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Hereditas(Beijing) ›› 2017, Vol. 39 ›› Issue (4): 333-345.doi: 10.16288/j.yczz.16-425

• Original article • Previous Articles     Next Articles

Identification and analysis of ZFPM2 as a target gene of miR-17-92 cluster in chicken

Xiaofei Zhang1,2,3(),He Song1,2,3,Jing Liu1,2,3,Wenjian Zhang1,2,3,Xiaohong Yan1,2,3,Hui Li1,2,3,Ning Wang1,2,3()   

  1. 1. Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin 150030, China
    2. Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China
    3. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China
  • Received:2016-12-21 Revised:2017-01-19 Online:2017-04-20 Published:2017-03-20
  • Supported by:
    the National Natural Science Foundation of China(31372299);the China Agriculture Research System(CARS-42)

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Abstract:

The miR-17-92 cluster plays important roles in a variety of physiological and pathological processes in mammals. Previously, we showed that miR-17-92 cluster promotes chicken preadipocyte proliferation; however, the mechanism for its action is unknown. In order to explore the mechanism by which miR-17-92 cluster promotes chicken preadipocyte proliferation, CCK8 proliferation assay was performed to determine the effect of ZFPM2 knockdown on chicken preadipocyte proliferation. The results showed that ZFPM2 knockdown significantly promoted chicken preadipocyte proliferation (P<0.01). Consistent with the CCK8 results, the mRNA levels of cell proliferation marker genes, i.e., Cyclin D1, PCNA and Ki67, were markedly increased in the si-ZFPM2-transfected preadipocytes (P<0.01 or P<0.05). Bioinformatics analysis showed that there were two potential miRNA binding sites for the four individual members of miR-17-92 cluster in the ZFPM2 3′UTR, one for miR-17-5p and miR-20a and the other for miR-19a and miR-19b. To test whether ZFPM2 is a target for the miR-17-92 cluster, the ZFPM2 3′UTR reporter (psi-CHECK2-ZFPM2-3′UTR-WT) and its mutant reporter (psi-CHECK2-ZFPM2-3′UTR-MUT) were constructed. Reporter assays showed that overexpression of miR-17-92 cluster significantly inhibited the luciferase reporter activity of psi-CHECK2-ZFPM2-3′UTR-WT (P<0.01), as compared with control vector (empty pcDNA3.1). Transfection of miR-17-5p, miR-19a and miR-20a inhibitors increased the reporter activities of psi-CHECK2-ZFPM2-3′UTR-WT (P<0.01 or P<0.05). In contrast, transfection of miR-17-5p, miR-19a, and miR-20a inhibitors had no obvious effect on reporter activity of psi-CHECK2-ZFPM2-3′UTR-MUT. Further qRT-PCR analysis showed that miR-17-5p, miR-20a and miR-19a inhibitors significantly elevated the endogenous ZFPM2 mRNA expression (P<0.01 or P<0.05). Cotransfection of either miR-17-5p or miR-19a inhibitor and siZFPM2 showed that both inhibitors tended to reduce only slightly the promoting effect of siZFPM2 on chicken preadipocyte proliferation. Taken together, these data demonstrated that ZFPM2 is a target of miR-17-5p, miR-20a, miR-19a, and miR-19b, and that miR-17-92 cluster promotes chicken preadipocyte proliferation at least in part by targeting ZFPM2 and inhibiting its expression.

Key words: miR-17-92 cluster, ZFPM2, chicken preadipocyte, cell proliferation