miRNA-17-5p在慢加急性肝衰竭合并乙型病毒性肝炎患者中的表达及与自噬相关蛋白表达的关系

Expression of miRNA-17-5p in patients with acute-on-chronic liver failure complicated with hepatitis B and its relationship with autophagy related protein expression

目的:探讨微小RNA-17-5p(miRNA-17-5p)在慢加急性肝衰竭合并乙型肝炎(HBV-ACLF)患者中的表达水平及其与自噬相关蛋白Beclin1、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)表达的相关性。方法:选取2019年2月至2020年5月HBV-ACLF住院患者82例为HBV-ACLF组,选取同期住院治疗的慢性乙型肝炎(CHB)患者79例作为CHB组,选取同期健康体检者86例作为对照组。采用实时荧光定量PCR(qRT-PCR)法检测血清miRNA-17-5p水平;酶联免疫吸附(ELISA)法检测血清Beclin1、LC3-Ⅱ、总胆红素、白蛋白水平;PCR结合荧光探针的体外扩增技术测定血清HBV DNA载量;Pearson法分析HBV-ACLF患者血清miRNA-17-5p与Beclin1、LC3-Ⅱ、总胆红素、白蛋白、HBV DNA载量的相关性;受试者工作特征曲线(ROC)分析血清miRNA-17-5p、Beclin1、LC3-Ⅱ水平对HBV-ACLF的诊断价值;多因素Logistic回归分析影响HBV-ACLF发生的因素。结果:HBV-ACLF组患者血清Beclin1、LC3-Ⅱ、总胆红素水平高于CHB组和对照组,miRNA-17-5p、白蛋白低于CHB组和对照组(P<0.05);CHB组患者血清Beclin1、LC3-Ⅱ、总胆红素水平高于对照组,miRNA-17-5p、白蛋白低于对照组(P<0.05);HBV-ACLF组患者血清HBV DNA载量高于CHB组(P<0.05)。HBV-ACLF组患者血清miRNA-17-5p水平与Beclin1、LC3-Ⅱ呈负相关(r=-0.580、-0.511;均P<0.05);Beclin1、LC3-Ⅱ与总胆红素、HBV DNA载量均呈正相关,与白蛋白呈负相关(P<0.05);miRNA-17-5p与总胆红素、HBV DNA载量均呈负相关,与白蛋白呈正相关(P<0.05)。血清miRNA-17-5p、Beclin1、LC3-Ⅱ水平诊断HBV-ACLF的曲线下面积(AUC)分别为0.862、0.784、0.886,特异性分别为88.4%、80.2%、81.4%,敏感度分别为74.4%、63.4%、81.7%;三者联合诊断的AUC为0.915,特异性为88.4%,敏感度为82.9%。Beclin1、HBV DNA载量是影响HBV-ACLF发生的独立危险因素(P<0.05),miRNA-17-5p是影响HBV-ACLF发生的保护因素(P<0.05)。结论:HBV ACLF患者血清miRNA-17-5p表达水平降低,与Beclin1、LC3-Ⅱ呈明显负相关,且三者均对HBV-ACLF有一定的诊断价值。

Objective:To investigate the expression level of microRNA-17-5p(miRNA-17-5p)in patients with acute-on-chronic liver failure complicated with hepatitis B(HBV-ACLF)and its correlation with autophagy related protein Beclin1 and microtubule associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ).Methods:A total of 82 patients with HBV-ACLF from February 2019 to May 2020 were selected as HBV-ACLF group,and 79 patients with chronic hepatitis B(CHB)in the same period were selected as CHB group;at the same time,86 healthy people were selected as the control group.The level of serum miRNA-17-5p was detected by real-time fluorescence quantitative PCR(qRT-PCR);the levels of serum Beclin1,LC3-Ⅱ,total bilirubin and albumin were detected by enzyme-linked immunosorbent assay(ELISA);the serum HBV DNA load was measured by PCR combined with fluorescence probe amplification in vitro;Pearson method was used to analyze the corre-lation between serum miRNA-17-5p and Beclin1,LC3-Ⅱ,total bilirubin,albumin and HBV DNA load in HBV-ACLF patients;receiver operating characteristic curve(ROC)was used to analyze the diagnostic value of serum miRNA-17-5p,Beclin1 and LC3-Ⅱlevels in HBV-ACLF;multivariate Logistic regression analysis was used to analyze the factors influencing the occurrence of HBV-ACLF.Results:The levels of serum Beclin1,LC3-Ⅱand total bilirubin in HBV-ACLF group were higher than those in CHB group and control group,while the miRNA-17-5p and albumin were lower than those in CHB group and control group(P<0.05);the levels of serum Beclin1,LC3-Ⅱand total bilirubin in CHB group were higher than those in control group,while miRNA-17-5p and albumin were lower than those in control group(P<0.05);the serum HBV DNA load in HBV-ACLF group was higher than that in CHB group(P<0.05).The serum miRNA-17-5p level of HBV-ACLF patients was negatively correlated with Beclin1,LC3-Ⅱ(r=-0.580,-0.511;P<0.05),total bilirubin and HBV DNA load;Beclin1 and LC3Ⅱwere positively correlated with total bilirubin and HBV DNA load,but negatively correlated with albumin(P<0.05);miRNA-17-5p was negatively correlated with total bilirubin and HBV DNA load,but positively correlated with albumin(P<0.05).The area under the curve(AUC)of serum miRNA-17-5p,Beclin1 and LC3-Ⅱlevels in the diagnosis of HBV-ACLF was 0.862,0.784 and 0.886,the specificity was 88.4%,80.2%,81.4%,and the sensitivity was 74.4%,63.4%and 81.7%,respectively;the AUC of the combined diagnosis was 0.915,the specificity was 88.4%,and the sensitivity was 82.9%.Beclin1 and HBV DNA load were independent risk factors of HBV-ACLF(P<0.05),and miRNA-17-5p was a protective factor(P<0.05).Conclusion:The expression level of serum miRNA-17-5p in patients with HBV-ACLF is decreased,and it is negatively correlated with Beclin1 and LC3-Ⅱ.All of them have certain diagnostic value for HBV-ACLF.