peptidase18(USP18)was the key mediator of refractory to interferon-α therapy. Recently, a novelclass of cytokines was discovered and named interferon-λ which belong totype Ⅲ interferon. Latest in vitro study found that interferon-λ could activateJAK-STAT pathway and exert antiviral activity in the cells which wasrefractory to interferon-α treatment. Whether the negative regulator ofinterferon-α therapy USP18could impaire interferon-λ antiviral activity iscurrently unknown. The aim of the present study is to explore the effect andclinical significance of USP18on the antiviral signal transduction pathway of IFN-α and IFN-λ.Methods: The new resuscitated Huh-7cells were plated and cultured at5%CO2,37℃in6-well culture plates with HD-DMEM mediumsupplemented with10%fetal bovine serum (FBS) and1%penicillin andstreptomycin. When the logarithmic phase cells fused completely to60%-70%confluence, they were divided to control group, IFN-α group and IFN-λ group.1. IFN-α group stimulated with IFN-α2a (20IU/ml), IFN-λ groupstimulated with IFN-λ1(10ng/ml) and the control group was treated withsterile saline. The cells were harvested after stimulated for6h,12h,24h,48hrespectively.2. IFN-α group stimulated with IFN-α2a (20IU/ml) for12h, after that thecells were washed three times with PBS and maintained in HG-DMEMwithout IFN for16h, then restimulated with IFN-α2a (20IU/ml) for12h beforeharvested. IFN-λ group stimulated with IFN-λ1(10ng/ml) for12h, after thatthe cells were washed three times with PBS and maintained in HG-DMEMwithout IFN for16h, then restimulated with IFN-λ1(10ng/ml) for12h beforeharvested. The control group was treated with equal amount of sterile saline.3. IFN-α group stimulated with IFN-α2a (20IU/ml) for12h, after that thecells were washed three times with PBS and maintained in HG-DMEMwithout IFN for16h, then restimulated with IFN-λ1(10ng/ml) for12h beforeharvested. IFN-λ group stimulated with IFN-λ1(10ng/ml) for12h, after thatthe cells were washed three times with PBS and maintained in HG-DMEMwithout IFN for16h, then restimulated with IFN-α2a (20IU/ml) for12h beforeharvested. The control group was treated with equal amount of sterile saline.RT-qPCR was applied to determine the mRNA expression of USP18,STAT1, PKR and MxA. The protein levels of USP18, P-STAT1, STAT1,PKR and MxA were determined by Western-blot.Results:1The changes of all target indicators in Huh-7cells at different stimulatetime point (6h,12h,24h,48h).1.1Changes of mRNA and protein expression in Huh-7cells of USP18. The mRNA expression of USP18were up-regulated both in IFN-α groupand IFN-λ group along with the stimulation of IFN. But the IFN-α groupshowed a more significant increase at24h and48h compared with IFN-λgroup (5.33±0.47VS4.60±0.42,5.41±0.46VS4.76±0.78, all P<0.01).Simultaneously, the mRNA expression of USP18both in IFN-α group andIFN-λ group were more than control group (P<0.01). Meanwhile, with theprolonging of stimulation, the protein expression of USP18were increase bothin IFN-α group and IFN-λ group. And the IFN-α group showed a moreobvious increase at48h compared with IFN-λ group (1.61±0.04VS1.46±0.03,P<0.05).1.2Changes of mRNA and protein expression in Huh-7cells of STAT1,PKR, MxA and P-STAT1.1.2.1With the prolonging of stimulation, the mRNA expression ofSTAT1, PKR and MxA were up-regulated in both group and the IFN-α groupreached their peak expression at12h compared with IFN-λ group (5.61±0.44VS4.84±0.33,5.42±0.37VS4.68±0.15,3.82±0.35VS3.23±0.46, all P<0.01).Then, the mRNA expression in the IFN-α group began to decrease while theIFN-λ group continued to increase. And the IFN-λ group reached their peakexpression at24h compared with IFN-α group (5.30±0.15VS5.95±0.37,4.81±0.43VS5.81±0.40,3.37±0.46VS4.63±0.62, all P<0.01). Afterwards,the mRNA expression of both group began to decline, but mRNA expressionin the IFN-λ group still exceeded IFN-α group at48h. The kinetic of proteinexpression was consistent with mRNA expression.1.2.2With the prolonging of stimulation, the phosphorylated STAT1protein showed an increase in both group and the IFN-α group reached theirpeak expression at12h compared with IFN-λ group (1.19±0.02VS0.63±0.02,P<0.05). Then, the IFN-α group began to decrease while the IFN-λ groupcontinued to increase and the IFN-λ group reached their peak expression at24h compared with IFN-α group (0.71±0.01VS1.25±0.02, P<0.05).2The changes of all target indicators in Huh-7cells of different treatment(Huh-7cells in control, IFN-α, IFN-λ group were stimulated with sterile saline, IFN-α2a (20IU/ml), IFN-λ1(10ng/ml) respectively for12h, after that the cellswere washed three times with PBS and maintained in HG-DMEM withoutIFN for16h, then restimulated with sterile saline, IFN-α2a (20IU/ml), IFN-λ1(10ng/ml) respectively for12h before harvested).2.1The expression of USP18mRNA in IFN-α group were higher thanIFN-λ group (4.04±0.39VS3.25±0.26, P<0.05). On the contrary, the mRNAexpression of STAT1, PKR, MxA were lower than IFN-λ group (4.71±0.28VS6.54±0.26,3.71±0.25VS5.41±0.32,2.91±0.23VS4.31±0.19, all P<0.05).2.2The expression of USP18protein in IFN-α group were higher thanIFN-λ group (1.93±0.15VS1.82±0.14, P<0.05). Similar with the mRNAexpression, the protein levels of STAT1, P-STAT1, PKR, MxA were lowerthan IFN-λ group (1.13±0.16VS1.61±0.17,1.12±0.18VS1.49±0.15,1.31±0.13VS1.91±0.16,1.09±0.19VS1.59±0.10, all P<0.05).3The changes of all target indicators in Huh-7cells of different treatment(Huh-7cells in control, IFN-α, IFN-λ group were stimulated with sterile saline,IFN-α2a (20IU/ml), IFN-λ1(10ng/ml) respectively for12h, after that the cellswere washed three times with PBS and maintained in HG-DMEM withoutIFN for16h, then restimulated with sterile saline, IFN-λ1(10ng/ml), IFN-α2a(20IU/ml) respectively for12h before harvested).3.1There was no statistical significance between IFN-α group and IFN-λgroup on the mRNA expression of USP18(3.54±0.28VS3.46±0.31, P>0.05).Whereas, the mRNA expression of STAT1, PKR, MxA were lower than IFN-λgroup (4.31±0.20VS4.76±0.26,4.31±0.20VS4.76±0.26,3.49±0.27VS3.80±0.20, all P<0.05).3.2The expression of USP18protein in IFN-α group was lower thanIFN-λ group (1.76±0.16VS1.90±0.12, P<0.05). But there was no statisticalsignificance of P-STAT1expression between the two groups (1.29±0.15VS1.30±0.19, P>0.05). The protein levels of PKR and MxA in IFN-α group werelower than IFN-λ group (1.40±0.10VS1.49±0.14,1.23±0.11VS1.42±0.14, allP<0.05). Conclusions:1IFN-λ antiviral signaling was not affected by USP18in Huh-7cells.2IFN-λ could still activate the antiviral signaling in the IFN-α refractoryHuh-7cells.3IFN-λ induced strong but delayed kinetics of STAT1phosphorylationsignal and antiviral protein induction, while IFN-α induced more rapid buttransient expression of these genes.4Repeated administration of IFN-α dramatically up-regulated theexpression of USP18and reduced the synthesis of antiviral protein whichinduce a refractory state in Huh-7cells.5Repeated administration of IFN-λ slightly up-regulated the expressionof USP18, and the synthesis of antiviral protein did not reduce. |
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