Expression and Functional Analysis of Hepcidin from Mandarin Fish (Siniperca chuatsi)
Abstract
:1. Introduction
2. Results
2.1. Sequence Analysis of Sc-Hep
2.2. Sc-Hep Expression
2.3. Relationship Between Sc-Hep and miR-19c-5p
2.4. Sc-Hep DNA Methylation Analysis
2.5. Expression of Iron Balance-Related Genes
2.6. Expression of Immune-Related Genes
2.7. Analysis of Protein–Protein Interactions and Iron Balance Mechanism
3. Discussion
4. Materials and Methods
4.1. Fish and Sample Preparation
4.2. Total RNA Extraction and cDNA Synthesis
4.3. DNA Extraction and Identification of Sc-Hep DNA Sequences
4.4. Analysis of Sc-Hep Gene And Deduced Protein Sequence
4.5. qPCR Analysis of the Sc-Hep Gene
4.6. DNA Methylation Analysis of the Sc-Hep Promoter
4.7. qPCR Analysis of miRNA and Dual-Luciferase Reporter Assay
4.8. Statistical Analysis
Author Contributions
Funding
Conflicts of Interest
Abbreviations
Sc-hep | Siniperca chuatsi hepcidin |
ISKNV | infectious spleen and kidney necrosis virus |
LPS | lipopolysaccharide |
Poly I:C | Polyinosinic–polycytidylic acid |
FS | ferrous sulfate |
PBS | phosphate buffered saline |
PF | cultured with the live prey fish |
AF | fed with artificially domesticated fish |
BLM | basolateral membrane |
HAMP | hepcidin |
FPN1 | ferroportin 1 |
TFR1 | transferrin receptor 1 |
TF | transferrin |
FTN | ferritin |
IL-1β | interleukin-1 beta |
IL8 | interleukin-8 |
SOCS3 | suppressor of cytokine signaling 3 |
TNF-α | tumor necrosis factor-alpha |
TLR22 | toll-like receptor 22 |
PPI | protein–protein interaction |
C/EBP | CCAAT-enhancer-binding protein |
HNF | Hepatocyte nuclear factor |
NFKB1 | Nuclear factor NF-kappa-B p105 subunit |
USF | Upstream stimulatory factor |
GATA | GATA-binding factor |
STAT | Signal transducer and activator of transcription |
TFAP2A | Transcription factor AP-2 alpha |
NC | Negative control |
WT | Wild-type |
MT | Mutated-type |
Appendix A
Transcription Factors | Putative Binding Site |
---|---|
TATA box | −32 |
CEBPA | −1738, −1619, −1500, −1102, −1024, −896, −790, −607, −136, −107 |
CEBPB | −1738, −1042, −896, −607, −136, −107 |
HNF1A | −1793, −1605, −201 |
HNF1B | −199, −330 |
HNF3A | −911 |
HNF3B | −1753, −1247, −597 |
HNF4A | −1158 |
HNF4G | −741, −1442 |
NFKB1 | −164, −140 |
USF1 | −1661, −1533, −1226, −783, −569 |
USF2 | −1699, −904, −783, −655 |
GATA1 | −1737, −1364, −986, −945, −321 |
GATA3 | −943 |
GATA4 | −324 |
STAT1 | −288 |
STAT3 TFAP2A | −1548, −288 −1434 |
GenBank ID | Gene Name | Primer | Purpose |
---|---|---|---|
MK111643 | Sc-hep-F | AAGGAGCTGACAAGAGTCAT | Clone the cDNA sequence of Sc-hep |
Sc-hep-R | GCTGCAACATTTTCTAATAAG | ||
Sc-hep-qPCR-F | TGCTCACCTTTATTTGCCTTCA | Real-time quantitative PCR | |
Sc-hep-qPCR-R | CGCTTCTGTCTGTTGTTATACG | ||
MK605396 | Sc-FPN1-qPCR-F | CAGCCTCACCAGCGTTCTT | |
Sc-FPN1-qPCR-R | CTCCGACTCTATCACATTCTCCT | ||
MK605397 | Sc-FTN-qPCR-F | TCCTACACCTTCCTTGCTCTG | |
Sc-FTN-qPCR-R | GACCACCTCTCCAATCCTCTC | ||
MK605398 | Sc-TFR1-qPCR-F | GAATGCCTCTGCTGTGTTGAT | |
Sc-TFR1-qPCR-R | GCCTGTCGTGATGGTCTGA | ||
AY647430.1 | Sc-IL-1β-qPCR-F | TTCAGTTCAGGAGGACGGATG | |
Sc-IL-1β-qPCR-R | TGTGGCAAGACAGGTAGAGATT | ||
JN180845.1 | Sc-IL8-qPCR-F | GAAGAGCAGCAGAGTTATCATCA | |
Sc-IL8-qPCR-R | ATCTCAGTCTCCTCGCAGTG | ||
JN969981.1 | Sc-TLR22-qPCR-F | CCTACGCCTACTACTTCTTCTTG | |
Sc-TLR22-qPCR-R | GGTCTTCCTGCTTCCATAGATG | ||
MK605399 | Sc-TNF-α-qPCR-F | GCATACACAACCGCACTA | |
Sc-TNF-α-qPCR-R | CTTCCATTCCAGCACCAA | ||
KT895250.1 | Sc-SOCS3-qPCR-F | TCCTCACCACACTCCACAAG | |
Sc-SOCS3-qPCR-F | CACATTGGATACGCAGGTTCTT | ||
FJ436084 | β-actin-qPCR-F | GTGCGTGACATCAAGGAGAAG | |
β-actin-qPCR-R | GGAAGGAAGGCTGGAAGAGG | ||
Sc-hep-met-F1 | TGATGTGATTATATGTTAAGTATTTGTAAATATTA | Methylation | |
Sc-hep-met-R1 | ACTAATATATTAATACTATATATTTATACAAAC | ||
Sc-hep-met-F2 | ATTGTAAATGTTTTTTTAGATGTTGTTTTAATAG | ||
Sc-hep-met-R2 | ATCATCAAACTCAAAAATCTAAATACAAATAATAC | ||
Sc-hep-DNA-F1 | GGCAGGCTGTAGTTAATGTGT | Clone the DNA sequence of Sc-hep | |
Sc-hep-DNA-R1 | AGTTCTCCTGACTGATGGTTGA | ||
Sc-hep-DNA-F2 | CACACTCAACCATCAGTCAGG | ||
Sc-hep-DNA-R2 | CAGTTTCCCACTACAAATGTTATGC | ||
Sc-hep-3’-UTR-WT-F | CTCGAGCAGTGTTGCAGTTGCAGTGGCCGT | qPCR analysis of miRNA and Dual-Luciferase reporter assay | |
Sc-hep-3’-UTR-WT-R | GTCGACAATTTTGCAGGAAACATTAAATGAAC | ||
Sc-hep-3’-UTR-MT-F | CTCGAGGGAGAGCTCATCATCATCGTCACTGAAGTGCAAGAGCTGGA | ||
Sc-hep-3’-UTR-MT-R | GTCGACAACCGTACACGAAACATTAAATGAATCACTTCCTGTGTGC | ||
miR-196b-F | TAGGTAGTTTCAAGTTGTTGGG | ||
miR-196d-F | TAGGTAGTTTTATGTTGTTGGG | ||
miR-23b-5p-F | GGGTTCCTGGCGTGCTGATTT | ||
miR-92b-5p-F | AGGTGTGGGATGTTGTGCAGTGTT | ||
miR-19c-5p-F | AGTTTTGCAGGATTGCATCCGG | ||
U6-F | TTGGAACGATACAGAGAAGATTAGCA | ||
Universe-R | GCTGTCAACGATACGCTCG |
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Shen, Y.; Zhao, Z.; Zhao, J.; Chen, X.; Cao, M.; Wu, M. Expression and Functional Analysis of Hepcidin from Mandarin Fish (Siniperca chuatsi). Int. J. Mol. Sci. 2019, 20, 5602. https://doi.org/10.3390/ijms20225602
Shen Y, Zhao Z, Zhao J, Chen X, Cao M, Wu M. Expression and Functional Analysis of Hepcidin from Mandarin Fish (Siniperca chuatsi). International Journal of Molecular Sciences. 2019; 20(22):5602. https://doi.org/10.3390/ijms20225602
Chicago/Turabian StyleShen, Yawei, Ziwei Zhao, Jinliang Zhao, Xiaowu Chen, Ming Cao, and Minglin Wu. 2019. "Expression and Functional Analysis of Hepcidin from Mandarin Fish (Siniperca chuatsi)" International Journal of Molecular Sciences 20, no. 22: 5602. https://doi.org/10.3390/ijms20225602