hsa-miR-34a-5p 通过靶向 HNF4α 减轻肝缺血/再灌注损伤。
hsa-miR-34a-5p Ameliorates Hepatic Ischemia/Reperfusion Injury Via Targeting HNF4α.
机构信息
Department of General Surgery, Shangrao Municipal Hospital, Shangrao, Jiangxi Province, China.
Department of General Surgery, Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China.
出版信息
Turk J Gastroenterol. 2022 Jul;33(7):596-605. doi: 10.5152/tjg.2022.21169.
BACKGROUND
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.
RESULTS
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.
CONCLUSION
miR-34a-5p possibly protected the liver from I/R injury through downregulating Hepatocyte nuclear factor 4α to inhibit the JNK/P38 signaling pathway.
背景
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。
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