Department of General Surgery, Shangrao Municipal Hospital, Shangrao, Jiangxi Province, China.
Department of General Surgery, Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China.
Turk J Gastroenterol. 2022 Jul;33(7):596-605. doi: 10.5152/tjg.2022.21169.
To investigate the relationship between the expression level of hsa-miR-34a-5p and liver injury and to further explore its regulatory signaling pathways Methods: Liver tissue and blood were collected from 60 patients undergoing hepatectomy. We constructed a rat HIRI model and treated it with an intraperitoneal injection of agomir-miR-34a-5p or agomir-normal control (NC) for 7 days after the surgery. The pathological changes of agomir-miR-34a-5p or agomir-normal control (NC) groups were compared. 7702 and AML12 cells were transfected with mimics NC or miR-34a-5p mimics and then treated with H2O2 for 6 hours. Cell apoptosis was detected by flow cytometry, Western blot, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, respectively. Furthermore, the target genes of miR- 34a-5p were identified by luciferase reporter gene assay and were verified in vitro.
The relatively high miR-34a-5p expression group revealed a lower level of alanine aminotransferase and aspartate aminotrans- ferase compared with the relatively low miR-34a-5p expression group. HIRI+agomir-miR-34a-5p rats exhibited significantly higher miR-34a-5p expression, lower serum alanine aminotransferase, aspartate aminotransferase, alleviated hepatic necrosis, reduced hepa- tocyte apoptosis, and decreased expression of apoptosis-related proteins, when compared with HIRI+agomir-NC rats (P < .05). After hydrogen peroxide treatment, alpha mouse liver-12 cell (AML-12) and normal liver cell line LO2 (LO2) cells transfected with miR-34a-5p mimics had significantly lower apoptosis rate compared with miR-34a-5p mimics NC group (P < .05). Hepatocyte nuclear factor 4α was identified as a miR-34a-5p target gene. Hepatocyte nuclear factor 4α expression was significantly downregulated in AML12 and HL-7702 (7702) cells transfected with miR-34a-5p (P < .05). Moreover, AML12 and 7702 cells transfected with miR-34a-5p signifi- cantly showed higher c-Jun N-terminal kinase (JNK), P38, cleavage cas-3, and BCL2 associated X (Bax) protein levels compared with AML12 and 7702 cells transfected with agomir-NC.
miR-34a-5p possibly protected the liver from I/R injury through downregulating Hepatocyte nuclear factor 4α to inhibit the JNK/P38 signaling pathway.
为了研究 hsa-miR-34a-5p 的表达水平与肝损伤的关系，并进一步探讨其调控信号通路。方法：收集 60 例行肝切除术患者的肝组织和血液。构建大鼠 HIRI 模型，并在手术后第 7 天给予腹腔注射激动剂 miR-34a-5p 或激动剂正常对照（NC）。比较激动剂 miR-34a-5p 或激动剂正常对照（NC）组的病理变化。将 7702 和 AML12 细胞分别用对照 NC 或 miR-34a-5p 模拟物转染，然后用 H2O2 处理 6 小时。分别用流式细胞术、Western blot 和末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记法检测细胞凋亡。此外，通过荧光素酶报告基因检测鉴定 miR-34a-5p 的靶基因，并在体外进行验证。结果：相对高 miR-34a-5p 表达组的丙氨酸氨基转移酶和天冬氨酸氨基转移酶水平低于相对低 miR-34a-5p 表达组。与 HIRI+agomir-NC 大鼠相比，HIRI+agomir-miR-34a-5p 大鼠的 miR-34a-5p 表达显著升高，血清丙氨酸氨基转移酶、天冬氨酸氨基转移酶水平降低，肝坏死减轻，肝细胞凋亡减少，凋亡相关蛋白表达降低（P <.05）。经过氧化氢处理后，转染 miR-34a-5p 模拟物的 alpha 小鼠肝-12 细胞（AML-12）和正常肝细胞系 LO2（LO2）细胞的凋亡率明显低于 miR-34a-5p 模拟物 NC 组（P <.05）。肝细胞核因子 4α被鉴定为 miR-34a-5p 的靶基因。转染 miR-34a-5p 的 AML12 和 HL-7702（7702）细胞中肝细胞核因子 4α的表达明显下调（P <.05）。此外，转染 miR-34a-5p 的 AML12 和 7702 细胞中 c-Jun N-末端激酶（JNK）、P38、cleavage cas-3 和 BCL2 相关 X（Bax）蛋白水平明显高于转染 agomir-NC 的 AML12 和 7702 细胞。结论：miR-34a-5p 可能通过下调肝细胞核因子 4α 抑制 JNK/P38 信号通路来保护肝脏免受 I/R 损伤。
