HDAC2的表达水平评分与胆道闭锁肝纤维化进展及预后的关系

Relationship between HDAC2 expression score and progression and prognosis of liver fibrosis in biliary atresia

目的探究组蛋白脱乙酰酶(histone deacetylase,HDAC)2在胆道闭锁(biliary atresia,BA)患儿肝组织中表达水平、诊断价值及其与BA肝纤维化进展和预后的关系。方法回顾性分析2019年1月至2021年12月因黄疸在天津市儿童医院普外科行手术治疗的患儿44例,包括BA患儿34例(BA组)和其他胆汁淤积性肝脏疾病10例(DC组)。使用生物信息学分析HDAC中HDAC1~HDAC11在BA中的表达情况。取BA组和DC组患儿肝组织样本,通过免疫组织化学染色和实时荧光定量PCR检测HDAC2在两组患儿肝组织中表达水平,对HDAC2的表达进行评分。使用受试者操作特征(receiver operating characteristic,ROC)曲线评估HDAC2评分对BA的诊断价值。分析BA患儿HDAC2表达与一般临床资料的关系。所有BA患儿在Kasai术后均进行了随访,记录术后6个月时的黄疸清除率(jaundice clearance rate,JCR)和自体肝生存率(survival with native liver rate,SNL)。采用Cox比例风险回归、Kaplan-Meier法分析HDAC2表达与JCR和SNL的关系。结果生物信息学分析显示,与DC组相比,BA组中HDAC的mRNA表达含量丰富,其中HDAC2差异具有统计学意义(log2FC=0.61,P=0.002)。免疫组织化学染色结果显示,BA组肝组织中HDAC2表达定位于多数肝细胞和胆管上皮细胞的细胞核内,而DC组肝组织内仅少量肝细胞和胆管上皮细胞核有表达。实时荧光定量PCR结果显示,BA组肝组织中HDAC2[(15.58±2.32)比(1.42±0.42),P<0.001]、ACTA2[(2.561±0.328)比(1.150±0.196),P<0.001]和KRT19[(2.324±0.367)比(1.116±0.164),P=0.005]mRNA相对表达量高于DC组。DC组与BA组的HDAC2评分之间差异存在统计学差异[3.0(3.0~5.0)分比8.5(6.0~10.0)分,P<0.001];BA组纤维化分级Ⅰ、Ⅱ级与Ⅲ、Ⅳ级的HDAC2评分之间差异有统计学意义[5.5(5.0~8.0)分比9.0(7.0~10.0)分,P=0.005]。HDAC2评分诊断BA的ROC曲线下面积为0.925(95%CI:0.846~1.000,P<0.001)。HDAC2高表达患儿和低表达患儿的SNL差异有统计学意义(P=0.007)。多变量Cox回归分析显示,Kasai手术日龄较大(HR:3.764,95%CI:1.239~11.428,P=0.019)和HDAC2高表达(HR:4.883,95%CI:1.296~18.392,P=0.019)是BA预后不良独立危险因素。结论HDAC2的表达水平评分具有潜在诊断价值,与BA肝纤维化程度有关;同时其高表达可以提示不良预后,为BA的诊断、预后评估带来新思路。

Objective To explore the expression level of histone deacetylase 2(HDAC2)in liver tissues of children with biliary atresia(BA),to examine its diagnostic value and to elucidate its relationship with the progression and prognosis of liver fibrosis(LF).Methods From January 2019 to December 2021,retrospective analysis was performed for 44 children operated for jaundice at Tianjin Children's Hospital.They were assigned into two groups of BA(n=34)and disease control(DC,n=10).DC group was composed of controls with other cholestatic liver diseases.The expressions of histone deacetylase 1-11(HDAC1-11)were analyzed by bioinformatics.Liver tissue samples were harvested from both groups and the expression levels of HDAC2 in liver tissue were measured by immunohistochemical stain and real-time fluorescent quantitative polymerase chain reaction(PCR)for formulating an expression scale of HDAC2.The value of HDAC2 score as a diagnostic aid of BA was assessed by receiver operating characteristic curves(ROC).The relationship between HDAC2 expression and general profiles was examined.Follow-ups were conducted after Kasai procedure and jaundice clearance rate(JCR)and survival with native liver rate(SNL)recorded at Month 6 postoperatively.The relationship between HDAC2 expression and JCR/SNL was examined by COX proportional risk regression and Kaplan-Meier analysis.Results Bioinformatic analysis showed a greater mRNA expression of HDACs in BA versus DC.And significant differences existed in HDAC2(Log2FC=0.61,P=0.002).Immunohistochemical stain showed that HDAC2 expression in BA liver tissue was localized in nuclei of most hepatocytes and biliary epithelial cells while only few hepatocytes and biliary epithelial cell nuclei were expressed in DC liver tissue.PCR results indicated HDAC2 in liver tissue[(15.58±2.33)vs(1.42±0.43),P<0.001];mRNA relative expression levels of ACTA2[(2.561±0.328)vs(1.150±0.196),P<0.001]and KRT19[(2.324±0.367)vs(1.116±0.164),P=0.005]were higher in BA group than those in DC group.Inter-group statistical difference existed between HDAC2 score[3.0(3.0-5.0)vs 8.5(6.0-10.0),P<0.001);fibrosis gradeⅠ-ⅡversusⅢ-ⅣHDAC2 score[5.5(5.0-8.0)vs 9.0(7.0-10.0),P=0.005].ROC curve for diagnosing BA by HDAC2 score alone had an area under the curve of 0.925(95%CI:0.846-1.000,P<0.001).Significant difference existed in SNL between children with high HDAC2 expression and those with low expression(P=0.007).Multivariate Cox regression analysis revealed that older operative day age(HR:3.764,95%CI:1.239-11.428,P=0.019)and high HDAC2 expression(HR:4.883,95%CI:1.296-18.392,P=0.019)were independent risk factors for a poor prognosis.Conclusions The expression score of HDAC2 has some potential diagnostic value and is correlated positively with the degree of LF in BA.And its high expression implies at a poor prognosis.It brings new rationales for diagnostic and prognostic assessments of BA.