Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (15): 3169-3179.doi: 10.3864/j.issn.0578-1752.2020.15.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Prediction and Verification of MicroRNAs Targeting Porcine Endoplasmic Reticulum Stress Pathway

ZHU JingJing(),ZHOU XiaoLong,WANG Han,LI XiangChen,ZHAO AYong,YANG SongBai()   

  1. 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
  • Received:2019-08-30 Accepted:2020-03-11 Online:2020-08-01 Published:2020-08-06
  • Contact: SongBai YANG E-mail:2018120012009@stu.zafu.edu.cn;sbyang@zafu.edu.cn

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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.

Key words: pig, endoplasmic reticulum stress signaling pathway, miRNA, ATF6

Table 1

Primers sequences for luciferase reporter gene vector construct"

名称 Names 引物序列 Primer sequence (5′—3′) 片段长度 Size (bp)
ATF6-m142-3′UTR F:CCGCTCGAGCCGTGGAGTGATTATGTG 413
R:ATTGCGGCCGCCTTCCTGAAACTTTACCCT
ATF6-m145-3′UTR F:CCGCTCGAGCCTGGCTTGAATCTTCCC 301
R:ATTGCGGCCGCGTTGTGAGTTCGATCCCT
ATF6-m150-3′UTR F:CCGCTCGAGTTTTGTCAGTCCTGGGTC 464
R:ATTGCGGCCGCAAGATACCTTTGTCATTC
ATF6-m199a-3′UTR F:CCGCTCGAGTCTTTCTGCCTTCTTGGT 176
R:ATTGCGGCCGCGGAAGATGTAGGGTAATGTG
PERK-m145/150-3′UTR F:CCGCTCGAGCATGGAATAGCCCACCTC 777
R:ATTGCGGCCGCTTGGGAAAGTACCGACCT
IRE1-m142/145/199-3′UTR F:CCGCTCGAGATCGAACCCGAGCCACTG 1006
R:ATTGCGGCCGCAGACGCCATCATCAATCA
IRE1-m150-3′UTR F:CCGCTCGAGAGCCAAGTGCCTTGAGCTG 256
R:ATTGCGGCCGCACAGGCTGACATCAAACAGGA
GRP78-m145/199-3′UTR F:CCGCTCGAGACTGCTCTGCTAGTGTTG 349
R:ATTGCGGCCGCACCAGTGTAAATAACAAA
XBP1-m142/145/150/199-3′UTR F:CCGCTCGAGCTGCTTTCAACCAGCCACT 596
R:ATTGCGGCCGCCCCTCAGGTAGGCATTCT

Table 2

The sequences of qRT-PCR primers"

名称 Names 引物序列 Primer sequence (5′—3′) 退火温度 Annealing temperature (℃) 片段长度 Size (bp)
ATF6 F:CTAAAGCGAGTCCTCTACG 60 241
R:GCCATGCCTGATTTCACA
XBP1 F:GGATTCTGACGGTGTTGA 60 161
R:GGAGGCTGGTAAGGAACT
PERK F:AGGAGCAAACGGAGCACG 60 187
R:GTCGGTGAGGATGAGGATGG
IRE1 F:GGACTGGCGGGAGAACAT 60 272
R:CGTGGTAGTAGGGCTGGAA
GAPDH F:GGACTCATGACCACGGTCCAT 60 220
R:TCAGATCCACAACCGACACGT

Fig. 1

Volcano plot of miRNA-sequencing data from PRV infected PK15 cells"

Fig. 2

The intersection prediction of miRNAs target ATF6, IRE1, PERK, GRP78 and XBP1"

Fig. 3

Identification of luciferase reporter gene vector by XhoⅠ and NotⅠ double digestion"

Fig. 4

Influence of miR-142-5p mimics, miR-145-5p mimics, miR-150 mimics and miR-199a-5p mimics on luciferase expression of recombinant vector"

Fig. 5

The expression of ATF6, IRE1, PERK and XBP1 mRNA after over-expression miRNAs"

Fig. 6

Western blotting analysis of ATF6 in PK15 cells transfected with miR-142-5p mimics A. shows the Western blotting analysis of ATF6 protein in PK15 cells transfected with miR-142-5p mimics; B. shows the expression level of ATF6 protein detected by Western blotting after miR-142-5p mimics transfecting into PK15 cells"

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