microRNA-146在肝移植排斥反应中的调控作用及其机制

doi:10.3877/cma.j.issn.2095-3232.2018.04.018

目的

探讨microRNA（miR）-146在肝移植排斥反应中的调控作用及其机制。

方法

采用"二袖套法"建立两组原位肝移植模型，同种异体移植模型组（实验组）以Lewis大鼠为供体，BN大鼠为受体；同系移植模型组（对照组）以Lewis大鼠为供体，Lewis大鼠为受体，每组3只大鼠。移植术后7 d取肝脏组织进行病理组织学检测以及microRNA高通量测序；利用生物信息学网站对microRNA靶基因进行GO以及KEGG信号通路分析。

结果

病理学检测显示实验组肝脏组织排斥活动指数（RAI）为8.4±0.5，诊断为重度急性排斥反应；对照组RAI为1.5±0.5，诊断为无排斥反应。高通量测序显示可能与排斥反应高度相关的22种microRNA，其中21种microRNA在实验组表达均较对照组上调。21种上调的microRNA中有2种microRNA属于miR-146家族的成员，分别是miR-146a-5P和miR-146b-5P，其在实验组的表达量分别是对照组的1.1倍和2.3倍。GO和KEGG信号通路分析显示，miR-146a-5P和miR-146b-5P通过靶基因中白介素-1受体相关激酶1(IRAK1)、活化T细胞核因子5（NFAT5）和肿瘤坏死因子受体相关蛋白6（TRAF6）参与多条和免疫相关的信号通路。

结论

miR-146a-5P和miR-146b-5P的上调与同种异体肝移植大鼠排斥反应发生相关，其机制可能是通过调控IRAK1、NFAT5和TRAF6的表达介导多条免疫相关信号通路而发挥作用。

关键词: 微RNAs, 肝移植, 移植物排斥, miR-146

Objective

To investigate the regulatory effect and mechanism of microRNA-146 (miR-146) in graft rejection after liver transplantation (LT).

Methods

Orthotopic LT models of two groups were established using two-cuff technique. In the allotransplantation model group (experimental group), Lewis rats were used as the donors and BN rats as the recipients. In the isotransplantation model group (control group), Lewis rats were used as both the donors and recipients, with 3 rats in each group. At 7 d after LT, liver tissues were collected for histopathological examination and microRNA high-throughput sequencing. The GO and KEGG signaling pathways of miR-146 target genes were analyzed using bioinformatics website.

Results

Histopathological examination revealed that the rejection activity index (RAI) of liver tissues in the experimental group was 8.4±0.5 and the rats were diagnosed with severe acute rejection. In the control group, the RAI was 1.5±0.5 and the rats were diagnosed with no graft rejection. High-throughput sequencing showed that among 22 microRNAs which were probably closely associated with graft rejection, the expression levels of 21 were significantly up-regulated in the experimental group compared with those in the control group. Two of the 21 up-regulated microRNAs, miR-146a-5P and miR-146b-5P, were members of miR-146 family, and their expression levels were respectively 1.1 and 2.3 times of those in the control group. GO and KEGG signaling pathway analysis revealed that miR-146a-5P and miR-146b-5P participated in multiple immune-related signaling pathways via the target genes interleukin-1 receptor-associated kinase 1 (IRAK1), nuclear factor of activated T-cells 5 (NFAT5) and tumor necrosis factor receptor-associated protein family 6 (TRAF6).

Conclusions

The up-regulated expression levels of miR-146a-5P and miR-146b-5P are correlated with the incidence of graft rejection in rat with allogeneic LT. The mechanism is probably mediating multiple immune-related signaling pathways through regulating the expression of IRAK1, NFAT5 and TRAF6.

Key words: MicroRNAs, Liver transplantation, Graft rejection, miR-146

图1 实验组和对照组大鼠肝移植术后肝脏组织病理学变化情况（HE染色×50）

图2 实验组和对照组大鼠肝移植术后肝脏组织中22种microRNA的聚类分析结果

表1 实验组与对照组大鼠肝移植术后肝脏组织中差异表达的microRNA

microRNA_ID	所属家族	上调/下调	log2(倍数变化)
rno-let-7i-5p	let-7家族	上调	1.1
rno-miR-10b-5p	miR-10家族	下调	-1.2
rno-miR-128-3p	miR-128家族	上调	1.1
rno-miR-142-3p	miR-142家族	上调	2.4
rno-miR-142-5p	miR-142家族	上调	2.3
rno-miR-146a-5p	miR-146家族	上调	1.1
rno-miR-146b-5p	miR-146家族	上调	2.3
rno-miR-150-5p	miR-150家族	上调	1.9
rno-miR-155-5p	miR-155家族	上调	4.0
rno-miR-17-5p	miR-17家族	上调	1.5
rno-miR-106b-3p	miR-17家族	上调	1.2
rno-miR-181a-5p	miR-181家族	上调	1.2
rno-miR-181b-5p	miR-181家族	上调	1.8
rno-miR-221-3p	miR-221家族	上调	1.8
rno-miR-223-3p	miR-223家族	上调	1.5
rno-miR-23a-3p	miR-23家族	上调	1.1
rno-miR-27a-3p	miR-27家族	上调	1.0
rno-miR-27a-5p	miR-27家族	上调	1.2
rno-miR-342-3p	miR-342家族	上调	1.4
rno-miR-363-3p	miR-363家族	上调	3.0
rno-miR-425-5p	miR-425家族	上调	1.2
rno-miR-7a-5p	miR-7家族	上调	1.4

表1 实验组与对照组大鼠肝移植术后肝脏组织中差异表达的microRNA

microRNA_ID	所属家族	上调/下调	log2(倍数变化)
rno-let-7i-5p	let-7家族	上调	1.1
rno-miR-10b-5p	miR-10家族	下调	-1.2
rno-miR-128-3p	miR-128家族	上调	1.1
rno-miR-142-3p	miR-142家族	上调	2.4
rno-miR-142-5p	miR-142家族	上调	2.3
rno-miR-146a-5p	miR-146家族	上调	1.1
rno-miR-146b-5p	miR-146家族	上调	2.3
rno-miR-150-5p	miR-150家族	上调	1.9
rno-miR-155-5p	miR-155家族	上调	4.0
rno-miR-17-5p	miR-17家族	上调	1.5
rno-miR-106b-3p	miR-17家族	上调	1.2
rno-miR-181a-5p	miR-181家族	上调	1.2
rno-miR-181b-5p	miR-181家族	上调	1.8
rno-miR-221-3p	miR-221家族	上调	1.8
rno-miR-223-3p	miR-223家族	上调	1.5
rno-miR-23a-3p	miR-23家族	上调	1.1
rno-miR-27a-3p	miR-27家族	上调	1.0
rno-miR-27a-5p	miR-27家族	上调	1.2
rno-miR-342-3p	miR-342家族	上调	1.4
rno-miR-363-3p	miR-363家族	上调	3.0
rno-miR-425-5p	miR-425家族	上调	1.2
rno-miR-7a-5p	miR-7家族	上调	1.4

图3 miR-146a-5P和miR-146b-5P的共同靶基因

表2 miR-146a-5P和miR-146b-5P靶基因的信号通路分析

基因	信号通路
IRAK1	NF-kβ信号通路、Toll样受体信号通路，神经营养因子信号通路
TRAF6	丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路，NF-kβ信号通路，内吞作用，Toll样受体信号通路，NOD样受体信号通路，RIG-I样受体样受体信号通路，神经营养因子信号通路
NFAT5	自然杀伤细胞介导的细胞毒性，T细胞受体信号通路，B细胞受体信号通路，Wnt信号通路，轴突导向，VEGF信号通路

表2 miR-146a-5P和miR-146b-5P靶基因的信号通路分析

基因	信号通路
IRAK1	NF-kβ信号通路、Toll样受体信号通路，神经营养因子信号通路
TRAF6	丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路，NF-kβ信号通路，内吞作用，Toll样受体信号通路，NOD样受体信号通路，RIG-I样受体样受体信号通路，神经营养因子信号通路
NFAT5	自然杀伤细胞介导的细胞毒性，T细胞受体信号通路，B细胞受体信号通路，Wnt信号通路，轴突导向，VEGF信号通路

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Regulatory effect and mechanism of microRNA-146 in graft rejection after liver transplantation

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Regulatory effect and mechanism of microRNA-146 in graft rejection after liver transplantation