| Hepatitis C virus(HCV)infection is a major cause of chronic liver disease.According to World Health Orgnization(WHO),there are 185 million people infected with HCV,approximately 3% of the world’s population.In recent years,the rate of HCV infection increases in China,and 220 thousand new cases were reported in 2013,Prospective studies have shown that 80% of cases of acute hepatitis C progress to chronic infection,More than 20~30years later,these will develop complications of chronic liver disease,such as liver cirrhosis,liver failure and hepaocellular carcinoma(HCC).HCV-associated death may result in 3 fold-increased burden to HCV patients in 2025 years,lead to a major global health problem.Liver fibrosis is the key process from chronic HCV infection to liver cirrhosis and end-stage liver disease.Therefore,early diagnosis and effective therapeutic target are crucial in the management of CHC(chronic hepatitis C).Currently liver biopsy is the gold standard for fibrosis assessment,but has a number of limitations including invasiveness,sampling error,and not repeated and accepted by patients.Therefore,we need to investigate some novel non-invasive(blood)biomarkers for the assessment of fibrosis and fibrosis progression in HCV-related hepatic fibrosis.MicroRNAs(miRNAs,miRNAs/miR)are class of short non-coding RNAs,that can bind to the 3′-untranlated regions(3′UTR)in target mRNA molecules,causing translation repression or the cleavage of the target mRNAs.miRNAs play essential roles in a variety of cellular processes such as cell growth,differentiation,and cell proliferation.Growing evidence has demonstrated that certain miRNAs integrate pro-fibrogenic and pro-inflammatory signals in HSCs,thereby controlling the expression of various extracellular matrix genes.Specific blocking or promote relatedmiRNAs expression may reverse the formation of liver fibrosis.However,the role and mechanisms of miRNAs in HCV related fibrosis remain largely unknown.In the present study,we chose clinical cases and constructed cell model of HCV-related liver fibrosis to explore the role of miRNAs in HCV-related hepatic fibrosis and the target regulated gene or relative signaling pathway,and to clarify the mechanism of miRNA and its target gene on the progression of HCV fibrosis.Furthermore,we evaluated the diagnosis value of miRNAs for liver fibrosis in patients with chronic hepatitis C,which might benefit to develop a sensitive and specific biomarker for early diagnosis of stages of liver fibrosis.This will provide scientific evidence of the gene diagnosis and therapy for HCV-related hepatic fibrosis.Part 1 Screening,validation and predictiontarget genes of hepatic microRNAs profile in HCV-related liver fibrosisObjective: To establish the change of miRNAs in serum of patients with HCV-related liver fibrosis,screen and validate the expression profiles of dysregulated miRNAs in HCV-related hepatic fibrosis.Method: Six-one patients with chronic HCV infection and 20 age-and gender-matched healthy subjects,all from the Third Hospital of Hebei Medical University were included in this study.Serum was isolated from peripheral blood.Chronic HCV infection was confirmed using serology and liver biopsy.Hepatic fibrosis was evaluated and divided into mild(F<2,n=27)and moderate to severe(2≤F<4,n=34)according to METAVIR scoring system.Normal liver tissues were obtained from five cases of liver transplant donors as healthy control.Serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were measured by the Olympus biochemical autoanalyzers.Liver tissue was processed for hematoxylin and eosin(HE)staining,Masson trichromatism to observe the stage of fibrosis.The serum miRNAs profiles of HCV related fibrosis were determined using LC Sciences micro RNA.Real-time quantitative polymerase chain reaction(qRT-RCR)was performed to validate the result of microarray.And hepaticmiRNA expression was stained through in situ hybridization assay.Three bioinformatic resources were performed to predict the target genes of dysregulated miRNAs.The validation of binding site between miRNA and target genes was determined by dual-luciferase reporter gene assay.Result:1 General characteristic of the patientsA total of 20 healthy control subjects included 8 male and 12 female,the average age was 48±9.7 years old;HCV patients with mild liver fibrosis(F<2)were 27 subjects,included 11 male and 16 female,the average age was47.44±11.8 years old;and HCV patients with moderate to severe liver fibrosis(2≤F<4)were 34 subjects,included 15 male and 19 female,the average age was 52.40±7.8 years old.There was no significant difference in age between the three groups(F=2.006,P =0.141).2 Serum ALT and AST level in healthy control and HCV patientsThe median of serum ALT level from healthy control group,HCV patients(F<2)group and HCV patients(2≤F<4)group were(25.5,48,31)U/L,respectively.The median of serum AST level from healthy control group,HCV patients(F<2)group and HCV patients(2≤F<4)group were(23.5,38,44)U/L,respectively.There were significant difference in ALT and AST between the three groups(Z=6.262,P=0.044;Z=29.511,P=0.000).Furthermore,post-hoc comparison showed that serum ALT in HCV patients(F<2)group and HCV patients(2≤F<4)group was higher than that in healthy control group(P=0.032,P=0.005).Serum AST in HCV patients(F<2)group and HCV patients(2≤F<4)group was significantly higher than that in healthy control group(P=0.000,respectively).3 Liver histolopathological featuresThe healthy controls showed normal liver histology.The liver sections of HCV patients(F<2)group showed mild ballooning of hepatocyte,no or mild proliferation of fibrous tissue in portal area.While HCV patients(2≤F<4)exhibited moderate to severe steatosis,perivenular fibrosis as well as septum formation progressing to bridging fibrosis.4 The results of microRNA ArrayThe microarray showed that there were 41 deregulated miRNAs in serum of HCV-related fibrosis,in which 30 miRNAs were upregulated in HCV-related fibrosis including has-miR-7977,has-miR-1273g-3p;11miRNAs downregulated including has-miR-191-5p,has-miR-4289.5 The validation of serum miRNA and the correlation with stages of liver fibrosisUsing qRT-RCR analysis,we revealed that the serum level of miR-1273g-3p in HCV patients was significantly increased compared with healthy control subjects(P<0.05),and the increased serum levels of miR-1273g-3p were closely correlated with the stage of liver fibrosis(r=0.601,P=0.000).In addition,the expression of miR-1273g-3p in hepatocytes cytoplasm was significantly increased with the progression of the liver fibrosis.6 Prediction of miR-1273g-3p target genes and dual-luciferase reporter gene assayThree bioinformatic resources were performed to predict the target genes of miR-1273g-3p.Among the predicted targets of miR-1273g-3p,the phosphatase and tensin homolog deleted on chromosome 10(PTEN)was predicted as potential target genes of miR-1273g-3p.We performed luciferase reporter assays the interaction between miR-1273g-3p and 3′UTR of PTEN mRNA.Conclusion:1 Serum miRNAs in patients with HCV-relaterd fibrosis were significantly changed,suggesting that miRNA might involve in the development of HCV-induced fibrosis.2 Serum miR-1273g-3p level was significantly increased in HCV patients,and further increased with development of moderate to severe liver fibrosis,and was positively correlated with the stage of liver fibrosis.3 miR-1273g-3p may directly bind to the predicated sites of the PTEN3′UTR,suggesting the interaction between miR-1273g-3p and the 3′UTR ofPTEN mRNA.Part 2 miR-1273g-3p and HSC activation in vivoObjective: To observe the change of miR-1273g-3p in activated hepatic stellate cells(HSC)in vivo and clarify the role of miR-1273g-3p on the activation of HSC.Method: HepG2 cell was transfected by pcDNA3.1-HCV core plasmid,and HepG2-Core cell line which could stably expression HCV core was screened and validated.The mRNA and protein expressions of HCV core were detected by q RT-RCR and Western blot assay.The TGF-β1 level in cell culture supernatants was measured by enzyme linked immunosorbent assay(ELISA).Human hepatic stellate cell LX-2 was treated with cell culture supernatants from HepG2-Core cell,and the expression of miR-1273g-3p and PTEN were detected,respectively.The expression of α-SMA,Col1A1 and TGF-β1 were detected by q RT-RCR,Western blot assay and fluorescent immunocytochemistry(IFC),respectively.Cell counting Kit 8(CCK-8)was used to detect cell proliferation.The LX-2 cell apoptosis was determined by flow cytometry.Result:1 Establish a HepG2-Core cell lineHepG2-Core cells were confirmed to express the mRNA and protein of HCV core,and the TGF-β1 level in cell culture supernatants from HepG2-Core cell was significantly increased compared to HepG2-NC cell.2 Cell culture supernatants from HepG2-Core cell promoted the activation of HSC and suppressed the apoptosis of HSCCell culture supernatants as conditioned media from HepG2-Core induced significant increased expression of α-SMA,Col1A1 and TGF-β1 at mRNA and protein levels in LX-2 cells.This promoted the activation,proliferation of HSC,and inhibited the apoptosis of HSC.3 miR-1273g-3p was upregulated in activated HSC,and the target gene of PTEN was downregulatedmiR-1273g-3p was significantly upregulated in LX-2 cell treated with thecell culture supernatants of HepG2-Core cells,and the target gene PTEN was downregulated with the activation of HSC.Conclusion:1 Cell culture supernatants as conditioned media from HepG2-Core cells which could stably express HCV core protein,and promote the activation,proliferantion of HSCs,and inhibite HSC apoptosis.2 The target gene PTEN was downregulated while miR-1273g-3p was upregulated in activated HSCs.Part 3 The effects and mechanisms of miR-1273g-3p and PTEN on the function of hepatic stellate cellsObjective: To investigate the effects and mechanisms of miR-1273g-3p mimic/inhibitor or overexpression PTNE on the function of HSCs.Method: 50μM miR-1273g-3p mimic and 100 μM inhibitor were transfected to LX-2 cell lines.Overexpress of PTEN and control were transfected to LX-2 cells;CCK-8 was used to measure cell ability,and qRT-RCR and Western blot were performed to evaluate the mRNA and protein expression of miR-1273g-3p,target genes PTEN and fibrogenics genes.Result:1 Effect of overexpression or inhibition of miR-1237g-3p on the activation,proliferation and apoptosis of LX-2 cellsInhibitor of miR-1273g-3p suppressed the mRNA and protein expression of α-SMA,Col1A1 and TGF-β1.CCK-8 showed that inhibition miR-1273g-3p led to reduce activation of LX-2.The rate of apoptosis of LX-2was increased(P<0.01)after inhibitor of miR-1273g-3p by flow cytometry.Overexpression of miR-1273g-3p showed the contrary changes.2 Inhibition of miR-1273g-3p increased the gene expression of PTEN signaling downstream and fibrogenesismiR-1273g-3p inhibition could reduce the expression of miR-1273g-3p,and increase the protein expression of PTEN,but mRNA not change.The downregulation of miR-1273g-3p caused a significant reduction in p-AKT protein and AKT mRNA.We also demonstrated the downregulation of BCL-2,and the simultaneous upregulation of several proapoptotic factors BAD,BAX,caspase 9/3 and PARP in LX-2 cells transfected with the miR-1273g-3p inhibitor compared with their expression in cells transfected with the negative control.Transfection of the miR-1273g-3p mimic caused the opposite changes.3 Overexpression of PTEN inhibited the expression of downstream genes in the PTEN/AKT signaling pathway and fibrogenesisPTEN overexpression could significantly increase the expression of PTEN,and reduce the expression of p-AKT mRNA,protein and those of the antiapoptosis protein BCL-2,and elevated the expression of the proapoptosis genes BAD,BAX,caspase 9/3 and PARP.The overexpression of PTEN also significantly suppressed the expression of the fibrotic gens α-SMA,Col1A1,TGF-β1 and caused a significant increase in the percentage of apoptotic cells compared with that in the control group.Conclusion:1 The inhibition of miR-1273g-3p significantly decreased the activation proliferation,and promoted the apopotsis of HSC.2 Inhibition of miR-1273g-3p suppressed PTEN expression and negatively regulated the AKT pathway in LX-2 cells.3 Overexpression of PTEN inhibited the activation,proliferation of HSC,and promoted the apopotsis of HSC.Part 4 Evaluation of microRNA-1273g-3p in diagnosis of liver fibrosis inpatients with chronic hepatitis CObjective: The relation and diagnostic performance of measuring miR-1273g-3p was compared with LSM,APRI and FIB-4,and the value of using miR-1273g-3p to predict the fibrosis stage in CHC patients was also determined.Method:112 Patients with CHC infection underwent liver biopsy,and venous blood samples were obtained to test biochemical assays,the platelet count and underwentliver stiffness measurement(LSM)using FibroTouch within 1 week before or after the liver biopsy.Hepatic fibrosis was evaluatedand divided into mild(F<2,n=29),moderate to severe((2≤F<4,n=47)and(F=4,26)according to METAVIR scoring system.Blood platelet counts(PLT)were analyzed by an automated haematology analyser,Serum ALT,AST,Total bilirubin(TBIL)were masured using an enzymatic method with an automatic chemical analyzer.AST-to-PLT ratio index(APRI)and fibrosis index based on the 4 factor(FIB-4)were calculated.Correlations between variables were calculated using Spearman rank order correlations and the diagnostic performance of miR-1273g-3p,LSM,APRI,FIB-4 were evaluated by receiver operating characteristic(ROC)curves.Results:1 General characteristic of the patientsA total of 112 CHC patients,HCV patients with mild liver fibrosis(F<2)were 39 subjects,included 17 male and 22 female,the average age was43±12.42 years old;HCV patients with moderate to severe liver fibrosis(2≤F<4)were 47 subjects,included 22 male and 25 female,the average age was 52.29±9.54 years old.And HCV patients with cirrhosis(F=4)were 26 subjects included 17 male and 9 case female,the average age was 55.19±10.18.There was significant difference in age between the three groups(χ2=12.294,P=0.000).Furthermore,post-hoc comparison showed that age in HCV patients(F=4)group and HCV patients(2≤F<4)group was no significant(P=0.274),but higher than that in(F<2)group(P=0.000,respectively).2 Biochemical assaysThe median of serum ALT level from HCV patients(F<2)group and HCV patients(2≤F<4)group and HCV patients(F=4)group were(31.5,33,38.4)U/L,respectively.The median of serum AST level from HCV patients(F<2)group,HCV patients(2≤F<4)group patients and HCV patients(F=4)group were(34,39,48.5)U/L,respectively.There was no significant difference in ALT and AST between the three groups(χ2=1.604,P=0.199;χ2=1.175,P=0.245,respectively).The median of serumTBIL level from HCV patients(F<2)group HCV patients(2≤F<4)group and HCV patients(F=4)group were(12.7,13.9,17.31)μmol/L,respectively.There was significantdifference in TBIL between the three groups(χ2=5.603,P=0.035).Serum TBIL in HCV patients(F=4)group was higher than(F<2)group and HCV patients(2≤F<4)group(P=0.002,P=0.004).The median of PLT level from HCV patients(F<2)group HCV patients(2≤F<4)and HCV patients(F=4)group were 183×109/L,149.6×109/L,125×109/L,respectively.There was significant difference in PLT between the three groups(χ2=13.518,P=0.000).Serum PLT in HCV patients(F=4)group and HCV patients(2≤F<4)group were lower than(F<2)group(P=0.001,P=0.000),Serum PLT in HCV patients(F=4)group was lower than HCV patients(2≤F<4,P=0.021).3 Correlations of non-invasive biomarkers with histological findingsReferring to the histologic fibrosis stage on liver biopsy,the Spearman’s correlation coefficient indicated that serum miR-1273g-3p levels gradually increased with increased fibrosis stage(r=0.657).The other three non-invasive diagnostic methods,LSM,APRI and FIB-4 had good consistency with liver biopsy results,and their correlation coefficients with fibrosis staging were0.815,0.417 and 0.522,respectively.4 Performance of miR-1273g-3p,LSM,APRI and FIB-4 in fibrosis stage assessmentThe areas under the ROC curves of miR-1273g-3p for F≥2 and F=4 stage samples were 0.841 and 0.933,respectively,which were lower than LSM(0.890,0.937),and higher than FIB-4(0.791,0.766)and APRI(0.719,0.760).Spearman analysis demonstrated that serum miR-1273g-3p levels were positively correlated with age,BMI,ALT,AST and TBIL(r=0.396,0.219,0.215,0.228 and 0.225,respectively),and negatively correlated with PLT(r=-0.318).Conclusion:1 The levels of miR-1273g-3p were increasing with the progression of HCV related liver fibrosis.2 miR-1273g-3p might serve as a biomarker for diagnosis of moderate to severe fibrosis and early cirrhosis. |
PDF Full Text Request