摘要
目的探讨过表达miRNA-29b-3p的外泌体通过DNA甲基化调控细胞上皮‑间充质转化,继而参与胆道闭锁(biliary atresia,BA)肝纤维化的发病机制。方法收集2018年12月至2021年12月就诊于合肥医科大学附属医院的30例4月龄以内的BA患儿和30例年龄匹配的非BA先天性胆总管囊肿(congenital choledochus cyst,CCC)患儿的外周血及病理检查后剩余肝组织,实时荧光定量PCR、蛋白质印迹法(Western blotting,WB)检测BA患儿和CCC患儿外周血和肝组织中miRNA-29b-3p表达和DNA甲基化水平。使用慢病毒感染人白血病细胞系(Jurkat细胞),获得miRNA-29b-3p模拟物以及阴性空载对照的稳转株。将感染后未经处理的细胞作为空白对照组,过表达miRNA-29b-3p慢病毒与细胞共培养后作为miR‑29b‑3p外泌体组,正常外泌体与细胞共培养后作为外泌体组,空载对照慢病毒与细胞共培养后作为阴性外泌体组,使用5‑氮杂胞苷(5‑azacytidine,5‑AzaC)抑制后的细胞作为5‑AzaC组。将每组细胞的外泌体分别与人正常肝细胞系(L02细胞)共培养24 h。通过双荧光素酶报告系统鉴定miRNA‑29b‑3p的靶基因。检测各组L02细胞中miRNA‑29b‑3p、DNA甲基化酶(DNA methylase,DNMT)3a、DNMT3b、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved cysteinyl aspartate specific proteinase 3,cleaved caspase‑3)、B淋巴细胞瘤‑2相关X蛋白(B lymphocytoma‑2 related X protein,Bax)、p21、Ki‑67、增殖细胞核抗原的mRNA及蛋白质表达水平,检测各组细胞中转录因子Snail、纤维连接蛋白(fibronectin,FN)和α‑平滑肌肌动蛋白(α‑smooth muscle actin,α‑SMA)的DNA启动子区CpG岛甲基化水平。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果PCR检测发现,BA患儿外泌体中miRNA‑29b‑3p相对表达水平为7.64±0.49,明显高于CCC患儿外泌体的1.00±0.04(t=13.59,P=0.016);BA患儿肝组织中miRNA‑29b‑3p相对表达水平为5.65±0.46,明显高于CCC患儿肝组织的1.00±0.05(t=9.90,P=0.041)。与外泌体组、空白对照组、阴性外泌体组相比,miR‑29b‑3p外泌体组的miRNA‑29b‑3p表达均高于其他组,miR‑29b‑3p外泌体组的DNMT3a、DNMT3b的mRNA、蛋白质表达均低于其他组(均P<0.05)。miRNA‑29b‑3p外泌体组cleaved caspase‑3、Bax蛋白表达水平高于空白对照组、阴性外泌体组,miRNA‑29b‑3p外泌体组Ki‑67蛋白表达水平低于空白对照组、阴性外泌体组(均P<0.05)。双荧光素酶实验显示,DNMT3a、DNMT3b是miRNA‑29b‑3p的直接靶标。加入miRNA‑29b‑3p模拟物或通过5‑AzaC抑制甲基化水平后,FN、Snail、α‑SMA的蛋白水平和mRNA水平均上调(均P<0.05)。甲基化检测结果表明,在L02细胞中,miR‑29b‑3p外泌体组与空白对照组、外泌体组以及阴性外泌体组相比Snail、FN启动子区DNA甲基化水平降低(均P<0.05),α‑SMA启动子区甲基化水平升高,加入5‑AzaC后Snail、FN启动子区DNA甲基化水平进一步降低(均P<0.05)。结论BA患儿外周血外泌体中miRNA‑29b‑3p高表达,过表达miRNA‑29b‑3p的外泌体使肝细胞Snail、FN的DNA启动子区低甲基化,促使Snail、FN高表达,从而发生细胞上皮‑间充质转化,进一步导致α‑SMA表达增高,可能加速了BA纤维化的进展。
Objective To investigate the role of the overexpressed exosomal miRNA‑29b‑3p in regulating epithelial‑mesenchymal transition(EMT)via DNA methylation and its involvement in the pathogenesis of liver fibrosis in biliary atresia(BA).Methods Peripheral blood and residual liver tissues after pathological examination were collected from 30 children with BA within 4 months of age and 30 age‑matched children with non‑BA congenital choledochal cyst(CCC)in the Affiliated Hospital of Zunyi Medical University from December 2018 to December 2021.Real‑time fluorescence quantitative PCR,and Western blotting(WB)were used to detect miRNA‑29b‑3p expressions and DNA methylation levels in peripheral blood and liver tissues of children with BA and children with CCC.The human leukemia cell line Jurkat was transfected with mimic or negative lentivirus to obtain stable cell lines overexpressing exosomal miRNA‑29b‑3p or negative control,respectively.Cells induced with 5‑azacytidine(5‑AzaC)were set as the 5‑AzaC group.Exosomes from cells in each group were co‑cultured with the human normal hepatocyte cell line(L02 cells)for 24 h.The target genes of miRNA‑29b‑3p were identified by dual‑luciferase reporter assay.Protein and mRNA levels of miRNA‑29b‑3p,DNA methylases(DNMT3a,DNMT3b),activated cysteinyl aspartate‑containing protein hydrolase 3(cleaved claspase‑3),B lymphocytoma‑2 related X protein(Bax),p21,Ki‑67 and proliferating cell nuclear antigen(PCNA)were detected by WB and PCR,respectively.The methylation level of CpG islands in the DNA promoter region of transcription factors Snail,fibronectin(FN),andα‑smooth muscle actin(α‑SMA)were detected.Intragroup comparisons were performed using the independent sample t‑test.Comparisons between multiple groups were analyzed by one‑way ANOVA.Results qRT‑PCR revealed significantly higher miRNA‑29b‑3p levels in BA‑derived exosomes(7.64±0.49 vs.1.00±0.04,t=13.59,P=0.016)and liver tissues(5.65±0.46 vs.1.00±0.05,t=9.90,P=0.041)than children with CCC.Compared with the exosome group,blank control group,and negative exosome group,miRNA‑29b‑3p level was significantly higher in the miR‑29b‑3p exosome group,while the mRNA and protein levels of DNMT3a and DNMT3b were significantly lower(all P<0.05).Cleaved caspase‑3 and Bax were upregulated,while Ki‑67 was downregulated in the miR‑29b‑3p exosome group than the exosome group,blank control group,and negative exosome group(all P<0.05).Dual‑luciferase assay confirmed DNMT3a/DNMT3b were direct targets of miRNA‑29b‑3p.The mRNA and protein levels of FN,Snail,andα‑SMA were significantly elevated after transfection of miRNA‑29b‑3p mimic or treatment of 5‑AzaC(all P<0.05).Methylation analysis demonstrated significantly reduced Snail/FN promoter methylation(all P<0.05),but elevatedα‑SMA promoter methylation in miR‑29b‑3p exosome‑treated L02 cells.Treatment of 5‑AzaC further decreased Snail/FN methylation(all P<0.05).Conclusions BA infants exhibit high exosomal miRNA‑29b‑3p in the peripheral blood.Overexpressed exosomal miR‑29b‑3p induces hypomethylation in the Snail/FN promoters,upregulating their expressions to drive EMT andα‑SMA‑mediated fibrogenesis,potentially accelerating BA‑related liver fibrosis.
作者
赵肖欢
廖昱
黄露
郑泽兵
汤成艳
祝代威
杜青
龚元
刘远梅
金祝
Zhao Xiaohuan;Liao Yu;Huang Lu;Zheng Zebing;Tang Chengyan;Zhu Daiwei;Du Qing;Gong Yuan;Liu Yuanmei;Jin Zhu(Department of Pediatric Surgery,Affiliated Hospital of Zunyi Medical University,Guizhou Children's Hospital,Zunyi 563000,China)
出处
《中华小儿外科杂志》
北大核心
2025年第7期604-612,共9页
Chinese Journal of Pediatric Surgery
基金
贵州省科技计划项目[黔科合基础‑ZK(2023)一般555]
贵州省卫生健康高质量发展医学科研联合基金项目(2024GZYXKYJJXM0063)。
