Tomohiro Koga1,2, Kiyoshi Migita3, Tomohito Sato1, Shuntaro Sato4, Masataka Umeda1, Fumiaki Nonaka1,5, Shoichi Fukui1, Shin-Ya Kawashiri1, Naoki Iwamoto1, Kunihiro Ichinose1, Mami Tamai1, Hideki Nakamura1, Tomoki Origuchi1, Yukitaka Ueki6, Junya Masumoto7, Kazunaga Agematsu8, Akihiro Yachie9, Koh-Ichiro Yoshiura10, Katsumi Eguchi6, Atsushi Kawakami1. 1. Department of Immunology and Rheumatology, Division of Advanced Preventive Medical Sciences, Nagasaki, Japan. 2. Center for Bioinformatics and Molecular Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. 3. Department of Rheumatology, Fukushima Medical University School of Medicine, Fukushima, Japan. 4. Nagasaki University Hospital, Clinical Research Center, Nagasaki, Japan. 5. Department of Internal Medicine, Sasebo City General Hospital, Sasebo, Japan. 6. Center for Rheumatic Disease, Sasebo Chuo Hospital, Sasebo, Japan. 7. Department of Pathology, Ehime University Graduate School of Medicine and Proteo-Science Center, Toon, Ehime, Japan. 8. Department of Infectious Immunology, Shinshu University, Graduate School of Medicine, Matsumoto, Japan. 9. Department of Pediatrics, School of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan. 10. Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
Abstract
Objective: We sought to identify the microRNA (miRNA) profile and potential biomarkers in FMF and to clarify their gene targets to elucidate the pathogenesis of FMF. Methods: We performed an miRNA microarray using serum from FMF patients in attack and in remission. We then examined the expression of miRNAs in macrophages derived from THP-1 cells stimulated with toll-like receptor (TLR) ligands. Macrophages derived from THP-1 cells transfected with pre-miRNA were stimulated with lipopolysaccharides (LPSs) for the quantification of inflammatory cytokine production. To identify the target genes, we overexpressed their miRNA and performed a complementary DNA microarray. Transfection with reporter construct and the precursor miRNA was performed to confirm the suppression of target mRNA. Results: We found that miR-204-3p was greatly decreased in the serum from FMF patients in attack. The expression of miR-204-3p was suppressed by LPS stimulation in the macrophages derived from THP-1 cells and the inhibition of miR-204-3p significantly induced the production of TLR4-related cytokines. The bioinformatic analysis showed that miR-204-3p is predicted to target genes implicated in the TLR pathway through the regulation of PI3Kγ signalling. The reporter assay revealed that miR-204-3p directly suppressed the luciferase activity of 3'-UTR of PIK3CG reporter construct. The inhibition of PI3Kγ resulted in decreased amounts of IL-6 and IL-12p40 in monocytes from FMF patients. Conclusion: These data suggest that serum miR-204-3p has potential as a useful biomarker in FMF patients and that miR-204-3p serves as a suppressor of inflammatory cytokine production in FMF by targeting the PI3Kγ pathway.
Objective: We sought to identify the microRNA (miRNA) profile and potential biomarkers in FMF and to clarify their gene targets to elucidate the pathogenesis of FMF. Methods: We performed an miRNA microarray using serum from FMFpatients in attack and in remission. We then examined the expression of miRNAs in macrophages derived from THP-1 cells stimulated with toll-like receptor (TLR) ligands. Macrophages derived from THP-1 cells transfected with pre-miRNA were stimulated with lipopolysaccharides (LPSs) for the quantification of inflammatory cytokine production. To identify the target genes, we overexpressed their miRNA and performed a complementary DNA microarray. Transfection with reporter construct and the precursor miRNA was performed to confirm the suppression of target mRNA. Results: We found that miR-204-3p was greatly decreased in the serum from FMFpatients in attack. The expression of miR-204-3p was suppressed by LPS stimulation in the macrophages derived from THP-1 cells and the inhibition of miR-204-3p significantly induced the production of TLR4-related cytokines. The bioinformatic analysis showed that miR-204-3p is predicted to target genes implicated in the TLR pathway through the regulation of PI3Kγ signalling. The reporter assay revealed that miR-204-3p directly suppressed the luciferase activity of 3'-UTR of PIK3CG reporter construct. The inhibition of PI3Kγ resulted in decreased amounts of IL-6 and IL-12p40 in monocytes from FMFpatients. Conclusion: These data suggest that serum miR-204-3p has potential as a useful biomarker in FMFpatients and that miR-204-3p serves as a suppressor of inflammatory cytokine production in FMF by targeting the PI3Kγ pathway.
Authors: Suhas Sureshchandra; Anthony Raus; Allen Jankeel; Brian Jin Kee Ligh; Nicole A R Walter; Natali Newman; Kathleen A Grant; Ilhem Messaoudi Journal: Sci Rep Date: 2019-05-24 Impact factor: 4.379