Expression And Clinical Significance Of RIPK3 MRNA In Acute-on-Chronic Hepatitis B Liver Failure And Hepatocellular Carcinoma

Part Ⅰ Expression of RIPK3 mRNA in Patients with Acute-on-Chronic Hepatitis B Liver Failure and Its Association with Poor PrognosisBackgroundAcute-on-chronic liver failure(ACLF)refers to a rapid and acute deterioration of liver function in patients with chronic liver diseases.In Asian,hepatitis B virus(HBV)infection is the main cause of ACLF and acute-on-chronic hepatitis B liver failure(ACHBLF)accounts for more than 70%of ACLF.ACHBLF usually progress rapidly and result in multiple organ failure within 4 weeks.Previous studies found that the short-and medium-term mortality of ACHBLF was about 50-90%,which resulted in the death of more than 120,000 patients every year.Although liver transplantation remains the only effective therapy for patients with ACHBLF,it cannot be widely applied because of the shortage of liver donors.Consequently,new prognostic markers are urgently needed for mortality prediction and severity discrimination in patients with ACHBLF.Necroptosis played an important role in viral defense and inflammatory diseases.Meanwhile,necroptosis was also found to be involved in the pathogenesis and progression of severe liver diseases.In the process of necroptosis,receptor-interacting protein kinase 3(RIPK3)plays an important role.ObjectiveIn this study,we detected RIPK3 mRNA level in peripheral blood mononuclear cells(PBMCs)of patients with acute-on-chronic hepatitis B liver failure and collected various clinical and prognostic indicators.The relationship between mRNA levels and the severity and prognosis of patients with acute-on-chronic hepatitis B liver failure was studied.This study might help to clarify the role of necroptosis in acute-on-chronic hepatitis B liver failure.MethodsOne hundred and twenty-two patients with ACHBLF,131 patients with CHB and 35 healthy controls(HCs)were retrospectively enrolled from July 2014 to November 2015 at the Department of Hepatology,Qilu Hospital of Shandong University.Using quantitative real-time polymerase chain reaction(RT-qPCR),we measured RIPK3 mRNA levels in peripheral blood mononuclear cells(PBMCs).ELISA was performed to measure serum levels of TNF-α,MLKL and caspase 8.Then,we attempted to investigate the role of necroptosis in ACHBLF and evaluate the predictive value of RIPK3 mRNA levels in PBMCs for the prognosis.Statistical analyses of the data were performed with SPSS version 19.0.Quantitative variables were expressed as median(centile 25;centile 75).Categorical variables were expressed as number(%).Mann-Whitney U-test was performed to compare the quantitative variables.Chi-square test was applied to compare the categorical data.Spearman test was applied for correlation analysis.Diagnostic value of RIPK3 mRNA and MELD score in predicting 3-month mortality for ACHBLF patients was assessed by the area under the receiver operating characteristic curve(AUC).From the receiver operating characteristic(ROC)curve coordinates,the optimal cut-off point associated with the maximum of the Youden index was determined.Youden index-based cut-off point was determined.Sensitivity,specificity,positive predictive value(PPV)and negative predictive value(NPV)was used to assess the diagnostic accuracy.Survival curves of ACHBLF patients were drawn using the Kaplan-Meier method.All statistical analyses were two-sided and P value<0.05 was considered statistically significant.Results1.RIPK3 mRNA levels in patients with ACHBLF were significantly higher than those with CHB(P<0.001)and HCs(P<0.001).Meanwhile,RIPK3 mRNA levels in patients with CHB were significantly higher than those in HCs(P<0.001),2.RIPK3 mRNA levels were positively correlated with TBIL(r=0.377,P<0.001),Cr(r=0.214,P<0.05),INR(r=0.364,P<0.001),and MELD scores(r=0.406,P<0.001),respectively.3.We compared RIPK3 mRNA levels between patients with LC and those without LC,HE or ascites.Patients with LC,HE or ascites had significantly higher RIPK3 levels than those witho-ut(P<0.05).4.We found that the TNF-α levels in patients with ACHBLF were significantly higher than those with CHB(P<0.001)and HCs(P<0.001).There was no significant difference between patients with CHB and HCs(P=0.058).The MLKL levels were significantly higher in patients with ACHBLF than those with CHB(P<0.001)and HCs(P<0.001).Meanwhile,Patients with CHB showed significantly higher MLKL levels than HCs(P<0.001).In addition,patients with ACHBLF showed decreased levels of caspase-8 than those with CHB(P<0.001)and HCs(P<0.001).However,no significant difference was found between patients with CHB and HCs(P=0.705).Importantly,RIPK3 mRNA levels in patients with ACHBLF were positively correlated with TNF-α levels(r=0.412,P<0.001)and MLKL levels(r=0.625,P<0.001),respectively.By contrast,RIPK3 mRNA levels were negatively correlated with caspase-8 levels(r=-0.279,P<0.05).5.Univariate and multivariate regression analysis were performed to investigate the association between clinicopathological parameters and the outcomes of ACHBLF patients.Univariate analysis identified that the outcomes of ACHBLF patients were significantly associated with TBIL(OR=1,007,95%CI:1.004-1.010,P<0.05),INR(OR=4.269,95%CI:2.254-8.086,P<0.05),PTA(OR=0.865,95%CI:0.811-0.922,P<0.05),ascites(OR=2.775,95%CI:1.329-5.795,P<0.05),LC(OR=2.445,95%CI:1.175-5.087,P<0.05),HE(OR=4.740,95%CI:2.065-10.877,P<0.05),RIPK3 mRNA levels(OR=1.106,95%CI:1.063-1.151,P<0.05),or MELD score(OR=1.207,95%CI:1.110-1.312,P<0.05).Then,multivariate regression analysis was performed with those variables.The RIPK3 mRNA level(OR=1.082,95%CI:1.037-1.129,P<0.05)was identified as an independent predictive factor for the outcome of ACHBLF patients.6.In this study,the 3-month mortality of ACHBLF patients was 46.7%(57/122).After 3-month follow-up,patients with ACHBLF were then divided into survivors and non-survivors.The 3-month mortality was significantly associated with ALT(P=0.002),TBIL(P<0.001),PTA(P<0.001),INR(P<0.001),LC(P=0.016),HE(P<0.001),ascites(P=0.006)or MELD scores(P<0.001).Meanwhile,The RIPK3 mRNA levels in survivors were significantly higher than those in non-survivors(P<0.001).7.We performed the receiver operating characteristic(ROC)curve to evaluate the predictive value of RIPK3 mRNA levels for predicting 3-month mortality in patients with ACHBLF.As shown in Fig.5c,the AUC of RIPK3 mRNA levels(0.810,95%CI:0.729-0.876)was higher than that of MELD scores(0.766,95%CI:0.681-0.838).With a cut-off point of 8.81,RIPK3 mRNA levels had a sensitivity of 80.7%,a specificity of 69.2%,a positive predictive value of 69.7%,and a negative predictive value of 80.4%.ConclusionIn conclusion,this study revealed that RIPK3 mRNA levels in PBMCs were significantly associated with poor prognosis of ACHBLF.It implied that necroptosis might play an important role in the pathogenesis of ACHBLF.Part Ⅱ Expression of RIPK3 mRNA in Patients withHepatocellular Carcinoma and Its Value in Early DiagnosisBackgroundHepatitis B virus(HBV)infection is a health problem worldwide.Without effective intervention,the chronic HBV infection can progress into life-threatening situations,such as liver cirrhosis and hepatocellular carcinoma(HCC).From the global perspective,HBV is a leading cause of HCC.Although exciting advances have been made in the HCC treatment and the expected survival time has been extended in the past decades,the prognosis is still poor for the patients with end-stage HCC.Therefore,strategies for accurate screening and diagnosis are crucial in the HBV-associated HCC management.Serum biomarkers are of great value in early detection of HCC,of which alpha-fetoprotein(AFP)is the most widely used.However,due to the unsatisfied sensitivity of AFP test alone,alternative serum biomarkers and combined detections are urgently needed for clinical practice.Receptor interacting protein kinase 3(RIPK3)is a key regulator of necroptosis,which is a type of regulated cell death despite apoptosis.Upon activation of caspase-8,RIPK1 and RIPK3 are cleaved and apoptosis is initiated,while caspase-8 inhibition causes the assembly of RIPK1 and RIPK3,leading to the initiation of necroptosis.Mixed lineage kinase domain-like protein(MLKL)has been considered to be a major downstream target of the necrosome,which triggers the production of reactive oxygen species(ROS)and cytotoxic influx of calcium ions.Although it is acknowledged that aberrant cell death contributes to the hepatocarcinogenesis,the necroptosis of different cell types can play varied roles in the onset and progression of HCC.Particularly,the significance of non-hepatocytes necroptosis in HCC is largely unknown.Furthermore,the clinical relevance of the key necroptosis regulator RIPK3 in HCC remains to be explored.ObjectiveIn this study,we investigated the expression of RIPK3 mRNA in the peripheral blood mononuclear cells(PBMCs)in patients with HBV-associated HCC and explored the diagnostic value of RIPK3 mRNA level for HCC.MethodsIn our study,80 patients with HBV-associated HCC,27 patients with HBV-associated LC patients,27 patients with chronic hepatitis B(CHB)and 26 healthy controls(HCs)were enrolled from October 2018 to October 2019 at the Department of Hepatology,Qilu Hospital of Shandong University.Venous peripheral blood of five milliliters was collected from each participant,using EDTA as an anticoagulant agent.PBMCs were isolated by gradient centrifugation from the blood via Ficoll-Plaque Plus(GE Healthcare,Uppsala,Sweden),followed by three times washes with phosphate-buffered saline and stored at-20℃ until use.The mRNA level of RIPK3 was determined by Quantitative real-time polymerase chain reaction(RT-PCR).Serum levels of TNF-α,MLKL and caspase-8 were determined by Enzyme-linked immunosorbent assay(ELISA).The lower detection limits of the TNF-α,MLKL and caspase-8 were 2 ng/L,10 ng/L and 0.05ng/mL,respectively.All statistical analysis of the data were performed using the IBM SPSS version 22.0(SPSS Inc.,Chicago,IL,USA).The Kolmogorov-Smirnov test was performed to determine whether data were complied with normal distribution.Quantitative variables were expressed as median and quartiles.Mann-Whitney U test was performed to compare the quantitative variables and Spearman test was applied for correlation analysis.Diagnostic performance was assessed by the receiver operating characteristic(ROC)curve.All statistical analysis was two-sided and P value<0.05 was considered statistically significant.Results1.An increasing trend of RIPK3 expression corresponding to the disease progression was observed.The RIPK3 mRNA level was significantly increased in HCC patients compared with LC patients(P=0.005),CHB patients(P<0.001)and HCs(P<0.001).Meanwhile,expression of RIPK3 was significantly higher in CHB patients than healthy controls(P<0.001),which was consistent with our previous study.2.Among the patients with HCC,the correlation between the expression of RIPK3 and clinical parameters was analyzed.The expression of RIPK3 was significantly higher in HCC patients with ascites compared to whose without ascites(P=0.021).More importantly,an increased expression of RIPK3 was observed in the HCC patients in advanced TNM stages.The mRNA level of RIPK3 of HCC patients in stage Ⅲ was significantly higher than those in stage Ⅱ(P--0.049),and HCC patients in stage Ⅳ was significantly higher than those in stage Ⅲ(P=0.002).Furthermore,Spearman correlation analysis showed that the mRNA level of RIPK3 was positively correlated with total bilirubin(TBIL)(r=0.244,P=0.029),and INR(r=0.236,P=0.035),and not significantly correlated with ALT(r=0.196,P=0.081),albumin(ALB)(r=0.196,P=0.081)and AFP(r=-0.113,P=0.347).3.The serum level of TNF-α was significantly increased in patients with HCC compared to the LC patients(P=0.006),CHB patients(P=0.001)and HCs(P<0.001).However,the serum level of TNF-α was not significantly different between LC group and CHB group(P=0.250).Correspondingly,a positive correlation between the serum level of TNF-α and the expression of RIPK3 was observed(r=0.371,P<0.001).MLKL is a major downstream effector of RIPK3.Similarly,the serum level of MLKL was significantly increased in patients with HCC compared to the LC patients(P=0.001),CHB patients(P<0.001)and HCs(P<0.001).The serum level of MLKL was not significantly different between LC group and CHB group(P=0.869).Significantly positive correlation between the serum level of MLKL and the expression of RIPK3 was observed(r=0.571,P<0.001).The presence of caspase-8 inhibits the formation of necrosome.Interestingly,the serum level of caspase-8 was significantly lower in patients with HCC compared to the LC patients(P<0.001),CHB patients(P=0.001)and HCs(P<0.001).The serum level of caspase-8 was not significantly different between LC group and CHB group(P=0.952).A negative correlation between the serum level of caspase-8 and the expression of RIPK3 was observed(r=0.571,P<0.001).4.The AUC of RIPK3 mRNA level in discriminating HCC from LC was 0.683(P=0.005,95%CI 0.573-0.794)with a sensitivity of 0.663 and a specificity of 0.593,which was comparable to AFP test(AUC 0.701,P=0.002,95%CI 0.603-0.799,sensitivity 0.650,specificity 0.778).However,the combination of RIPK3 mRNA level and AFP test greatly improved the diagnostic accuracy(AUC 0.817,P<0.001,95%CI 0.737-0.896,sensitivity 0.675,specificity 0.852).5.We evaluated the diagnostic accuracy of the RIPK3 mRNA level in discriminating HCC from CHB.The AUC of RIPK3 mRNA level was 0.746(P<0.001,95%CI 0.649-0.844)with a sensitivity of 0.763 and a specificity of 0.667,which was better than AFP test(AUC 0.636,P=0.035,95%CI 0.526-0.747,sensitivity 0.588,specificity 0.741).Similarly,the combination of RIPK3 mRNA level and AFP test improved the diagnostic accuracy(AUC 0.775,P<0.001,95%CI 0.674-0.876,sensitivity 0.663,specificity 0.815).6.We also evaluated the diagnostic accuracy of the RIPK3 mRNA level in discriminating HCC from HCs.The AUC of RIPK3 mRNA level was 0.985(P<0.001,95%CI 0.966-1.000)with a sensitivity of 0.925 and a specificity of 0.962,which was much better than AFP test(AUC 0.807,P<0.001,95%CI 0.727-0.887,sensitivity 0.675,specificity 0.962).The AUC of combination of RIPK3 mRNA level and AFP test was 0.990(P<0.001,95%CI 0.974-1.000,sensitivity 0.975,specificity 0.999).ConclusionIn conclusion,the current study demonstrates that the mRNA expression level of RIPK3 is elevated in the PBMCs of patients with HBV-associated HCC.The RIPK3 mRNA level is positively correlated with the clinical parameters and the tumor staging.And the RIPK3-related cytokines are aberrantly altered in the sera of patients with HBV-associated HCC.The combination of RIPK3 mRNA level and AFP test can improve the diagnosis of HBV-associated HCC.These findings uncovered the clinical relevance of RIPK3 expression and provide stepstones for novel diagnostic strategies development in HBV-associated HCC.