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

Tumor necrosis factor alpha (TNF-α) mediated inflammatory response plays indispensable roles in organisms defending against the invaded bacteria, during which microRNAs have been found crucial by controlling multiple TNF-α-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-α 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-α 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.