Introduction

Despite the availability of an effective vaccine since 1986,hepatitis B virus(HBV)infection still is a signi ficant threat to public health.HBV infection is the most common cause of acuteon-chronic liver failure(ACLF)in China[1,2].According to the guidelines of the Asian Paci fic Association for the Study of the Liver[3],ACLF is characterized by severe deterioration in liver function manifesting as hepatic encephalopathy,gastrointestinal bleeding,hepatorenal syndrome,jaundice,coagulopathy and ascites.The 28-day mortality of ACLF is 33%,15 times higher than chronic hepatitis B(CHB)and chronic asymptomatic HBV carriers(ASC)[4].Moreover,HBV-ACLF is a life-threatening condition and its exact pathophysiology and progression remain unclear.Alanine aminotransferase(ALT)/aspartate aminotransferase(AST)and prothrombin activity(PTA)and the model for end-stage liver disease(MELD)score all provide insights into liver function and the severity of liver injury in clinical settings.However,enzyme levels(ALT/AST)are not speci fic and convey limited information,and the MELD score requires a complicated evaluation with PT/INR,total bilirubin(TBil),creatinine(Cr),and sodium.Thus,more sensitive and convenient biomarkers are needed to diagnose ACLF in patients with HBV infection.microRNAs(miRNAs)are small noncoding RNAs of 19–23 nucleotide lengths that were discovered in 1993[5],and play major roles in the regulation of gene expression.miRNAs regulate expression via control of mRNA stability or translation processes which consequently affect cell differentiation,development,and apoptosis[6–8].A single miRNA likely regulates multiple genes,and a single gene may also be regulated by multiply miRNAs[9,10].miRNA expression may also be tissue-or organ-speci fic,which suggests that miRNA biomarkers are more likely to be highly speci fic for certain conditions.Further,miRNAs can be detected in blood plasma or serum and exhibit high stability[11,12].They are not signi ficantly affected by RNase activity and remain stable after exposure to harsh conditions.Serum miRNA analyses represent a promising approach to monitoring liver function.To evaluate the potential prognostic value of miRNAs in HBVACLF,we measured the circulating miRNAs in our study population of HBV-ACLF,CHB and ASC by different liver in flammation.We expected that the levels of these miRNAs in patients with ACLF would presumably be higher compared with CHB and ASC.

电子商务行业目前仍处于快速发展阶段，进入门槛较低，市场竞争激烈，根据《2016年中国淘宝村研究报告》显示，截至2016年8月底，在全国共发现1311个淘宝村和135个淘宝镇。竞争压力大是限制网店业务发展的首要问题，如低价竞争、恶性竞争、假冒伪劣产品等都可能给网商们带来无形的压力。尤其对于青岩刘而言，大多数网商属于业内中小卖家，在管理、人才、资金、流量和创新等方面都存在着一定的不足与缺陷，从而导致其竞争力和抗风险能力较弱。网商稳定性不足、流动性大等因素都不利于青岩刘电商产业的建设与发展。

Since a small change in miRNA expression can affect expression of hundreds of target genes and signi ficantly alter the transcriptome[13],disruption of miRNA regulatory networks has been implicated in a number of diseases[14].The presence of miRNAs in serum and their potential use as biomarkers was first reported by Lawrie et al.[14].To explore the clinical applicability of miRNAs as non-invasive markers of HBV-ACLF,we investigated the expression pro file of miRNAs in selected serum candidate miRNA biomarkers(146a-5p,197-3p,223-3p,328-3p,338-3p,122-3p and let7a-5p)[15–20].These biomarkers were evaluated in serum samples from the same patient cohort.

Methods

Patients and samples

One hundred and twenty-six HBV-infected patients were enrolled in the present study including 55 HBV-ACLF,41 CHB,and 30 ASC.Patients were recruited from Xiangya Hospital,Changsha,China,from September 2013 to November 2014.Informed consent was obtained from participants.The European Association for the Study of the Liver guidelines on the prevention and treatment for chronic hepatitis B were used as the diagnostic criteria for cohort identi fication[15].CHB is de fined as chronic HBV infection with clinical evidences of liver diseases such as biochemical,virological and histological features together with exclusion of other causes.Patients with CHB can be divided into HBeAg-positive and HBeAgnegative.ASC is de fined as HBsAg-positive,anti-HBe-positive with persistent normal serum ALT and HBV-DNA＜2000 IU/mL,and no evidence of liver injury[3].Patients exhibiting the following were excluded from the study:alcoholic liver disease,drug-induced liver injury,acute fatty liver,pregnancy,autoimmune liver disease,hepatolenticular degeneration,and liver failure after transplantation.

miRNA pro file and reagents

We investigated the expression pro file of miRNAs in serum from select candidates.miRNA biomarkers(146a-5p,197-3p,223-3p,328-3p,338-3p,122-3p,let7a-5p)were analyzed in comparison with HBV-related disease symptoms.The miRNeasy Serum Kit,miRNeasy Serum Spike-in control,QuantiTect Reverse Transcription Kit,miScriptSYBRGreen PCR Kit,and miRNA primers were purchased from Qiagen,Hilden,Germany.

下一秒，所有的想念与期待，怦然爆发。场景里绿色的松树，彩色的礼品盒通通变成了五彩的背景色。Merry Christmas的歌曲，萦绕回荡耳边。抱着一袋子礼物，她跑出来跳上了车。这样会很唐突吧？但她真的好想去见酒刀先生。

Serum sample collection and RNA isolation

All serum samples were from fresh blood and stored at−80°C.Total small RNA was extracted from 200 μL of serum using the miRNeasy serum kit(Qiagen)according to the manufacturer’s protocol.Extracted serum RNA concentrations were from 25 to 45 ng/μL.

Quantitative PCR

此外，从RTA签署实施的年代上看，近年来签订的RTA质量水平较过去2010年以前签署的RTA有了明显提升。2014年底生效实施的韩国—澳大利亚RTA，对WTO+领域的条款覆盖率达到了历年实施RTA的最高标准，超过了92%，且WTO+承诺率也高达79%，WTO-X的覆盖率和承诺率也处于高水平，分别为42%和24%。

Statistical analyses

All statistical analyses were performed in SPSS 18.0(SPSS Science,Chicago,IL).Patients’clinical and biochemical indices were expressed as mean±standard deviation(SD).The expression differences of miRNAs among groups were tested with ANOVA and paired group differences were assessed by Student’s t test.Spearman correlations were used to assess correlations between variables.The receiver operating characteristic(ROC)curve and area under the ROC curve(AUROC)analyses were used to evaluate the difference in serum miRNA levels among ACLF and non-ACLF patients.Univariate and multivariate analyses were also conducted to test associations between the prognosis(dependent variable)and independent variables,followed by multivariate logistic regression analysis and calculation of the Youden’s index and 95%con fidence interval(CI).Youden’s index was used to identify the optimal cutoff point.All tests were conducted as two tailed and P＜0.05 was considered statistically signi ficant.

Results

The levels of these miRNAs are more sensitive than commonly used markers,such as ALT and AST,to evaluate the severity of liver diseases[35].Here,we further investigated the reliability of laboratory tests associated with ACLF prognosis using statistical analyses.Our results indicated that the prognosis of ACLF is associated with INR,Na+,hepatic encephalopathy,gastrointestinal bleeding,spontaneous peritonitis,hepatorenal syndrome,miR-122-3p,miR-146a-5p,and miR-328-3p.Models like MELD can help quickly evaluate and predict the severity and prognosis of HBV-ACLF.Our newly established model meets the criteria for outcome prediction due to early applicability and a high level of accuracy.Our results indicated that MELD accurately categorized patients based on their risk scores.The AUROC for MELD was 0.779,which indicated that the MELD scoring system was useful in forecasting survival in ACLF patients.However,our new model exhibited a substantially higher AUROC value of 0.847.Moreover,statistical analyses indicated that the new scoring model had a higher predictive capability than MELD.Therefore,the prognostic value of our model should be taken into account in future studies on ACLF prognosis.

通过以上分析可知,March-Like算法不仅能够覆盖March C+算法能够覆盖的故障类型,还能够检测到March C+算法不能覆盖到的写破坏耦合故障、读破坏耦合故障和写干扰故障,整体上提高了算法的故障覆盖率。

Serum miRNA expressions

The expression levels of miR-146a-5p,miR-122-3p and miR-328-3p were signi ficantly elevated in patients with ACLF compared with that in HBV carriers and CHB(P＜0.05,Fig.1).The levels of miR-197-3p between the ASC and ACLF groups,and between the CHB and ACLF groups were statistically signi ficant(P＜0.05).However,no statistically signi ficant difference was found for miR-197-3p between the ASC and CHB groups.miR-338-3p expression was upregulated in CHB patients compared to ASC patients(P＜0.05),but was not signi ficantly different among other pairs(Fig.1).

实施“四化”建设标准化采购是油田改造和优化传统物资采购供应模式的一次重大变革，也是油田在开创高质量发展中，点石成金的“必备武器”和“胜利攻略”。

ROC curves of miRNAs

There are some limitations to the present study.First is the sample size.Our data need to be validated by larger-scale studies.Second,patients recruited regionally from the same hospital might have similar genetic backgrounds,which may in fluence the miRNA expression results.Multiple centers across several regions should be conducted.Third,the exact role of miR-146a-5p,miR-122-3p,and miR-328-3p in the pathogenesis of HBV-ACLF needs to be further explored.

Table 1 Clinical characteristics in patients with different stages of liver disease progression.

ASC:chronic asymptomatic HBV carrier;CHB:chronic hepatitis B;ACLF:HBV-associated acute-on-chronic liver failure;WBC:white blood cell;PLT:platelet;TBil:total bilirubin;ALT:alanine aminotransferase;AST:aspartate aminotransferase;ALB:albumin;GLO:globulin;HBV:hepatitis B virus;HBsAg:hepatitis B surface antigen;HBeAg:hepatitis B e antigen;MELD:model for end-stage liver disease;NS:not signi ficant;P＜0.05

Variables ASC(n=30) CHB(n=41) ACLF(n=55) P value Age(yr) 34.2±12.3 42.4±13.3 44.6±11.3 0.000 Gender(M/F) 19/11 28/13 47/8 NS WBC(109/L) 4.2±2.0 5.3±2.1 7.1±3.6 NS PLT(1012/L) 122.0±23.4 102.2±12.4 85.7±43.0 NS TBil(μmol/L) 89.0±19.2 76.0± 21.9 356.9± 173.6 NS ALT(U/L) 18.0±13.0 121.0±101.0 381.7±411.3 0.032 AST(U/L) 20.0±8.0 148.0±96.0 369.8±454.6 0.042 ALB(g/L) 37.1±2.3 34.1±3.1 30.5±4.0 NS GLO(g/L) 22.4±2.1 23.1±4.4 28.0±8.0 NS ln(HBV-DNA)(IU/mL) 5.3±2.4 6.7±1.2 4.8±2.0 0.013 HBsAg(IU/mL) 21,126.5±27,004.0 12,637.9±15,749.0 10,185.2±21,624.0 NS HBeAg(+/−) 9/21 34/7 18/37 NS MELD 0.2±0.5 13.4±5.9 28.4±5.3 NS Na+(mmol/L) 143.5±5.0 135.4±4.5 137.8±4.1 NS

Fig.1.Comparison of serum miRNAs expression pro files in chronic asymptomatic HBV carrier(ASC),chronic hepatitis B(CHB)patients,and acute-on-chronic liver failure(ACLF)patients by real-time quantitative polymerase chain reaction.The 7 miRNAs expression levels of patients were normalized to miR-39.∗:P ＜ 0.05.

Risk factors for the development of ACLF

ACLF patients were followed for up to 6 months to assess longterm prognosis.The outcomes(i.e.,recovery and mortality)were recorded for each patient.As shown in Table 2,13 patients died during follow-up,the remaining 42 patients were still alive at study end.The deaths were related to complications from liver disease,as hepatic encephalopathy,hepatorenal syndrome,gastrointestinal bleeding,and spontaneous bacterial peritonitis.We analyzed baseline clinical and laboratory variables as possible predictors of prognosis with univariate logistic analysis.The analyses indicated that INR,Na+,hepatic encephalopathy,gastrointestinal bleeding,hepatorenal syndrome,spontaneous peritonitis,miR-122-3P,miR-146a-5p,and miR-328-3p are related to the development of ACLF(P＜0.05).

Analysis of independent factors with multivariate regression analysis

We used the data from the univariate analyses to further analyze the correlation of clinical variables by multivariate regression analysis.Multivariate analyses results indicated that Na+,INR,gastrointestinal bleeding,and miR-122-3p are all related to the prognosis and therefore,are useful parameters in the new model(Table 3).

A new model to predict the development of ACLF

Fig.2.ROC curves of the miR-146a-5p(A),miR-328-3p(B)and miR-122-3p(C).The area under ROC curve(sensitivity,speci ficity)of the miR-146a-5p,miR-328-3p,and miR-122-3p were 0.729(89.1%,52.4%),0.721(76.8%,66.7%),and 0.52(50.0%,59.1%),respectively.

Table 2 Complications and laboratory tests associated with prognosis in ACLF via univariate analyses.

P＜0.05.

Variables Recovery(n=42) Mortality(n=13) t/χ2 P value Age(yr) 45.1±11.5 42.3±8.6 −1.369 0.346 Male 36 11 1.412 0.221 WBC(109/L) 6.4±2.3 8.5±5.7 1.233 0.076 PLT(1012/L) 84.8±42.8 88.9±45.4 −2.96 0.768 Cr(μmol/L) 113.6 ± 60.4 96.6 ± 22.1 0.989 0.327 ALT(U/L) 300.2±300.9 406.1±254.1 −1.146 0.257 AST(U/L) 381.3±490.7 293.4±265.4 0.616 0.540 ALB(g/L) 30.3±4.3 31.6±4.3 −0.696 0.489 GLO(g/L) 27.8±6.8 29.6±11.1 −0.713 0.479 INR 1.9±0.7 2.4±0.8 −2.515 0.015 Na+(mmol/L) 138.2±3.9 136.8±2.4 −1.315 0.041 MELD 28.2±4.4 29.4±1.1 0.493 0.050 ln(HBV-DNA) 4.5±2.1 5.3±1.5 1.582 0.137 miR-122-3p 1.8±3.0 3.3±4.8 3.853 0.000 miR-146a-5p 1.1±1.8 0.7±0.7 4.132 0.000 miR-328-3p 2.9±3.2 2.3±1.5 5.788 0.000 Complications Hepatic encephalopathy 5 8 13.549 0.000 Gastrointestinal bleeding 0 4 15.12 0.000 Spontaneous peritonitis 9 5 4.253 0.000 Hepatorenal syndrome 4 9 19.607 0.000 Ascitic fluid 40 13 0.642 0.423

PSF and OY proposed the study.WY and flperformed the research and wrote the first draft.FXY,WDX and LBJ collected samples.WY and TDM analyzed the data.All authors contributed to the design and interpretation of the study and to further drafts.OY is the guarantor.

Table 3 Multivariable Cox regression analysis of complications and laboratory tests associated with prognosis in ACLF.

INR:international normalized ratio;GB:gastrointestinal bleeding;OR:odds ratio;95%CI:95%con fidence interval.

Variables βStandard deviation Wald value P value OR 95%CI Na+ 0.402 0.206 3.796 0.049 1.324 0.098–2.241 INR −1.72 0.645 7.104 0.080 0.179 0.051–0.634 GB −4.963 1.693 8.592 0.030 0.070 0.000–0.193 miR-122-3p −0.278 0.124 5.038 0.025 0.075 0.594–0.964 Constant terms 50.449 27.482 3.370 0.066 8.120 –

Fig.3.ROC curves of the new model(A)versus MELD model(B)as predictor of long-term prognosis with data gathered after 6-month follow-up in ACLF patients in the validation cohort.The area under ROC curve(sensitivity,speci ficity)of the new model and MELD were 0.847(75.0%,95.3%)and 0.779(73.8%,97.0%),respectively.

Discussion

Although the role of miRNAs has been explored extensively in many liver diseases,surprisingly few studies have examined its role in patients with HBV-ACLF.Song et al.[22]reported the utility of miR-210 as a marker of liver in flammation in patient with CHB.Zheng et al.[23]demonstrated that miR-130a upregulation was a good prognostic indicator of HBV-ACLF.

HBV-ACLF is associated with high mortality[17].Although liver transplantation can signi ficantly improve the survival rate,donor shortage remains a critical issue.Liver damage caused by the host’s immune attack may contribute to the production of miRNAs in the serum of patients with CHB and ACLF.miRNAs play important roles in immune response processes and in liver-related diseases.The close relationship between HBV reactivation and miRNA regulation suggests that the alteration of miRNA expression pro files may reflect the progression of liver disease.In this study,we evaluated the relationship between different types of chronic HBV infection and serum levels of miRNAs and further evaluated the potential prognostic value of miRNAs for HBV diseases.We explored the clinical applicability of miRNAs as non-invasive circulating HBV infection biomarkers.We selected 7 miRNAs(146a-5p,197-3p,223-3p,328-3p,338-3p,122-3p and let7a-5p)as biomarkers for this study based on previous results.These miRNAs have been previously demonstrated to be involved in the pathogenesis of liver injury or liver failure caused by diverse etiologies[24–28].

Our results indicated that serum miRNAs were biomarkers of HBV infection.In particular,levels of miR-146a-5p,miR-122-3p,and miR-328-3p in the CHB and ACLF groups were signi ficantly higher than those in the ASC group.miR-146a has emerged as a master regulator of the immune system and is involved in innate and adaptive immunity,various viral infections,cancers,and some nonimmune human diseases[18,19].Moreover,the correlation between miR-146a levels and liver diseases has been reported previously.miR-146a upregulation by viral infection may contribute to the functional impairment of HBV-speci fic T cells and viral persistence[20].In addition,miR-146a was upregulated in HBV-loaded Huh-7 hepatocytes,HBV-infected mice,and patients with HBV infection[29].Our results are thus in concordance with these previous observations suggesting the possible reliability of miRNAs as liver disease diagnostic tools.

miR-122,a liver-speci fic miRNA,comprises approximately 70%of the total miRNA population in the adult liver[21].miR-122 is upregulated in patients with severe ACLF and coagulopathy[30].Moreover,Wang et al.demonstrated that miR-122 is signi ficantly increased in the early stages of CHB,and may be used as a reliable and sensitive biomarker for clinical diagnosis in liver failure[31].

To our knowledge,this is the first study to identify serum miR-328-3p as a potential predictor for the prognosis of ACLF.The expression of serum miRNA-328-3p has also been shown to be signi ficantly associated with acute myeloid leukemia[32]and in the motility of HCC cells[33].Further,circulating miR-328 could be potential indicators for acute myocardial infarction as they are associated with increased risk of mortality or development of heart failure[34].

miRNAs in serum RNA samples from ASC,CHB and ACLF patients were measured using the SYBR Green(Qiagen)array miRNA real-time quantitative polymerase chain reaction(q-PCR)pro filing platform(Applied Biosystems,FosterCity,CA,USA).q-PCR was carried out on an Applied BioSystems PCR machine(StepOnePlusTM Real-Time PCR).To evaluate miRNA ampli fication efficacy,standard curves were generated using 10-fold serial dilutions of the reverse transcribed cDNA by total RNA.Since U6 and other house-keeping miRNAs are unstable in serum,there is no current consensus on the use of house-keeping miRNAs for q-PCR analysis.Therefore,the expression levels of target miRNAs were directly normalized to total RNA.Moreover,we used spike-in-controls(miR-39,Qiagen)in order to normalize the expression levels of target miRNA[16].The reactions were incubated in a 96-well optical plate at 95°C for 15 min,followed by 45 cycles of denaturation at 94°C for 15 s,annealing at 55°C for 30 s,and extension at 70 °C for 30 s.All reactions were performed in triplicate and no-template negative controls were included.The speci ficity of each PCR product was validated by performing melt curve analysis following PCR analysis.Melt curve samples were analyzed in triplicate and the cycle threshold(Ct)was de fined as the number of cycles required for the fluorescent signal to reach the threshold.We used the 2−△△Ct method for analyses of real-time q-PCR data,where ΔCt=mean Ct miRNA−mean Ct internal control[21].Brie fly,the relative abundance(as a fold-change in value)of each miRNA was calculated as the ratio from HBV infection to the value from controls(ASC).

The clinical characteristics of ASC(n=30),CHB(n=41),and ACLF patients(n=55)are presented in Table 1.

To determine which levels of the three putatively diagnostic miRNAs could distinguish ACLF from non-ACLF(ASC and CHB)more clearly,ROC analysis was performed on individual miRNAs.The sensitivity,speci ficity,and accuracy were determined using the Youden’s index.The cut-off for each miRNA was selected with the highest Youden’s index values.Among the three miRNAs,miR-146a-5p had the highest AUC of 0.729(Youden’s index of 0.2998,sensitivity of 89.1%and speci ficity of 52.4%)in separating non-ACLF controls from those with ACLF.miR-328-3p had an AUC of 0.721(Youden’s index of 0.9217,sensitivity of 76.8%and speci ficity of 66.7%).miR-146a-5p and 328-3p both had high values of specificity and sensitivity using cut-off points of 0.2998 and 0.9217 respectively,which suggests that when Youden’s index ＞ 0.2998 and 0.9217,ACLF patients can be distinguished from non-ACLF patients.In contrast,miR-122-3p had an AUC of 0.520,and there was no statistically signi ficant difference among groups(Fig.2).

Although great advances have been made in ACLF diagnosis and treatment recently,high mortality is still a signi ficant concern for ACLF patients.To our knowledge,serum miR-146a-5p,miR-122-3p,and miR-328-3p were reported for the first time as potential predictors for the prognosis of ACLF.These serum miRNAs of patients with ACLF may lay the foundation for additional studies on the role of miRNAs in the pathogenesis of ACLF,and provide the basal work for future study of the molecular mechanisms of ACLF.Importantly,we also have established and validated a new prognostic model for ACLF patients,and the new scoring model had a higher predictive capability than MELD model.The feasibility of this novel scoring system should be validated by larger prospective studies.

选择两种泄漏孔直径，分别为25 mm和219 mm，当泄漏孔直径达到管径时会造成管道破裂，每种泄漏孔直径的失效概率见表7所列。

Contributors

Our new model for prognosis in ACLF that was established on the basis of independent risk factors is as follows:Y=0.402× Na+−1.72× INR−4.963× gastrointestinal bleeding(Yes=0;No=1)−0.278×(miR-122-3p)+50.449.The AUROC of the model for the prognosis in ACLF was 0.847,the cut-off value was 101.249,the sensitivity was 75.0%,and the speci ficity was 95.3%.The alternative MELD model is as follows:R=0.378×ln(TBil)+1.12×ln(INR)+0.95×ln(Cr)+6.4.The AUROC of the MELD model for prognosis in ACLF was 0.779,the cut-off value was 31.265,the sensitivity was 73.8%,and the specificity was 97.0%(Fig.3).Our analyses suggested that the new model for ACLF prognosis established on the basis of independent risk factors had a better clinical value compared to the conventionally used MELD model(Fig.3).Using Kaplan–Meier analysis,a logrank test revealed a signi ficant difference between the new model described here and the MELD model,with the new model displaying a signi ficantly higher AUROC compared to the MELD model.

3) 试验和有限元模拟结果在峰值力和最大位移上差距明显，这是由于上面板在冲击时发生破裂以及面板与夹芯之间脱焊导致的，试验结果和模拟结果在能量吸收方面非常接近，略微的差别是由于面板破裂导致部分弹性变形能耗散所致。

Funding

This study was supported by a grant from the Clinical Research Foundation of Xiangya Hospital,Central South University(No.2016L10).

Ethical approval

This study was approved by the Ethics Committee of Xiangya Hospital,Central South University.

政府会计改革对于高校资产管理提出了更高的要求，从会计核算到业务管理都带来了很大的变化。将经营理念和成本意识引入资产管理，逐渐促进高校资产管理向现代企业管理方式转变，提高资产管理效率，为学校的事业发展提供有力保证。

Competing interest

No bene fits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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