猪流行性腹泻病毒通过miR-133c-3p/BCL2L2轴调控细胞凋亡

doi:10.11843/j.issn.0366-6964.2021.08.016

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (8): 2233-2243.doi: 10.11843/j.issn.0366-6964.2021.08.016

猪流行性腹泻病毒通过miR-133c-3p/BCL2L2轴调控细胞凋亡

郑红青¹, 吴旭锦^1*, 朱小甫¹, 尹宝英¹, 高军花², 李艳芝³, 植婵萍⁴

1. 咸阳职业技术学院畜牧兽医研究所, 咸阳市动物疫病分子生物学诊断技术研究重点实验室, 咸阳 712000;
2. 邢台市农业农村局, 邢台 054001;
3. 衡水职业技术学院, 衡水 053000;
4. 广东茂名农林科技职业学院, 茂名 525000

收稿日期:2021-02-04 出版日期:2021-08-23 发布日期:2021-08-21
通讯作者: 吴旭锦,主要从事病毒感染调控机制研究,E-mail:yaoyuanwxj@163.com
作者简介:郑红青(1983-),女,河北枣强人,博士,主要从事病毒感染调控机制研究,E-mail:huagaiyuxiang3@126.com;Tel:029-33680109
基金资助:
咸阳职业技术学院科研基金重大项目（2020KJA01）；咸阳市科技计划项目（2019k02-64）；陕西省教育厅专项科学研究计划项目（18JK1208）；陕西科技计划一般青年项目（2021JQ-900）；咸阳职业技术学院博士启动基金（2021BK04）；陕西省科技计划项目（2021NY-037）

Porcine Epidemic Diarrhea Virus Regulates Cell Apoptosis by miR-133c-3p/BCL2L2 Axis

ZHENG Hongqing¹, WU Xujin^1*, ZHU Xiaofu¹, YIN Baoying¹, GAO Junhua², LI Yanzhi³, ZHI Chanping⁴

1. Key Laboratory of Animal Epidemic Disease Diagnostic Laboratory of Molecular Biology in Xianyang City, Institute of Animal Husbandry and Veterinary Medicine, Xianyang Vocational Technical College, Xianyang 712000, China;
2. Xingtai Agricultural Information Center, Xingtai 054001, China;
3. Hengshui College of Vocational Technology, Hengshui 053000, China;
4. Guangdong Maoming Agriculture & Forestry Technical College, Maoming 525000, China

Received:2021-02-04 Online:2021-08-23 Published:2021-08-21

摘要/Abstract

摘要： 旨在探讨miR-133c-3p在猪流行腹泻病毒（PEDV）引起的细胞凋亡过程中所起的作用，并探讨其发挥作用的机制。以PEDV感染MARC-145细胞为模型，检测PEDV感染过程中细胞的凋亡情况以及6个与凋亡相关microRNAs的表达差异，RT-qPCR检测PEDV感染过程中与凋亡相关microRNAs的表达差异，合成miR-133c-3p的模拟物和抑制剂，转染MARC-145细胞，采用流式细胞术检测PEDV感染MARC-145细胞的凋亡情况，流式细胞术检测过表达和敲低miR-133c-3p后细胞的凋亡情况，采用生物信息学方法预测miR-133c-3p的靶基因，荧光素酶报告基因检测了miR-133c-3p和靶基因的结合情况，Western blot检测了过表达miR-133c-3p对BCL-w和PEDV蛋白表达水平的调控，用siRNA敲低BCL-w蛋白检测细胞凋亡情况。结果显示，PEDV的感染可以诱导MARC-145细胞凋亡（P<0.05），与凋亡相关的microRNAs，如miR-133c-3p和miR-149-5p的表达上调（P<0.05；P<0.001），miR-138-3p的表达下调（P<0.05）。其中，miR-133c-3p在病毒感染后升高了约5倍（P<0.01）。过表达miR-133c-3p后，细胞凋亡率显著升高（P<0.01）；敲低miR-133c-3p后，细胞凋亡率降低（P<0.05）。用生物信息学方法预测miR-133c-3p与BCL2L2的3'UTR区域有结合位点，荧光素酶报告基因试验显示miR-133c-3p可以和BCL2L2基因靶向结合（P<0.01），过表达miR-133c-3p后细胞内BCL-w蛋白的表达明显下调，且病毒的感染可以降低BCL-w的表达水平，敲低BCL-w后细胞凋亡率显著升高（P<0.01）。PEDV感染后可以通过上调miR-133c-3p的表达来下调BCL2L2的表达，从而促进细胞的凋亡。

关键词: 猪流行性腹泻病毒, 微小RNA133c-3p, BCL2样蛋白, 凋亡

Abstract: The purpose of this study was to investigate the role of miR-133c-3p in the process of cell apoptosis caused by porcine epidemic diarrhea virus (PEDV) infection, and to explore its mechanism. In this study, PEDV-infected MARC-145 cells were used as a model to detect the expression differences of 6 apoptosis-related microRNAs during PEDV infection. The expression levels of 6 apoptosis-related microRNAs were measured by RT-qPCR during PEDV infection. The apoptosis of PEDV-infected, miR-133c-3p transfected MARC-145 cells was detected by flow cytometry. The effect of miR-133c-3p on cell apoptosis was determined by flow cytometry. The target gene of miR-133c-3p was predicted by bioinformatics method. The binding of miR-133c-3p and the 3'UTR of target gene was determined by the luciferase reporter genet. The expression levels of BCL-w and PEDV protein were determined by Western blot when miR-133c-3p was over-expressed. The cell apoptosis rate was determined by flow cytometry when knock-down of BCL-w. The results showed that PEDV infection could induce apoptosis of MARC-145 cells, and the expression of microRNAs related to apoptosis, such as miR-133c-3p and miR-149-5p, were upregulated (P<0.05 or P<0.001), the expression of miR-138-3p was down-regulated (P<0.05). The apoptosis-related microRNAs miR-133c-3p and miR-149-5p were up-regulated (P<0.05 or P<0.001), and miR-138-3p was down-regulated (P<0.05). Among these, the expression of miR-133c-3p was upregulated almost 5 folds (P<0.01). The cell apoptosis rate was significantly increased after overexpression of miR-133c-3p (P<0.01) and the cell apoptosis rate was reduced after knock-down of miR-133c-3p (P<0.05). Bioinformatics methods were used to predict the binding sites between the miR-133c-3p and the 3'UTR of BCL2L2 gene. The result of luciferase reporter gene experiment showed that miR-133c-3p could bind to 3'UTR of BCL2L2 gene (P<0.01). The expression of BCL-w in cells was significantly down-regulated after over-expression of miR-133c-3p (P<0.01). PEDV could inhibit the expression level of BCL-w. Knock-down of BCL-w could induce cell apoptosis. PEDV infection could down-regulate the expression of BCL-w by up-regulating the expression level of miR-133c-3p, thereby promoting cell apoptosis.

Key words: porcine epidemic diarrhea virus, miR-133c-3p, BCL2L2, apoptosis

中图分类号:

S852.659.6

郑红青, 吴旭锦, 朱小甫, 尹宝英, 高军花, 李艳芝, 植婵萍. 猪流行性腹泻病毒通过miR-133c-3p/BCL2L2轴调控细胞凋亡[J]. 畜牧兽医学报, 2021, 52(8): 2233-2243.

ZHENG Hongqing, WU Xujin, ZHU Xiaofu, YIN Baoying, GAO Junhua, LI Yanzhi, ZHI Chanping. Porcine Epidemic Diarrhea Virus Regulates Cell Apoptosis by miR-133c-3p/BCL2L2 Axis[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(8): 2233-2243.

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猪流行性腹泻病毒通过miR-133c-3p/BCL2L2轴调控细胞凋亡

Porcine Epidemic Diarrhea Virus Regulates Cell Apoptosis by miR-133c-3p/BCL2L2 Axis

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