Chen Hao, Jiang Shuai, Wang Lin, Wang Lingling, Wang Hao, Qiu Limei, Song Linsheng
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China.
Fish Shellfish Immunol. 2016 Aug;55:577-84. doi: 10.1016/j.fsi.2016.06.036. Epub 2016 Jun 23.
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.
肿瘤坏死因子α（TNF-α）介导的炎症反应在生物体抵御入侵细菌的过程中发挥着不可或缺的作用，在此过程中，微小RNA通过控制多个与TNF-α相关的基因而被发现至关重要。在本研究中，cgi-miR-92d被注释为miR-17-92家族的成员，并且可以靶向太平洋牡蛎（Crassostrea gigas）中脂多糖（LPS）诱导的TNF-α因子（CgLITAF3）的编码区。据观察，cgi-miR-92d可被灿烂弧菌或LPS刺激强烈调节，而CgLITAF3则呈相反变化。随后在CgLITAF3的编码区发现了两个cgi-miR-92d的假定结合位点。cgi-miR-92d与CgLITAF3之间的相互作用随后在体外和体内均得到验证。结果，在cgi-miR-92d过表达时，CgLITAF3荧光素酶报告基因检测中观察到细胞发光显著降低。当cgi-miR-92d抑制剂与miRNA模拟物共转染时，发光降低得以恢复。此外，在灿烂弧菌攻击期间，当cgi-miR-92d在体内过表达时，CgLITAF3转录本显著下调。然后在HEK293T细胞中进行CgLITAF3的功能获得实验以验证其功能。结果，在实验过程中观察到TNF-α显著增加。同时，在体内cgi-miR-92的功能获得实验中，CgTNF也下调，CgTNF是牡蛎TNF超家族的成员，在病原体刺激后可被强烈诱导。总之，这些结果验证了CgLITAF3与cgi-miR-92d之间的相互作用，这可能在牡蛎炎症反应期间CgTNF表达的快速激活中起关键作用。
