Cgi-miR-92d indirectly regulates TNF expression by targeting CDS region of lipopolysaccharide-induced TNF-alpha factor 3 (CgLITAF3) in oyster Crassostrea gigas

• Published in: Fish & Shellfish Immunology
• Main Authors: Chen, Hao, Jiang, Shuai, Wang, Lin, Wang, Lingling, Wang, Hao, Qiu, Limei, Song, Linsheng
• Format: Article in Journal/Newspaper
• Language: English
• Published: 2016
• Subjects: Oyster; Immunomodulation; Mir-17-92 Family; Tumor Necrosis Factor; Lps-induced Tnf-alpha Factor; Inflammation; Crassostrea gigas
• Online Access: http://ir.qdio.ac.cn/handle/337002/135864; https://doi.org/10.1016/j.fsi.2016.06.036

Tumor necrosis factor alpha (TNF-alpha) mediated inflammatory response plays indispensable roles in organisms defending against the invaded bacteria, during which microRNAs have been found crucial by controlling multiple TNF-alpha-related genes. In the present study, cgi-miR-92d was annotated as a member of miR-17-92 family and could target the CDS region of lipopolysaccharide (LPS)-induced TNF-alpha factor (CgLITAF3) in oyster Crassostrea gigas. It was observed that cgi-miR-92d could be vigorously modulated by Vibrio splendidus or LPS stimulation while CgLITAF3 altered oppositely. Two putative binding sites of cgi-miR-92d were then found at CDS region of CgLITAF3. The interaction between cgi-miR-92d and CgLITAF3 was subsequently verified both in vitro and in vivo. As a result, a significant decrease of cellular luminescence was observed in CgLITAF3 luciferase reporter assay when cgi-miR-92d was overexpressed. The luminescent decrease was then recuperated when cgi-miR-92d inhibitor was co-transfected with miRNA mimics. Besides, CgLITAF3 transcripts were significantly down-regulated when cgi-miR-92d was overexpressed in vivo during V. splendidus challenge. Gain-of-function assay of CgLITAF3 was then conducted in HEK293T cells to verify its function. Consequently, a significant increase of TNF-alpha was observed during the assay. At the meantime, CgTNF was also down-regulated in gain-of-function assay of cgi-miR-92 in vivo, which was a member of TNF superfamily in oysters which could be robustly induced after pathogen stimulation. Together, these results verify the interaction between CgLITAF3 and cgi-miR-92d, which might dedicate crucially in the repaid activation of CgTNF expression during inflammatory response of oysters. (C) 2016 Elsevier Ltd. All rights reserved.