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ORIGINAL ARTICLE
Year : 2018  |  Volume : 131  |  Issue : 4  |  Page : 454-460

Exosomal MicroRNA-10a Is Associated with Liver Regeneration in Rats through Downregulation of EphA4


1 Department of Liver Surgery, Liver Transplantation Division, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
2 Department of Orthopedics, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
3 Laboratory of Liver Surgery, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China

Correspondence Address:
Prof. Yong Zeng
Department of Liver Surgery, Liver Transplantation Division, West China Hospital, Sichuan University, Chengdu, Sichuan 610041
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0366-6999.225057

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Background: MicroRNAs (miRNAs) have been reported to play vital roles in liver regeneration. Previous studies mainly focused on the functions of intracellular miRNAs, while the functions of circulating exosomal miRNAs in liver regeneration remain largely unknown. The aim of this study was to identify the key exosomal miRNA that played vital roles in liver regeneration. Methods: The Sprague–Dawley male rats were assigned to 70% partially hepatectomized group (n = 6) and sham surgery group (n = 6). The peripheral blood of both groups was collected 24 h after surgery. The exosomal miRNAs were extracted, and microarray was used to find out the key miRNA implicated in liver regeneration. Adenovirus was used to overexpress the key miRNA in rats, and proliferating cell nuclear antigen (PCNA) staining was applied to study the effect of key miRNA overexpression on liver regeneration. Western blotting was used to validate the predicted target of the key miRNA. Results: Exosomal miR-10a was upregulated more than nine times in hepatectomized rats. The level of miR-10a was increased in the early phase of liver regeneration, reached the top at 72 h postsurgery, and decreased to perioperative level 168 h after surgery. Moreover, enforced expression of miR-10a by adenovirus facilitated the process of liver regeneration as evidenced by immunohistochemical staining of PCNA. Erythropoietin-producing hepatocellular receptor A4 (EphA4) has been predicted to be a target of miR-10a. The protein level of EphA4 was decreased in the early phase of liver regeneration, reached the bottom at 72 h postsurgery, and rose to perioperative level 168 h after surgery, which was negatively correlated with miR-10a, confirming that EphA4 served as a downstream target of miR-10a. Moreover, inhibition of EphA4 by rhynchophylline could promote the proliferation of hepatocytes by regulating the cell cycle. Conclusion: Exosomal miR-10a might accelerate liver regeneration through downregulation of EphA4.

 

 Abstract in Chinese

外泌体中的miR-10a可以通过下调EphA4的方式促进肝再生

摘要

背景: 大量的研究证据显示microRNAs在肝再生过程中发挥着重要的作用。 然而之前的报道大都集中在关于细胞内microRNAs功能的研究, 对于循环系统中外泌体内的microRNAs在肝再生中的作用却研究较少。 本文的目的在于找到在肝再生过程中发挥重要作用的外泌体microRNA。

方法: 以大鼠70% 肝切除术后的肝再生模型作为实验组, 以单纯开关腹的假手术模型作为对照组。 术后24小时分别抽取实验组和对照组大鼠的外周血, 并提取其中的外泌体microRNAs。 通过microRNA芯片筛选出在对照组和实验组中显著差异性表达的microRNA。于70% 肝切除术后各设定的时间点经尾静脉抽取外周血并提取外泌体, 采用qPCR明确目标miRNA在术后的变化规律。 对大鼠行70% 肝切除术后经门静脉注射目标microRNA的过表达腺病毒载体和对照载体, 于术后各设定时间点分别处死大鼠, 完整切取肝脏组织, 获得肝重/体重比; 将所得肝组织行免疫组化检测PCNA (proliferating cell nuclear antigen), 明确增殖情况; 通过生物信息学分析和文献及数据库检索等方式, 筛选出目标miRNA的下游作用基因, 确定结合位点, 并利用WB检测所得肝脏标本中该下游蛋白表达量的差异。 体外实验培养人L-02肝细胞, 利用流式细胞术进一步验证下游基因的作用。

结果: 实验结果显示, 术后24小时实验组中的miR-10a比对照组增高9倍余。 在肝再生早期, miR-10a的表达水平已经开始增高, 在术后72小时达到峰值, 在此后逐渐下降, 直到术后168小时, 恢复到了术前水平。 PCNA免疫组化染色的结果也表明, 过表达miR-10a可以促进大鼠的肝再生。 我们通过软件预测和文献检索, 确定了EphA4是miR-10a的下游靶标。 同时, 我们也发现EphA4在肝再生的早期聚在下降, 在术后72小时达到最低值, 并于术后168小时组件恢复到了术前水平与miR-10a的表达呈负相关, 进一步证实了EphA4是miR-10a的下游标靶。 除此之外, 在体外实验中, 我们也发现, 运用钩藤碱抑制EphA4的表达后可以通过调节细胞周期的方式促进肝脏细胞的增殖。

结论: 外泌体中的miR-10a可以通过下调EphA4的方式促进肝再生。



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