| Backgroud and AimsChronic Hepatitis B virus(CHB)remains a public health problem that severely affects global health.Sustained viral infection can eventually cause progreossive liver diseases,such as cirrhosis,liver failure and liver hepatocellular carcinoma(HCC).Nucleos(t)ide analogues(NAs)are inhibitors of hepatitis B virus(HBV)DNA polymerase which directly blocking the viral replication,and would reduce liver inflammation,and prevent the development of liver disease.But Long-term indefinite therapy might bring huge economic burden,and longterm side effects at the same time.Since NAs does not eradicate covalently closed circular DNA or the integrated HBV genome,some people could face the risk of recurrence after withdrawal.Currently,there is still much controversy about NA discontinuation.Several mayor international guidelines,recommended that discontinuation could be considered when HBsAg disapear or seroconversion occurs.However there is a little possibility of HBsAg seroconversion with NUCs therapy in clinical practice.Therefore,with above concerns and high relapse rate of HBV during longterm NUCs treatment,it is important to investigate surrogate markers of clinical relapse.miRNAs are a class of on-protein-coding RNAs,which regulate gene expression at the post-transcriptional level.Inceasing evidence suggests that microRNAs(miRNAs)are essential for the regulation of various biological processes such as cell development,differentiation,proliferation,apoptosis and metabolic fuction.miRNA-122 is the most frequent miRNA in the adult liver,and play a central part in liver biology and disease.Recently studies have shown miRNA-122 can inhibit intracellular HBV replication.Moreover,several recent studies demonstrated that miRNAs not only exist in cells but also in the circulation.,increassing expression of miR-122 in the blood circulation is associated with HBV or HCV infection,drug-induced liver damage,alcoholic hepatitis,and HCC.Indeed,Muscle damage does not lead to increased levels of miR-122,which means miR-122 is a more specific biomarker than ALT,the traditional liver injury biomarker.Moreover,miR-122 may be elevated earlier than serum transferase levels and serum miR122 increased significantly more than ALT in the late response in animal models of alcoholic hepatitis and drug-induced liver damage,which means miR-122 can be a more sensitive biomarker in liver inflammation and necrosis.Furthermore,some reports showed that In the process of chronic HBV infection,circulating miR-122 levels may be positively correlated with serum ALT level,HBe status,HBV DNA and HBsAg level.Terefore,our study followed up the changes in clinical biochemical and virological indicators of patients who dicontinuation of NAs treatment to find out the risk factors that predict clinical recurrence after drug withdrawal.By detecting the expression levels of serum miR-122 after drug withdrawal,to explore whether serum miR-122 expression levels can be used as a more sensitive surrogate marker of early clinical relapse.Given these questions,we conducted our study.Materials and Method Clinical characteristics36 CHB patients who enrolled in a prospective,single-center,observational study and underwent cessation of NA therapy(clinical trial number:ChiCTR-ONRC-12002315)were included in this study.This study was conducted in compliance with the Declaration of Helsinki and approved by the ethics committee(study iden-tifier NFEC-201209-K3).According to the guidelines of the Chinese"Chronic hepatitis B prevention and treatment guidelines criterias:these subjiects were divided into two groups of study subjects:Clincal relapse(CR,n=23),Sustained viral Responses(SVR,n=13).The study included patients with CHB who received NUCs therapy at Nanfang Hospital,Southern Medical University,between January 2003 and July 2011.Patients voluntarily discontinued NUCs,and were prospectively followed-up between November 1,2012,and June 31,2017.Serological Assays and HBV-DNA AssaysLocal standardized automated techniques were used to perform biochemical tests.Quantitative analysis of HBsAg(lower limit of detection[LLOD],0.05 IU/mL),HBeAg,and anti-HBe was performed using the Architect assay(Abbott Laboratories,Chicago,IL).Data on pretreatment serum HBV DNA levels were retrospectively collected(by an HBV-specific polymerase chain reaction assay with a LLOD of 1000 copies/mL;Daan Gene,Sun Yat-sen University,Guangzhou,China)).Serum HBV DNA levels at NA cessation and follow-up were determined using the Cobas HBV-specific TaqMan polymerase chain reaction assay with a LLOD of 20 IU/mL or 100 copies/ml(Roche Diagnostics,Basel,Switzerland).RNA extraction,reverse transcription,and quantitative real-time PCRmiR-122 levels were assayed in the stored frozen serum samples collected on every visiting timepoint.miRNA in serum was extracted by miRNeasy Serum/Plasma kit(Qiagen,Hilden,Germany)according to the manufacturer’s instruction.The cDNA synthesis was performed with 100ng total miRNA using the miScript Reverse transcription Kit(Qiagen).The expression of miR-122 was determined by real-time RT-PCR,which was performed with miScript SYBR Green PCR Kit(Qiagen)by using commercially available qPCR primer(Qiagen)on a LightCycler 480(Roche Diagnostics,Switzerland).The data were presented as values normalized against 106 copies of C.elegans miR-39 miRNA transcripts.Statistical analysisAll of the tests were two-tailedand a p-value of<0.05 calculated by SPSS Statistics 22.0 and GraphPad Prism 6.0 was considered statistically significant.ReslutsClinical characteristicIn Total,36 non-cirrhotic Asian patients with CHB who stoped NAs therapy,divided into two groups,Clincal relapse(CR,n=23),and Sustained Responses(SR,n=13),base on the difinition of clinical relapse.At the baseline,we can not find significant difference about age,gender,therapy time,consolidation treatment,HBcrAg levels,Fibroscan parameters.There are significant diferences in HBsAg between CR group and SR group at baseline,4th week,8th,12th week,24th week.And during the follow up times,we can see signficant difference of HBV DNA levels in CR and SR at 4th week,8th week,12th week,24th week.Moreover,we can only observe the difference of ALT between two groups at week 24.Dynamic change in serum miRNA-122 levels after cessation of NA therapySerum miRNA-122 levels in the CR group at 8th week 12th week and 24th week after withdrawal NAs were significantly higher than those at the baseline the(Respectively,p=0.0012,P=0.006,P<0.001),However,there was no significant difference in serum miR-122 levels between week 4 and baseline after discontinuation NAs therapy in the clinical relapse group,In the sustained response group(SR),serum miRNA-122 levels were no significant difference between the baseline and the miR-122 levels at 4th week,8th week,12th week,24th week,after drug withdrawal.There was no significant difference between SR group and CR group at baseline and at week 4.The expression levels of serum miRNA-122 in the clinical relapse group at 8th week,12th week and 24th week after withdrawal NAs was significantly higher than that in the continuous response group(P=0.001,P=0.004,P=0.002)The correlation between serum miRNA-122 and HBV DNA,ALT,HBsAgSpearman analysis reverevealed a positive correlation between serum miRNA-122 levels and ALT levels at week 24 in all patients who were discontinued(r=0.637,p=0.002).At the same time,miRNA-122 level was positively correlated with HBV DNA levels at week 12(r=0.416,p=0.0141)and week 24(r=0.693,p=0.0036)after NAs therapy withdrawal.Beside,serum miRNA-122 leves was positively correlated with HBsAg at all follow-up weeks after cessation of NAs therapy,the baseline(r=0.650,p=0.0013),week 4(r=0.421,p=0.0012),week 8(r=0.819,p<0.0001),week 12(r=0.827,p<0.0001),week 24(r=0.794,p=0.0004)And we also find there was a positive correlation between serum miRNA-122 level and HBcrAg at baseline(r=0.4997,p=0.006),and week 12(r=0.2974,p=0.0272)after drug withdrawal.The value of serum miRNA-122 expression level in predicting clinical relapse afer withdraw long-term NAs drugs.Univariate analysis showed that serum miR-122 level at 8th week(HR:2.111,CI:1.265-3.521),serum miR-122 level at 12th week(HR:2.243,CI:1.265-3.977)and serum miR-122 level at 24th week(HR:2.742,CI 1.384-5.434)were independent predictors of clinical relapse.Multivariate analysis showed that patients older than 35 years old(HR:5.341,CI:1.185-24.08)and serum miR-122 level at 24th week(HR:4.770,CI:0.003)were risk factors for clinical recurrence after NAs cessation.ROC curve generated to assess the usefulness of miRNA-122,HBsAg,HBcrAg,which showed that the AUC of serum miRNA-122 expression level at week 8 was 0.856,CI:0.571-0.951,p=0.001,and the AUC of serum miR-122 expression level at week 12,0.903,CI:0.733-1.00,p<0.001.The combination of serum miR-122 expression and HBcrAg at week 12,AUC was 0.931,CI(0.817-1.00),p<0.001,and the combination of two fators improved the sensitivity,specificity,positive predictive value and negative predictive value.ConclusionBaseline HBsAg titers,age>35 years,and serum miRNA-122 level were independent predictors of clinical relapse.The serum miRNA-122 level was highly expressed in clinical replapse group,which increased with the time course of drug withdrawal.Serum miRNA-122 expression levels of CHB patients after discontinuation of NAs was positively correlated with HBsAg,ALT,HBV DNA and HBcrAg levels.The combination of miRNA-122 at 12th week and HBcrAg at 12th week were effective indicators for predicting clinical relapse.These may suggest that the expression level of serum miR-122 can be used as a potential biomarker for early prediction of clinical relapse in CHB patients after cessation of treatment with long-term NAs drugs. |
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