Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (9): 1624-1634.doi: 10.3864/j.issn.0578-1752.2019.09.013

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

CEBPα and p53 Regulate Kiss1 Gene Expression in Porcine Ovary Granulosa Cells

XIN XiaoPing1,WANG JiaYing1,ZHANG AiLing2,ZHONG YuYi1,HE YingTing1,CHEN ZanMou1,ZHANG Zhe1,ZHANG Hao1,LI JiaQi1,YUAN XiaoLong1()   

  1. 1 Guangdong Provincial Key Lab of Agro-animal Genomics and Molecular Breeding/National Engineering Research Center for Breeding Swine Industry/College of Animal Science, South China Agricultural University, Guangzhou 510642
    2 Development Center of Applied Ecology and Ecological Engineering in Universities/Biology and Food Engineering Institute, Guangdong University of Education, Guangzhou 510310
  • Received:2018-04-18 Accepted:2019-03-18 Online:2019-05-01 Published:2019-05-16
  • Contact: XiaoLong YUAN E-mail:yxl@scau.edu.cn

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Abstract:

【Objective】The objectives of this study were to predict the potential transcription factors in the upstream region of Kiss1, and then to verify the regulatory role of these transcription factors on the expression of Kiss1 in the ovarian granulosa cells of pigs. This study could provide the basic data for the molecular mechanism of Kiss1 in granulosa cells of pigs. 【Method】 By using the bioinformatic software, we predicted the potential binding site of transcription factors in the upstream region of Kiss1 in NCBI database. In addition, we reviewed a lot of relevant papers and references, and then CEBPα and p53 were selected as potential binding transcriptional factors in the upstream region of Kiss1 gene. The primers were designed near the potential binding sites in the upstream region of Kiss1 genes, and verified the binding of transcriptional factors CEBPα and p53 to the upstream region of Kiss1 gene by ChIP. According to the mRNA sequences of CEBPα and p53 in NCBI database, Primer Premier 5 software was used to design primers, and CDS regions of CEBPα (containing KpnI and XhoI sites) and p53 (containing KpnI and Hind III sites) were amplified by PCR and identified by sequencing. Then the CDS regions were connected to the eukaryotic expression vector pcDNA3.1, and the constructed plasmids were extracted with endotoxin-freely, named pcDNA3.1-CEBPα and pcDNA3.1-p53. The interfering siRNA fragments of CEBPα and p53 were synthesized through the chemical method. Pig ovaries were collected from the slaughterhouse, and the ovarian granulosa cells were isolated and cultured. The eukaryotic expression vector or siRNA fragment was transfected into ovarian granulosa cells by cationic liposome, and the effects of p53 and CEBPα on the expression of Kiss1 were verified by the real-time fluorescence quantitative PCR and Western Blotting, respectively. 【Result】 The bioinformatic prediction indicated that there were putative binding sites of p53 (tumor protein p53, p53), CEBP (CCAAT/enhancer binding protein, CEBP), Stat4 (signal transducer and activator of transcription 4, Stat4) and other potential of transcription factor binding sites in the upstream (-850 -+221) of Kiss1. And the putative binding site of CEBPα (GenBank Gene ID: 397307, XM_003127015.4) were found in -744—-733 bp region and the putative binding site of p53 (GenBank Gene ID: 397276, NM_213824.3) were found in -533—-523 bp region. The results of ChIP showed that p53 and CEBPα were bounded at -533—-523 and -744—-733 of Kiss1, respectively. After overexpression of p53 or CEBPα, both mRNA and protein expression level of Kiss1 significantly decreased (P<0.05). Furthermore, both mRNA and the expression level of Kiss1 increased significantly (P<0.05) by interfering p53 or CEBPα. 【Conclusion】 In pigs, p53 and CEBPα could bind at the upstream region of Kiss1 to inhibit its expression in ovarian granulosa cells.

Key words: pig, ovarian granulosa cells, Kiss1, CEBPα, p53

Table 1

Primer sequences"

名称 Name 序列 Sequence 片段长度 Size (bp)
CDS-p53 F: GGGGTACCATGGAGGAGTCGCAGTCCGA 1161
R: CCAAGCTTTCAGTCTGAGTCAGGTCCTT
CDS-CEBPα F：GGGGTACCAGACCAAGACTTGCCCTCCAC 1227
R：CCCTCGAGTCTTCGGGTTTTGGTATCCTCA
qRT-Kiss1 F: AGGTACCTGTGGATCCTGTC 192
R: TTCTGCCATTATCCTATGTCTAC
qRT-p53 F: CGATGGCCAGGGATCTCTTC 151
R: TCGGCCAAGTTTAAGAGCGT
qRT-CEBPα F: CTGAGGTCTGCCAGAAGC 150
R: AACAGAAGAAGGAAGGGAGT
qRT-GAPDH F: TCCCGCCAACATCAAAT 163
R: CACGCCCATCACAAACAT
ChIP-kiss1-p53 F：CTCAGAGCCCATGGAGAATAG 135
R：TGCTTGCTCTCATAACCCTTAAC
ChIP-kiss1-CEBPα F：CACTGTGCAAGTGGTCTCGG 131
R：CCCGTTAGAATGTGCCTTCC
ChIP-GAPDH F：GATGTCCTGAGCCCCTACAG 102
R：GGTAGGTGATGGGGACTGAG
p53-siRNA GGAACTTGTTCAAGCAGCT
CEBPα-siRNA TCGACATCAGCGCCTACAT

Fig. 1

Identified the eukaryotic expression vector of p53 and CEBPα A. The enzyme digestion of eukaryotic expression vector pcDNA3.1-p53; B. The enzyme digestion of eukaryotic expression vector digestion pcDNA3.1- CEBPα"

Fig. 2

Bioinformatics prediction for the potential binding site of transcription factors in the upstream region of porcine Kiss1, and ChIP results of transcription factors binding to the upstream region of porcine Kiss1 gene M：DL5 000 DNA marker,from up to bottom：5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp. IgG: Negative control; Input and H3: Positive control. A: The analysis of the binding sites for the transcription factor of the upstream region of porcine Kiss1 gene. B: The ChIP results for the binding of p53 with the upstream region of porcine Kiss1 gene. C: The ChIP results for the binding of CEBPα with the upstream region of porcine Kiss1 gene"

Fig. 3

The effects of over-expressed p53 on the expression of Kiss1 A: Over-expression of p53 on the mRNA level of p53; B: Over-expression of p53 on the mRNA level of Kiss1; C,D: Over-expression of p53 on the protein level of Kiss1"

Fig. 4

The effects of interfering p53 on the expression of Kiss1 A: siRNAs interference of p53 on the mRNA level of p53; B: siRNAs interference of p53 on the mRNA level of Kiss1; C, D: siRNAs interference of p53 on the protein level of Kiss1"

Fig. 5

The effects of over-expressed CEBPα on the expression of Kiss1 A: Over-expression of CEBPα on the mRNA level of CEBPα; B: Over-expression of CEBPα on the mRNA level of Kiss1; C,D: Over-expression of CEBPα on the protein level of Kiss1"

Fig. 6

The effects of interfering p53 on the expression of Kiss1 A: siRNAs interference of CEBPα on the mRNA level of CEBPα; B: siRNAs interference of CEBPα on the mRNA level of Kiss1; C, D: siRNAs interference of CEBPα on the protein level Kiss1"

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