Intermittent Hypoxia Up-Regulates CCL2, RETN, and TNFα mRNAs in Adipocytes via Down-regulation of miR-452
Abstract
:1. Introduction
2. Results
2.1. Gene Expression of RETN, TNFα, and CCL2 Was Increased by IH in Mouse 3T3-L1 Adipocytes and Human SW872 Cells
2.2. The Promoter Activities of RETN, TNFα, and CCL2 Were not Increased by IH
2.3. The MiR-452 Level Was Significantly Decreased by IH
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Differentiation of 3T3-L1 Cells into Adipocyte-Like Cells
4.3. RT-PCR
4.4. Measurement of RETN, TNFα and CCL2 in Culture Medium by Enzyme-Linked Immunosorbent Assay (ELISA)
4.5. Construction of Reporter Plasmid and Luciferase Assay
4.6. MiRNA Extraction, Reverse Transcription, and Real-Time Quantitative PCR
4.7. MiR-452 Mimic Transfection
4.8. Data Analysis
Author Contributions
Funding
Conflicts of Interest
Abbreviations
ADIP | Adiponectin |
CCL2 | C-C motif chemokine ligand 2 |
DICER | Endoribonuclease Dicer |
DROSHA | Ribonuclease type III |
ELISA | Enzyme-linked Immunosorbent assay |
FCS | Fetal calf serum |
HUVEC | Human umbilical endothelial cells |
IBMX | methylisobutylxanthine |
IH | Intermittent hypoxia |
IL-6 | Interleukin-6 |
LEP | Leptin |
miRNA | MicroRNA |
RETN | Resistin |
RpS15 | Ribosomal protein S15 |
RT-PCR | Reverse transcriptase-polymerase chain reaction |
SAS | Sleep apnea syndrome |
TNFα | Tumor necrosis factor-α |
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Target mRNA/miR | Primer Sequence |
---|---|
Mouse Adip (NM_009065) | 5′-GGCTCTGTGCTGCTCCATCT-3′ |
5′-AGAGTCGTTGACGTTATCTGCATAG-3′ | |
Mouse Retn (NM_022984) | 5′-GTACCCACGGGATGAAGAACC-3′ |
5′-GCAGAGCCACAGGAGCAG-3′ | |
Mouse IL-6 (NM_031168) | 5′-ACAACCACGGCCTTCCCTACTT-3′ |
5′-CAGGATTTCCCAGCGAACATGTG-3′ | |
Mouse TNFα (NM_013693) | 5′-CCTCCCTCTCATCAGTTCTA-3′ |
5′-ACTTGGTGGTTTGCTACGAC-3′ | |
Mouse Ccl2 (NM_011333) | 5′-CCACTCACCTGCTGCTACTCAT -3′ |
5′-TGGTGATCCTCTTGTAGCTCTCC -3′ | |
Mouse Rig/RpS15 (NM_009091) | 5′-ACGGCAAGACCTTCAACCAG-3 |
5′-ATGGAGAACTCGCCCAGGTAG-3′ | |
Human ADIP (NM_001177800) | 5′-CATGACCAGGAAACCACGACT -3′ |
5′-TGAATGCTGAGCGGTAT -3′ | |
Human RETN (NM_020415) | 5′-TCCTCCTCCTCCCTGTCCTGG-3′ |
5′-CAGTGACATGTGGTCTGGGCG -3′ | |
Human IL-6 (NM_000600) | 5′-GGTACATCCTCGACGGCATC-3′ |
5′-GCCTCTTTGCTGCTTTCACAC-3′ | |
Human TNFα (NM_000594) | 5′-CTTCTCCTTCCTGATCGTGG-3′ |
5′-TCTCAGCTCCACGCCATT-3′ | |
Human CCL2 (NM_002982) | 5′-GTCTCTGCCGCCCTTCTGT-3′ |
5′-TTGCATCTGGCTGAGCGAG -3′ | |
Human DICER (NM_177438) | 5′-GAGCTGTCCTATCAGATCAGGG-3′ |
5′-ACTTGTTGAGCAACCTGGTTT-3′ | |
Human DROSHA (NM_013235) | 5′-GGCCCGAGAGCCTTTTATAG-3′ |
5′-TGCACACGTCTAACTCTTCCAC-3′ | |
Human β-actin (NM_001101) | 5′-GCGAGAAGATGACCCAGA-3′ |
5′-CAGAGGCGTACAGGGATA-3′ | |
Human miR-452 (NR_029973) | 5′-GCGAACTGTTTGCAGAGG-3′ |
5′-CAGTGCGTGTCGTGGAGT-3′ | |
Human U6 (NR_004394) | 5′-CTCGCTTCGGCAGCACA-3′ |
5′-AACGCTTCACGAATTTGCGT-3′ |
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Uchiyama, T.; Itaya-Hironaka, A.; Yamauchi, A.; Makino, M.; Sakuramoto-Tsuchida, S.; Shobatake, R.; Ota, H.; Takeda, M.; Ohbayashi, C.; Takasawa, S. Intermittent Hypoxia Up-Regulates CCL2, RETN, and TNFα mRNAs in Adipocytes via Down-regulation of miR-452. Int. J. Mol. Sci. 2019, 20, 1960. https://doi.org/10.3390/ijms20081960
Uchiyama T, Itaya-Hironaka A, Yamauchi A, Makino M, Sakuramoto-Tsuchida S, Shobatake R, Ota H, Takeda M, Ohbayashi C, Takasawa S. Intermittent Hypoxia Up-Regulates CCL2, RETN, and TNFα mRNAs in Adipocytes via Down-regulation of miR-452. International Journal of Molecular Sciences. 2019; 20(8):1960. https://doi.org/10.3390/ijms20081960
Chicago/Turabian StyleUchiyama, Tomoko, Asako Itaya-Hironaka, Akiyo Yamauchi, Mai Makino, Sumiyo Sakuramoto-Tsuchida, Ryogo Shobatake, Hiroyo Ota, Maiko Takeda, Chiho Ohbayashi, and Shin Takasawa. 2019. "Intermittent Hypoxia Up-Regulates CCL2, RETN, and TNFα mRNAs in Adipocytes via Down-regulation of miR-452" International Journal of Molecular Sciences 20, no. 8: 1960. https://doi.org/10.3390/ijms20081960