Effects And Mechanism Studying Of SOCS3-targeting MicroRNAs On HCV Replication

Hepatitis C virus(HCV)mostly infects the liver through the blood pathway,causing acute and chronic viral hepatitis.At present,about 180 million people worldwide were infected with HCV,and the positive rate of anti HCV in healthy people is 0.7%~3.1% in China.Most HCV infections tend to evolve into chronic infection,and some patients caused liver cirrhosis and hepatocellular carcinoma after years of development.Pegylated interferon(IFN)and ribavirin(RBV)are the current anti HCV classic treatment,but because of the serious side effects of IFN,different sensitivity to various factors such as interferon resistance limit the efficacy and scope of IFN therapy.In recent years,with the further researches on HCV,great achievements have been made in anti HCV treatment.For example,the application of direct-acting antiviral agents(DAAs),small protease inhibitors against HCV,have significantly increased SVR(sustained virological response)compared with the IFN and RBV combination therapy.However,because of the high cost of DAAs treatment,and side effects recently reported such as DAAs might increase the incidence and postoperative recurrence of hepatocellular carcinoma.Therefore,exploration of specific targets include HCV infection-related host factors are still hot topics in current HCV research.HCV is a flavivirus,single-stranded RNA virus.Many host factors were reported to participate in the process of HCV life cycle including invasion,replication,assembly and release.The relationship between the HCV life cycle and the host lipid metabolism,and HCV infection and innate immunity have attracted the researchers in recent years.microRNAs(miRNAs)are a family of endogenous non-coding small molecule RNAs.Numerous studies have shown that miRNAs could participate in many aspects of cell progress,including cell differentiation,proliferation,apoptosis,metabolism,cell cycle,and are closely related to the occurrence and development of inflammatory diseases,infectious diseases,metabolic diseases and tumors.The main biological function of miRNA is to regulate gene expression by binding to the 3'-UTR of its target molecule to inhibit translation or degradation of mRNA.A variety of miRNAs have been shown to have important role in the occurrence and development of HCV-HCC.In addition,miRNAs also play an important role in the replication,diagnosis and treatment of HCV infection.For example,miR-122 and miR-141 have been identifiedto play a role in promoting HCV replication.Miravirsen(SPC3649),a small complementary antagonist of miR-122,has entered the phase III clinical trials.Suppressor of cytokine signaling 3(SOCS3)is one of the important molecules of the SOCS family.A large number of studies have showed that SOCS3 plays an important role in the occurrence and development of various diseases.The current studies found that SOCS3 could negatively regulate the signal transduction produced by various cytokines and hormones(including IL-6,IL-10,LPS,IFN-alpha,leptin and insulin signal transduction etc.),which play an important role in the occurrence and development of inflammatory diseases,such as viral infection,obesity and cancer and other diseases.The role of SOCS3 in viral infection is mainly associated with its negative regulation of JAK-STATs signal pathway.JAK-STATs pathway is the anti-viral signal pathway of type I and type II IFN.The expression of SOCS3 is closely related to the degree of replication of HCV in cells.It has been shown that the expression of SOCS3 protein in the liver samples of HCV patients is increased,and the high level of SOCS3 expression is associated with the resistance of IFN in HCV patients.At the same time,the level of SOCS3 in liver tissue can reflect the effect of HCV infection treated with IFN.The polymorphism of the SOCS3 gene correlates with the host response to HCV antiviral therapy.It has been reported that SOCS3 was one of the important host factors for HCV replication in vivo.Many miRNAs play important role in the occurrence and development of various diseases by regulating the expression of SOCS3.However,few studys have been reported which miRNAs targeting SOCS3 affect HCV replication and anti-HCV of IFN.Based on the important role of SOCS3 in HCV replication,we explored that miRNAs targeting SOCS3 affect HCV replication in cells by inhibiting SOCS3 and promote the anti-HCV effect of IFN.First,we validated the effect of SOCS3 on HCV replication in vitro by using SOCS3 siRNA.si-SOCS3 and si-NC were transfected into Huh 7 cells,compared with si-NC,si-SOCS3 could inhibit HCV replication in cells.Further more,we found that IFN-? can enhance the inhibitory effect of si-SOCS3 on HCV replication.Next,mimicNC(mNC)was used as a positive control,miR-185-5p,miR-19b-3p,and miR-30c-5p were transfected in Huh 7 cells.The result shows that miR-185-5p,miR-19b-3p,and miR-30c-5p can significantly inhibit the expression of SOCS3.In a further experiment,plasmids containing wild type SOCS3 3'-UTR and mutant SOCS3 3'-UTR which has mutated the binding site with miR-185-5p,miR-19b-3p,and miR-30c-5p were constructed.The results of Dual luciferase reporter assay showed that SOCS3 was the target gene for miR-185-5p,miR-19b-3p and miR-30c-5p.Then,HCVcc system was used in Huh 7 cells,the results showed that miR-185-5p,miR-19b-3p,and miR-30c-5p can inhibit HCV replication while inhibiting the expression of SOCS3.Inhibition of HCV replication by miR-185-5p,miR-19b-3p,and miR-30c-5p were further enhanced after treatment with IFN-?.Previous studies have confirmed that SOCS3 is one of the important negative regulators of the JAK-STATs pathway.The effect of IFN-? against HCV is through the JAK-STATs pathway.IFN-? play its anti-HCV role by upregulating the expression of p-STAT1 downstream of the JAK-STATs pathway.Therefore,we transfected si-SOCS3,miR-185-5p mimic,miR-19b-3p mimic,miR-30c-5p mimic,and control,and found that si-SOCS3,miR-185-5p mimic,miR-19b-3p mimic,and miR-30c-5p mimic all significantly up-regulated the expression of p-STAT1 compared with the control.The above results further confirmed that IFN-? enhanced the effect of inhibiting HCV replication of miR-185-5p,miR-19b-3p,and miR-30c-5p by enhancing the JAK-STATs pathway.Based on the above data,miRNAs(miR-185-5p,miR-19b-3p and miR-30c-5p)targeting SOCS3 can inhibit HCV replication in cellular model by down regulating the expression of SOCS3.This inhibitory effect relieved or weakened the negative regulatory effect of SOCS3 on the JAK-STAT pathway,and significantly upregulated the expression of p-STAT1,which is one of the mediators of anti-HCV effect of IFN-?.Therefore,miR-185-5p,miR-19b-3p,and miR-30c-5p targeting SOCS3 inhibit HCV replication and synergize with IFN-? suppressing HCV replication.This study will provide more evidence for SOCS3 as a marker of IFN theraputic response,and lay the foundation for the development of potential therapeutics based on SOCS3,providing more basis for broadening IFN therapy for HCV infection.