105 ISJ 16: 105-112, 2019 ISSN 1824-307X RESEARCH REPORT Expression analysis of miR-2005 and its target genes in Apostichopus japonicus by Vibrio splendidus challenged XX Zhou, YQ Chang*, YY Zhan, XL Wang*, K Lin Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture, Dalian Ocean University, Dalian, 116023, China Accepted June 25, 2019 Abstract MicroRNAs (miRNAs) are important effectors in mediating host-pathogen interaction. MiR-2005 is observed to be involved in immune response processes in Apostichopus japonicus. In the present study, the putative target genes of miR-2005 in A. japonicus coelomocytes were predicted by bioinformatics analysis of transcriptome database and PCR approaches. A total of 506 potential targets were screened, and 187 targets were annotated. Several immune-related target genes were identified in this study, such as SLI3, CFHR5, FGL, A2ML, and Rab9al. The expression patterns of miR-2005 and its potential targets were validated by quantitative real-time PCR in Vibrio splendidus challenged A. japonicus. For further characterization, an overexpression experiment of miR-2005 at cellular levels was applied. Accordingly, significant negative correlation expression profiles were detected between miR-2005 and two candidates targets, suggesting that SLI3 and CHRP5 showed high possibility to be the targets of miR-2005 in A. japonicus. Altogether, this study will enhance our understanding in the context of miR-2005 modulating the interaction of A. japonicus after being challenged by V. splendidus. Key Words: miR-2005; Sea cucumber (Apostichopus japonicus); Vibrio splendidus; overexpression experiment; spatial expression Introduction The sea cucumber, Apostichopus japonicus with medicinal effects and rich nutritional value for human consumption, naturally distributed along the coasts of East Asian countries such as China, Japan, Korea and Russia (Sloan et al., 1984; Chang et al., 2009). Additionally, it is widely cultured as a significant aquaculture species in the above countries of East Asia. The echinoderm immune system initiates a response when challenged with pathogen-associated molecular patterns (PAMPs), which are recognized through molecules known as pattern recognition receptors (PRRs) (Kawai et al., 2010). The combination of PAMPs and PRRs activates many immune factors, such as antimicrobial ___________________________________________________________________________ Corresponding authors: Xiuli W ang Yaqing Chang Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea Ministry of Agriculture Dalian Ocean University 52, Heishijiao Street, Dalian, 116023, China E-mails: xiuliwang417@sina.com; yaqingchang@hotmail.com peptides, lectin, and complement through a series of cascade reactions to kill and eliminate invading pathogenic microorganisms, parasites and apoptotic bodies (Fukuzawa et al., 2008). The immune response of A. japonicus is shown to be based on coelomocyte activity (chemotaxis and phagocytosis) (He et al., 2016), some humoral immune factors (lectins, opsonins and some bactericidal substances) (Mai et al., 2009; Wei et al., 2015) could also involve in the immune response that resist the invasion of pathogens directly. Within such an intricate immune defense system, there are multiple layers of process for molecular regulation. MicroRNAs (miRNAs) are important effectors in complicated gene expression profiles through sequence-specific regulation, they could prove to be of significant functional importance in intricate molecular regulation. In a previous study, Lv et al. (2015) observed that miR-200 could modulate the lipopolysaccharides (LPS) priming and antimicrobial activities via augmenting toll-interacting protein (AjTollip). The negative expression profiles between miR-31 and its target Ajp105 were also observed (Lu et al., 2015). It’s indicated that reactive oxygen species (ROS) accumulation could be stimulated, whether by miR-31 overexpression or Ajp105 silencing. miR-137 and miR-2008 were predicated that could targeted the 3’-UTR of betaine-homocysteine mailto:xiuliwang417@sina.com mailto:yaqingchang@hotmail.com 106 S-methyltransferase (AjBHMT), which induces respiratory bursts and affects Hcy accumulation in coelomocytes (Zhang et al., 2015). In addition, Lv et al. (2017b) also found another target of miR-137, and identified the depressed expression profiles of miR-137 and its target gene 14-3-3ζ (Aj14-3-3ζ) in both LPS-exposed primary coelomocytes and Vibrio splendidus-challenged A. japonicus (Lv et al., 2017a). Moreover, several putative targets of miR-92a were also identified by Zhang et al. (2014). Studies of Drosophila and Euarchonta (Lu et al., 2008; Zhang et al., 2008) suggested that gain and loss of miRNAs can be affected by transposition and retrotransposition. Thus, miRNAs have been revealed as major regulators in various hosts-pathogen interaction processes of A. japonicus by repressing the transcription and post-transcriptional expression of target genes. However, although miRNA libraries were constructed for understanding the physiological process of A. japonicus (Li et al., 2012; Chen et al., 2013; Chen and Storey, 2014; Wang et al., 2014, 2015; Sun et al., 2016), the studies on miRNAs and their targets associated with immune response related skin ulceration syndrome of A. japonicus are relatively few. Although, miR-2005 is found present only in Strongylocentrotus purpuratus, Lytechinus variegatus and Patiria miniata based on miRBase records, the expression level of miR-2005 in sea cucumber was also discovered by Wang et al., (2014) and Zhong et al., (2015). Wang et al., (2014) indicated that miR-2005 was significantly up-regulated in the tube foot of A. japonicus, followed by coelomocytes and respiratory tree. It’s suggested that the expression level of miR-2005 was not only highly expressed in the external tissues, but also in the internal environment of A. japonicus. Moreover, miR-2005 was also observed to be involved in the immune response process in A. japonicus after the LPS injection (Zhong et al., 2015). In addition to miR-2005, the expressing levels of miR-2004, miR-133, and miR-137 that was altered along with the expression of A. japonicus complement C3 changing at diff erent time points after LPS injection was also observed (Zhong et al., 2015). According to the evidence resulting from Zhong et al., (2015), it provided further proof for the important regulatory role of miR-2005 in A. japonicus immune system. To sum up, miR-2005 may be involved in the development and immunity defense process of A. japonicus. However, the knowledge of the regulation mechanism of miR-2005 in immune response is still very vague. As previously documented, coelomocytes are recognized to be one of the main components in echinoderm animal immune responses. The coelomocytes of A. japonicus are proved as an appropriate target to explore immune-related genes and physiological mechanisms in response to pathogenic challenge (Dong et al., 2014). V. splendidus, as the Gram-negative bacteria, is identified as a major pathogen that can cause skin ulceration disease in A. japonicus (Zhang et al., 2006). Therefore, V. splendidus was chosen as the pathogen to use to understand the regulation mechanism of miR-2005 in immune response by stimulating A. japonicus coelomocytes. Fig. 1 Predicted target mRNAs of miR-2005. In this study, we report the identification of target genes of miR-2005 from the transcriptome database, including our transcriptome (unpublished) and that of Sun et al., (2013) (accession NO. GSE44995). Then, the relative expression of miR-2005 and its predictive targets were further investigated in V. splendidus-challenged A. japonicus coelomocytes. Subsequently, overexpression was conducted in vitro to obtained the connection of miR-2005 and putative target genes. The present study can strengthen the understanding of the regulatory role of miR-2005 in host–pathogen interactions. Additionally, this study could enhance the knowledge base of A. japonicus immunity upon being challenged by pathogens. Material and methods Target prediction of miR-2005 The 5’-untranslated regions (5’-UTRs) and 3’-untranslated regions (3’-UTRs) were regarded as the potential binding region between target mRNA and miRNA. The extracted UTRs were obtained by in-house Perl script according to the predicated open reading frame (ORF) as previous study (Zhou et al., 2018). The miR-2005 sequences, which were obtained from several previous works (Wang et al., 2014; Zhong et al., 2015), were used to predicate the target mRNA by using the RNA-Seq sequences containing the extracted UTRs. Three software packages, including RNAhybrid (Rehmsmeier et al., 2004), PITA (Kertesz et al., 2007) and miRanda-3.3 (Ellegren, 2008), were used to predict the target mRNAs, and the potential target genes were considered by the intersection of all three programs as previously described (Zhou et al., 2016). The parameters of PITA software were “-l 7-8”, and “-sc 140”, “-en -17” for miRanda-3.3, while the default parameters were used and filtered by ΔG <= 20 kcal/mol in the RNAhybrid. 107 Table 1 Primers used in qRT-RCR. Animals and challenge experiment Healthy sea cucumbers (average weight of 100 g) provided by the Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture (Dalian, Liaoning) were used in this study. For the challenge experiment, the pathogen bacteria Vibrio splendidus (D4501) were chosen to cultured and harvested in our laboratory as previously described by (Cheng et al., 2017). The V. splendidus were cultured at 28 °C overnight with shaking at 200 rpm. Then centrifuged at 4000 rpm for 1 min (4 °C) to harvest the bacteria as previously described by Cheng et al., (2017). For bacterial challenge experiment, the overnight cultured V. splendidus (D4501) was diluted with the phosphate buffered saline (PBS, 0.1 mM, pH 7.4) to achieve the working solution of V. splendidus (D4501) at the concentration of 107 CFU/ml. The challenge experiments were divided into a control group and a bacterial challenge group. A total of 60 sea cucumbers were divided into bacterial-challenged group and control group. The bacterial-challenge group (30 individuals) was injected with 100 l V. splendidus (D4501), while control group (30 individuals) was injected only in a tank containing PBS. The celomic fluids from five A. japonicus was collected at 0, 4, 8, 12, 24, 48 and 72 h post-injection. The samples centrifuged directly at 1000 × rpm for 5 min at 4 °C. After centrifugation, the supernatant was discarded, and snap-frozen in liquid nitrogen, then transferred to -80 °C until RNA extraction. The validation of interaction between miR-2005 and target genes To evaluate the regulatory relationships between miR-2005 and its predicted targets, the expression level of miR-2005 and five predicted target genes were assessed by qRT-PCR. Total RNAs were extracted from coelomic fluid using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and the corresponding cDNAs were synthesized with TransScript miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Co., Ltd., China). The SYBR® PrimeScriptTM miRNA RT-PCR kit (TaKaRa) was used for miRNA and miRNA quantification in the same samples by using described method with an ABI 7500 Real-time PCR machine (Applied Biosystems, Foster City, CA, USA). The specific primer sets for miR-2005 and potential targets are shown in Table 1. In our study, CYTB and RNU6B (Table 1) were served as a reference gene to normalize targets and miRNA, respectively. In brief, each reaction was performed in a final volume of 16 μl containing 2 μl of the cDNA as in a previous study (Zhou et al., 2016a). Three technical replications were performed for each qRT-PCR validation. PCR was conducted as follows: 94 °C for 30 s, 45 cycles of 94 °C for 5 s, and annealing temperature for 32 s. Primary coelomocytes culture and miR-2005 over-expression in vitro In this study, coelomocytes were cultured as described by Lu et al. (2015) and Shao et al. (2015). The A. japonicus were dissected and the coelomic Gene ID Gene name Primer Sequence (5’-3’) Application isotig15273 Serum lectin isoform 3 F: ATTGACAGACACCCTTCCAC R: GACTTCCTGACCTAACATCG Real-Time PCR isotig01560 Fibrinogen-like protein F: GCCAGGATGTTTATGACGCT R: TTGTTACCGAAGCCGTCTCT Real-Time PCR isotig15571 Alpha-2-macroglobulin-like F: CGGAGAGTAGGTCTGATGAT R: GAGTGACAAAGAGGGAGGTT Real-Time PCR comp279590_c0 Complement factor H-related protein 5 F: GCCAGTAGTATCCATCATCC R: CGTGCTCCAACAGATTAGTC Real-Time PCR comp275604_c0 Rab-9A-like F: AGCCAGTCCTTCCATACGAT R: CTCTTGAACCTCTCCTGTCCT Real-Time PCR miR-2005 F: AGTCCAATAGGGAGGGCATTGCAGT R: Universal miRNA qPCR Primer Real-Time PCR miR-2005M AGUCCAAUAGGGAGGGCAUUGCAG GCAAUGCCCUCCCUAUUGGACUUU miR-2005 mimics NCM UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT Negative control of miRNA mimics CYTB F: TGAGCCGCAACAGTAATC R: AAGGGAAAAGGAAGTGAAAG Reference gene RNU6B F: ACGCAAATTCGTGAAGCGTT R: Universal miRNA qPCR Primer Reference gene 108 fluids were filtered through a 300 Mesh CellCribble to filter out large tissue debris, and then the coelomic fluids were centrifuged at 900 g 16 °C for 10min with the same volume of anticoagulant solution (0.48 M NaCl, 0.019 M KCl, 0.02 MEGTA, and 0.068 M Tris-HCl, pH 7.6). The isotonic buffer (0.53 M NaCl, 0.001 M EGTA, and 0.01 M Tris-HCl, pH 7.6) was used to wash the harvested cells, and re-suspended in Leibovitz's L-15 cell culture medium (Invitrogen, USA) supplemented with Gentamycin sulfate (100 μg × ml-1), penicillin (100 U ml-1), streptomycin sulfate (100 μg × ml-1), and NaCl (0.39 M) to adjust the osmotic pressure. The coelomic cells that were diluted to 106 cells ml-1 were the suspended and dispensed into 24-well microplates and incubated at 16 °C for 12 h before the miR-2005 overexpression experiment. The miRNA mimics and negative control of miRNA mimics (NCM) were synthesized at GenePharma and are shown in Table 1. The RNase-free water was used to dissolve miR-2005 mimics and NCM to obtain a working solution of 20 μM. Then 2 μl of miR-2005 mimics and NCM were mixed with an equal volume of HiPerFect transfection reagents (GenePharma, Shanghai), and transfected into primary cultured cells. After 24 h post-transfection, the primary cultured cells were harvested and used for miR-2005 overexpression analysis. Results Prediction and analysis putative target genes of miR-2005 By using the query of the obtained UTR reads in our previous study (Zhou et al., 2018), the potential target genes of miR-2005 were screened using the program PITA, miRanda-3.3a and RNAhybrid In that order. The Veen plot shows the number of potential target genes that predicted by PITA, miRanda-3.3a and RNAhybrid. As shown in Fig. 1, 506 potential target genes of miR-2005 were predicted. The sequences of predicted target genes and the predicted binding sites for the miR-2005 are listed in Supplementary Table 1 and Supplementary Table 2, respectively. A total of 187 potential target genes were annotated (Supplementary Table 3). From the Fig. 2 Time-course expression patterns of miR-2005 and five predict target genes in A. japonicus after V. splendidus challenged at 0, 4, 8, 12, 24 and 48 h. 109 187 potential targets, five potential target genes (serum lectin isoform 3, SLI3; fibrinogen-like protein, FGL; alpha 2-macroglobulin like, A2ML; Complement factor H-related protein 5, CFHR5; and Rab-9A-like, Rab9al) were involved in immune defense against pathogens. Both of SLI and CFHR can possess the functions as opsonization (Boackle et al., 2003). FGLA, A2M and Rab9a are also play vital roles in many physiological and biochemical reactions, including blood clotting and regeneration (Lu et al., 2002; Wu et al., 2014), regulate the prophenoloxidase (proPO) activating system (Jiang et al., 2006), biogenesis of lysosome-related organelles and regulates the degradation of cytoplasmic contents in the lysosome (Nottingham et al., 2011). Expression profile of miR-2005 and its target genes in V. splendidus-challenged A. japonicus To further understand the putative role of miR-2005 in A. japonicus immune defense in vitro, we selected five immune-related targets (SLI3, CFHR5, FGL, A2ML, and Rab9al) of miR-2005 using qRT-PCR method combined with the result of the bioinformatics analysis result. As shown in Fig. 2, the first drastic cut was found in the expression of miR-2005 at 4 h (0.12-fold), and the second downward trend happened at 48 h. The expression pattern of SLI3, Rab9a and CHRP5 all had two peaks, reached at 4 h and 48 h. Functional analysis of miR-2005 and its target genes in vitro The primary coelomocytes of A. japonicus were cultured for gain-of-function experiment of miR-2005 (Fig. 3). As shown in Fig.4, the significant increase was obtained in the overexpression of miR-2005. The qRT-PCR results of SLI3, A2ML and CFHR5 are also shown in Fig. 4. The overexpression of miR-2005 decreased the mRNA expression levels of SLI3, A2ML and CFHR5 obtained in this study, and the downward trend of SLI3 and CFHR5 were more significant than A2ML. Discussion The echinoderm, with no specific immune cells, are mainly rely on the non-specific immune system that composed of cellular immunity and humoral immunity to complete the body's defense response. Many complex molecular regulatory mechanisms are involved in the cellular immunity and humoral immunity of sea cucumber, one of the process is the reprogramming of expression of immune-related genes. The universal transcripts have been demonstrated that are across 70-90 % of mammal genome. However, more than 98 % of total transcripts are represented by non-coding RNAs (ncRNAs) (Tay et al., 2014). Accumulative evidence indicated that miRNAs play essential role in multiple regulatory mechanisms of gene-expression to adapt and survive under pathogen infection (Li et al., 2012; Wang et al., 2014, 2015; Sun et al., 2016; Lv et al. 2017a,b; Chen et al., 2018). Thus, miRNAs have been discovered as major regulators in different hosts-pathogen interaction processes of A. japonicus by blocking the expression of target genes. Fig. 3 Primary coelomocytes culture. In this study, we recognized that miR-2005 played a regulatory role in the interactions between A. japonicus and V. splendidus. The putative target genes of miR-2005 in A. japonicus coelomocytes were predicted by bioinformatics analysis. Among the 506 potential target genes, five immune-related target genes (SLI3, CFHR5, FGL, A2ML, and Rab9al) of miR-2005 were found and selected to verified their relation expression patterns in A. japonicus under V. splendidus infection. Two highly confident target genes (SLI3 and CFHR5) are spotted in vitro and in vivo. Both lectin and complement can be activated by the combination of PAMPs and PRRs. Lectin that exists on the surface of host cells or bacteria are necessary for the specificity recognition between humoral factors and foreign substances. SLI and Ca2+ dependent lectin has been found in the sea urchin and sea cucumber, which is able to recognize and attack foreign substances, and similar homologous to vertebrate cell membrane or soluble lectin (Giga et al., 1987; Himeshima et al., 1994; Matsui et al., 1994; Liu et al., 2012). After A. japonicus infected by V. splendidus, the expression pattern of SLI3 was reached at 4 h and 48 h. Similar expression pattern have been also observed in Liu et al. (2012). The expression level of SLI significantly increased in A. japonicus after they were infected by Vibrio sp. for 3 days (Liu et al., 2012). It is suggesting that SLI may play important roles in the immune defense mechanism of sea cucumber against bacterial infection. Moreover, the complement system is also playing a necessary role in the innate defense against common pathogens. As a complement control protein, CFHR is a member of the complement activation family regulators. Five plasma proteins (CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5) that each member binds to the complement component 3b (C3b) can comprise factor H related proteins (Zhou et al., 2011; Skerka et al., 2013; Józsi et al., 2015). In human, CFHR5, composed of nine SCR domains, which is the longest CFHR protein in the CFHR family (Skerka et al., 2013). Although, CFHR5 shares high 110 Fig. 4 Relative expression of miR-2005, Rab-9a, SLI3, and CHRP5 in miR-2005 mimics transfected coelomocytes. NCM: Negative control of miRNA mimics; miR-2005M: miR-2005 mimics. * p < 0.05. sequence similarity with factor H. Unlike factor H (FH), CFHR5 can binding to activated C3 and potentially enhance C3 deposition (Zhu et al., 2018). One complement factor H gene and four-factor H-like genes were identified in Danio rerio (Sun et al., 2010), which highlights the possibility that all of the five genes were involved in the acute phase due to the fact that they were up-regulated by an LPS challenge (Sun et al., 2010). In our study, the expression pattern of CFHRs was similar to SLI3 that were reached at 4 h and 48 h after A. japonicus infected by V. splendidus. By using an overexpression experiment of miR-2005 at cellular levels, significant negative correlation expression profiles were detected between miR-2005 and SLI3 and CFHR5. It’s suggested that the abnormal expression of mir-2005 may have great effect in V. splendidus-challenged A. japonicus by targeting SLI3 and CFHR5. 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