The Roles of Two miRNAs in Regulating the Immune Response of Sea Cucumber

Abstract

MicroRNAs (miRNAs) have emerged as key regulators in many pathological processes by suppressing the transcriptional and post-transcriptional expression of target genes. MiR-2008 was previously found to be significantly up-regulated in diseased sea cucumber Apostichopus japonicus by high-through sequencing, whereas the reads of miR-137, a well-documented tumor repressor, displayed no significant change. In the present study, we found that miR-137 expression was slightly attenuated and miR-2008 was significantly enhanced after Vibrio splendidus infection or Lipopolysaccharides application. Further target screening and dual-luciferase reporter assay revealed that the two important miRNAs shared a common target gene of betaine-homocysteine S-methyltransferase (AjBHMT), which exhibited noncorrelated messenger RNA and protein expression patterns after bacterial challenge. In order to fully understand their regulatory mechanisms, we conducted the functional experiments in vitro and in vivo. The overexpression of miR-137 in sea cucumber or primary coelomocytes significantly decreased, whereas the inhibition of miR-137 increased the mRNA and protein expression levels of AjBHMT. In contrast, miR-2008 overexpression and inhibition showed no effect on AjBHMT mRNA levels, but the concentration of AjBHMT protein displayed significant changes both in vitro and in vivo. Consistently, the homocysteine (Hcy) contents were also accordingly altered in the aberrant expression analysis of both miRNAs, consistent with the results of the AjBHMT silencing assay in vitro and in vivo. More importantly, small interfering RNA mediated AjBHMT knockdown and Hcy exposure analyses both significantly increased reactive oxygen species (ROS) production and decreased the number of surviving invasive pathogen in sea cucumber coelomocytes. Taken together, these findings confirmed the differential roles of sea cucumber miR-137 and miR-2008 in regulating the common target AjBHMT to promote ROS production and the clearance of pathogenic microorganisms through Hcy accumulation.KEYWORDS Apostichopus japonicus; miR-137; miR-2008; betaine-homocysteine S-methyltransferase; homocysteine; ROS, genetics of immunity M ICRORNAS (miRNAs) are a class of endogenous small noncoding RNAs of $22 nucleotides, which typically function as repressors of target genes, resulting in direct messenger RNA (mRNA) degradation or translation inhibition (Bartel 2004). These RNAs play crucial roles in many cellular processes, such as proliferation, differentiation, apoptosis, and survival (Lai 2002;He and Hannon 2004;Miska 2005). The expression of up to 60% of all protein-coding genes is estimated to be under the control of miRNAs (Friedman 2009), and combinatorial regulation has been identified as a prominent feature of miRNA regulation. Thus, a given miR-NA might have multiple different mRNA targets, and a given mRNA might similarly be targeted by multiple miRNAs (Krek et al. 2005;Rajewsky 2006). Consistent with the crucial roles of miRNA in the normal functioning of euka...

microRNA-137 promotes apoptosis in ovarian cancer cells via the regulation of XIAP

“…p21Cip1/Waf1, a member of the Cip/Kip family of cyclin kinase inhibitors, is inhibited by 28 miRNAs at the translational level (Wu et al , 2010). Both miR-137 and miR-2008 promote ROS production and the clearance of pathogenic microorganisms by regulating the common target AjBHMT (Zhang et al , 2015). Our previous study showed that miR-24-3p and miR-27a-3p can enhance cell proliferation in glioma cells via cooperative regulation of MXI1 (Xu et al , 2013).…”

miR-31 Links Lipid Metabolism and Cell Apoptosis in Bacteria-Challenged Apostichopus japonicus via Targeting CTRP9

“…Ajβ-actin was used as a control, as described in our previous work (38). The recombinant plasmid (pGEX-4T-2-AjCTRP9) was transformed into Escherichia coli cells of Transetta (DE3) (TransGen, China), and the DNA was sequenced to ensure correct orientation.…”

Acute metabolic response of Portunus trituberculatus to Vibrio alginolyticus infection

“…However, recent studies have shown that betaine plays an immunostimulatory role in fish and chick (Klasing et al, 2002;Kumar et al, 2014). Notably, in sea cucumber, this alkaloid involved in the immune response and functions as a methyl donor which enables homocysteine re-methylated to form methionine via betaine homocysteine methyltransferase (Zhang et al, 2015). The accumulation of homocysteine may contribute to the increased intracellular reactive oxygen species generation and the reduced vibrio survival rate in sea cucumber (Zhang et al, 2015).…”

“…Notably, in sea cucumber, this alkaloid involved in the immune response and functions as a methyl donor which enables homocysteine re-methylated to form methionine via betaine homocysteine methyltransferase (Zhang et al, 2015). The accumulation of homocysteine may contribute to the increased intracellular reactive oxygen species generation and the reduced vibrio survival rate in sea cucumber (Zhang et al, 2015). In this study, the accumulated betaine might indicate the reduced re-methylation of homocysteine in the muscle, thus implying an enhanced ability of swimming crab to resist against V. alginolyticus.…”