Pathologic Characteristics And Screening Study Of MicroRNAs Biomarkers For Liver Injury Among Chronic Hepatitis B(CHB)Patients With Normal Alanine Amiotransferase(PNALT)

Currently,the main international therapeutic guidelines for chronic hepatitis B virus(CHB)infection considered that alanine aminotransferase(ALT)level is the most important biochemical indicator of hepatic histologic lesion.Generally,the elevated ALT level is a marker of hepatic necroinflammation and also an indicator of antiviral therapy,however,CHB patients with persisterntly normal ALT(PNALT)level do not need antiviral treatment because they have no or only minor necroinflammation change in liver.Nevertheless,recent studies have shown that 28%-37%PNALT CHB patients have severe liver damage,some even cirrhosis or liver cancer;In addition,some studies have shown that serum ALT levels of some CHB patients had poor correlation with liver histological lesions;there were greater limitations in the treatment of CHB patients only relying on ALT levels.Therefore,it is an urgent task to look for more sensitive and more specific biomarkers than ALT level.MicroRNAs(MiRNAs)has become a hotspot in the field of molecular biology in the past decade,and miRNAs are small noncoding single-chain RNAs with about 19-22 nucleotides in length,widely existed in eukaryotic cells,matured miRNAs negative regulated expression of target gene at the posttranscription level by pairing with 3'untranslated region(3'-UTR),and participated in almost all the important physiological processes.In addition,miRNAs can stably exist in plasma or serum to resist the degradation of RNA enzyme due to the special structure and existence mode,so the peripheral blood MiRNAs may become a new ideal molecule biomarker for many diseases.Recent studies have also provided clear evidence that miRNAs is abundant in the liver and regulates many liver diseases.However,it is still insufficient study on whether miRNAs can be used as molecular diagnostic marker and regulation mechanism in CHB-PNALT patients.Exosome is a tiny vesicle,about 30?120nm,containing a certain level of proteins and nucleic acids through vesicles(microvesicles,MVS)inverted generation,is a variety of cell-body endocrine vesicles,containing bioactive proteins,functional mRNA,miRNAs and other non-coding RNA speices.It is pointed out that the miRNAs produced after cell damage can be transferred to the blood circulation,which can mediate the reaction process of organism after the cell damage.The aim of this study was to clarify the incidence of pathological changes in liver tissues among CHB patients with PNALT and to analyse the correlation between clinical biochemical markers and the severity of hepatic lesions in this group,to screen clinical indicators that can respond to severe liver injury of these population;And then through high-throughput miRNAs array,look for more stable and sensitive miRNAs markers that can reflect the liver damage of CHB patients with PNALT,to provide a comprehensive theoretical basis for the timely diagnosis and treatment of these patients,who would otherwise missed the curative treatment window can benefit from optimal therapy;Finally,separated exsome from the supernatant of human hepatic stellate cells(HSCs),and detected differentially expressed miRNAs of plasma in exsome,to investigate the feasibility of plasma miRNAs as a diagnosis marker for SPNALT.Part I Clinical and Pathologic Feature of Chronic Hepatitis B(CHB)Patients with Normal Alanine Aminotransferase(PNALT)ObjectiveTo determine incidence and clinical biomarkers of marked necroinflammation and fibrosis feature among chronic hepatitis B(CHB)patients with normal alanine aminotransferase(PNALT).MethodsLiver biopsy was performed on 155 CHB patients with PNALT.Necroinflammation and fibrosis were graded and staged by the Knodell histologic activity index and the Ishak fibrosis score,respectively.Correlations between the available clinical parameters and necroinflammation and fibrosis were analysed by logistic regression,and the area under the receiver operating characteristic curve(AUC)was used to analyze the accuracy of potential predictors.ResultsMarked necroinflammation and fibrosis were found in 36.5%and 15.5%of CHB patients with PNALT,respectively.Following a univariate logistic regression analysis,multiple logistic regression analysis indicated that PLT(AUROC = 0.905,cut-off value=171.5*109/ml)and GGT(AUROC = 0.0.909,Cut-off value = 21.5U/L)levels serve as independent predictors of notable liver fibrosis.ConclusionsA considerable proportion of marked histological abnormalities existed in our cohort,who will benefit from optimal therapeutic strategies administered according to predictive indication by PLT and GGT levels.Part II Identify Plasma MicroRNAs as Potential Diagnostic Biomarkers for Significant Liver Injury among CHB Patients with PNALTObjectivePlasma microRNA(miRNAs)levels may be altered during pathological processes;therefore,they may potentially serve as biomarkers for the diagnosis and prognosis of human diseases.This study aimed to explore whether plasma miRNAs may serve as new biomarkers for liver injury among chronic hepatitis B(CHB)patients with normal or nearly normal alanine aminotransferase(ALT)levels.MethodsPlasma miRNAs from each of three independent groups(no prominent liver injury and persistently normal ALT levels,NPNALT;significant liver injury with persistently normal ALT levels,SPNALT;and Healthy)were profiled by miRNAs microarray analysis.Differentially expressed miRNAs were then validated by Taqman probe quantitative reverse-transcription polymerase chain reaction(qRT-PCR)assay.The area under the receiver operating characteristic(ROC)curve(AUC)was used to analyze the candidate miRNAs validated by qRT-PCR for diagnostic accuracy.ResultsEighteen differentially expressed miRNAs were identified by microarray analysis.Eleven miRNAs with elevated serum levels were validated by qRT-PCR analysis,and two of them were significantly different among the three groups at enlarged sample validation eventually.The AUC of has-miR-122-5P and has-miR-151-3P were 0.901(95%CI,0.825-0.982;sensitivity = 80%,specificity = 87%)and 0.844(95%CI,0.745-0.943;sensitivity = 76.7%,specificity = 87.3%),indicating liver injury,respectively.ConclusionsPlasma has-miR-122-5P and has-miR-151-3P may function as new biomarkers for SPNALT patients.With these two biomarkers,we may be able to identify a subset of patients who are experiencing liver injury but have normal ALT levels for further evaluation with a biopsy procedure.Part ? Existence of the Differentially Expressed MiRNAs for SPNALT in Insupernatant of Human Hepatic Stellate Cells.ObjectiveTo observe existence of the differentially expressed microRNAs for SPNALT in human hepatic stellate cells insupernatant,and then discusses the feasibility of plasma miRNAs as molecular diagnostic markers for SPNALT.MethodsExtracted supernatant of human hepatic stellate cells(HSC-LX2)by low speed rotation and high speed centrifugation;detect differentially expressed miRNAs of SPNALT were quantified by Taqman probe quantitative reverse-transcription polymerase chain reaction(qRT-PCR)assay among centrifugal exosomes after precipitation,not centrifugal supernatant and centrifugal supernatant three groups;using ANOVA and SNK statistic methods to analyze the data.ResultsMiR-122-5p,miR-140-3p,miR-151a-3p and miR-320d in HSC cells were quantified by Taqman real-time PCR,the values of ACt,all higher in the exosome,were 5.23±0.8,4.28± 1.11,6.62±0.78 and 0.78±0.87,respectively,the results had statistically significant(P<0.05).ConclusionMiR-122-5p,miR-140-3p,miR-151a-3p and miR-320d all exists in exosome of HSC-LX2 supernatant,thus miR-122-5p and miR-151a-3p might be the molecular diagnostic markers for SPNALT.