| 靶向猪内质网应激通路的microRNAs预测与验证 |
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| 引用本文： | 朱静静,周晓龙,汪涵,李向臣,赵阿勇,杨松柏. 靶向猪内质网应激通路的microRNAs预测与验证[J]. 中国农业科学, 2020, 53(15): 3169-3179. DOI: 10.3864/j.issn.0578-1752.2020.15.016 |
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| 作者姓名： | 朱静静 周晓龙 汪涵 李向臣 赵阿勇 杨松柏 |
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| 作者单位： | 浙江农林大学动物科技学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室,浙江临安 311300 |
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| 基金项目： | 浙江省自然科学基金(LY19C170002) |
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| 摘 要： | 【目的】预测并验证靶向猪内质网应激通路中关键基因的microRNAs(miRNAs),为进一步研究miRNAs对猪内质网应激信号通路调控提供理论基础。【方法】首先利用伪狂犬病毒(PRV)感染猪肾上皮(PK15)细胞,高通量测序检测差异表达的miRNAs。然后通过TargetScan预测靶向内质网应激通路关键基因ATF6、IRE1、PERK、GRP78、XBP1的miRNAs。构建含有候选miRNAs作用位点的双荧光素酶报告基因重组载体,并分别与miRNA-mimics共转染幼仓鼠肾(BHK-21)细胞,通过测定荧光素酶活性来验证内质网应激通路关键基因与候选miRNAs的靶标关系。然后在PK15细胞中分别过表达候选miRNAs,利用qRT-PCR和Western blot检测候选miRNAs对内质网应激通路关键基因mRNA和蛋白表达的影响。【结果】miRNA测序结果显示,PRV感染后共引起35条miRNAs差异表达。TargetScan预测显示,靶向ATF6、IRE1、PERK、GRP78、XBP1的交集miRNAs为miR-142-5p、miR-145-5p、miR-150和miR-1...
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| 关 键 词： | 猪 内质网应激通路 miRNA ATF6 |
| 收稿时间： | 2019-08-30 |
Prediction and Verification of MicroRNAs Targeting Porcine Endoplasmic Reticulum Stress Pathway |
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| ZHU JingJing,ZHOU XiaoLong,WANG Han,LI XiangChen,ZHAO AYong,YANG SongBai. Prediction and Verification of MicroRNAs Targeting Porcine Endoplasmic Reticulum Stress Pathway[J]. Scientia Agricultura Sinica, 2020, 53(15): 3169-3179. DOI: 10.3864/j.issn.0578-1752.2020.15.016 |
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| Authors: | ZHU JingJing ZHOU XiaoLong WANG Han LI XiangChen ZHAO AYong YANG SongBai |
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| Affiliation: | College of Animal Science and Technology, Zhejiang A&F University/Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Lin'an 311300, Zhejiang |
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| Abstract: | 【Objective】 This study was aimed to predict and validate microRNAs (miRNAs) that targeting key genes in the porcine endoplasmic reticulum (ER) stress pathway, so as to provide a theoretical basis for regulation of porcine ER stress signaling pathway by miRNAs. 【Method】 Firstly, the differential expression of miRNAs in PRV-infected PK15 cells in porcine ER were determined by high-throughput sequencing. TargetScan was used to predict miRNAs that targeting the genes ATF6, IRE1, PERK, GRP78, and XBP1 of ER stress pathways. Recombinant plasmids were constructed, including candidate miRNA target sites, and the regulation of ATF6, IRE1, PERK, GRP78, and XBP1 by candidate miRNAs was validated by co-transfecting dual-luciferase reporter gene vector construct with miRNA-mimics into BHK-21 cells. Then, the candidate miRNAs were over-expressed in PK15 cells, and then fluorescent quantitative PCR and Western blot were used to detect the effect of miRNAs on key genes expression at mRNA and protein levels. 【Result】 MiRNA sequencing results showed that 35 differential expression of miRNAs were determined in PRV-infected PK15 cells. The results of TargetScan prediction showed that the intersection of miRNAs targeting four or more genes of ATF6, IRE1, PERK, GRP78, and XBP1 were miR-142-5p, miR-145-5p, miR-150, and miR-199a-5p, and these intersections of miRNAs were selected as candidate miRNAs. The dual-luciferase reporter plasmids psiCHECK-2-ATF6-m142-3'UTR, psiCHECK- 2-ATF6-m145-3'UTR, psiCHECK-2-ATF6-m150-3'UTR, psiCHECK-2-ATF6-m199-3'UTR, psiCHECK-2-IRE1-m150-3'UTR, psiCHECK-2-IRE1-m142/145/199-3'UTR, psiCHECK-2-PERK-m145/150-3'UTR, psiCHECK-2-XBP1-m142/145/150/199- 3'UTR, psiCHECK-2-GRP78-m145/199-3'UTR were successfully constructed. The luciferase assay experiments showed that miR-142-5p mimics could significantly inhibit luciferase activity of psiCHECK-2-ATF6-m142-3'UTR dual-luciferase reporter recombinant vector. psiCHECK-2-IRE1-m142/145/199-3'UTR was co-transfected with miR-142-5p mimics, miR-145-5p mimics, and miR-199a-5p mimics, respectively. The luciferase activity of the over-expressing group were significantly lower than the negative control group. In addition, psiCHECK-2-XBP1-m142/145/150/199-3'UTR was co-transfected with miR-142-5p mimics and miR-199a-5p mimics, respectively, and the luciferase activity of the over-expressing group were also significantly lower than the negative control group. The luciferase assay experiments showed that miR-145-5p mimics could significantly inhibit luciferase activity of psiCHECK-2- PERK-m145/150-3'UTR dual-luciferase reporter recombinant vector. The results indicated that miR-142-5p, miR-145-5p and miR-199a-5p might targeted the genes ATF6, IRE1, and XBP1. Among them, miR-142-5p might target these three key genes to regulate the ER signaling pathway. Overexpression of miR-142-5p significantly down-regulated the levels of mRNA and protein by qRT-PCR and Western blot methods. The results showed that miR-142-5p might participate in the regulation of ER signaling pathway by targeting ATF6.【Conclusion】 In this study, miR-142-5p was validated to target the key gene ATF6 of the porcine ER stress pathway. Thereby, these results laid a foundation for further study of regulation of ER stress pathway through miR-142- 5p-ATF6 gene axis. |
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| Keywords: | pig endoplasmic reticulum stress signaling pathway miRNA ATF6 |
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