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Genomic Analysis of Mir-21-3P and Expression Pattern with Target Gene in Olive Flounder

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Genomic Analysis of Mir-21-3P and Expression Pattern with Target Gene in Olive Flounder eISSN 2234-0742 Genomics Inform 2017;15(3):98-107 G&I Genomics & Informatics https://doi.org/10.5808/GI.2017.15.3.98 ORIGINAL ARTICLE Genomic Analysis of miR-21-3p and Expression Pattern with Target Gene in Olive Flounder Ara Jo1,2, Hee-Eun Lee1,2, Heui-Soo Kim1,2* 1Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 46241, Korea, 2Genetic Engineering Institute, Pusan National University, Busan 46241, Korea MicroRNAs (miRNAs) act as regulators of gene expression by binding to the 3’ untranslated region (UTR) of target genes. They perform important biological functions in the various species. Among many miRNAs, miR-21-3p is known to serve vital functions in development and apoptosis in olive flounder. Using genomic and bioinformatic tools, evolutionary conservation of miR-21-3p was examined in various species, and expression pattern was analyzed in olive flounder. Conserved sequences (5’-CAGUCG-3’) in numerous species were detected through the stem-loop structure of miR-21-3p. Thus, we analyzed target genes of miR-21-3p. Among them, 3’ UTR region of PPIL2 gene indicated the highest binding affinity with miR-21-3p based on the minimum free energy value. The PPIL2 gene showed high expression levels in testis tissue of the olive flounder, whereas miR-21-3p showed rather ubiquitous expression patterns except in testis tissue, indicating that miR-21-3p seems to control the PPIL2 gene expression in a complementary repression manner in various tissues of olive flounder. Taken together, this current study contributes to infer the target gene candidates for the miR-21-3p using bioinformatics tools. Furthermore, our data offers important information on the relationship between miR-21-3p and target gene for further functional study. Keywords: conserved sequence, gene expression, microRNAs, minimum free energy, 3’untranslated regions Introduction and China [8]. Recently, due to lots of consumption, its economic values have increased in the fish markets [9]. MicroRNAs (miRNAs) are groups of endogenous small, However, olive flounder is sensitive to infection of many noncoding, single-stranded RNA molecules 20–24 nucleo- bacterial pathogens [10] and they can trigger significant tides in length [1]. The miRNAs bind to the 3´ untranslated economic losses. Therefore, there has been an increase in regions (UTRs) of its target mRNAs entirely or partly and studies that strive to understand the effects of bacterial they act as post-transcriptional inhibitors of gene expression infection in olive flounder via quantitative real-time poly- [2, 3]. This event causes specific mRNA degradation or merase chain reaction (qRT-PCR) [11]. Genetic variation translational repression [4]. Each single miRNA could target and population structure among olive flounder populations numerous mRNAs, and a single mRNA can be targeted by are estimated using several molecular approaches. Those several miRNAs, which allows to coordinate and control research results indicated proper yields and retaining genetic regulation of protein inhibition [5]. The miRNAs have played diversity [12]. Genetic variation is beneficial and important a role in a variety of biological processes such as cell for the long-term survival of natural populations, as it proliferation, cell survival, DNA repair, and immune res- ensures that a high level of fitness is maintained by giving ponse [6]. Therefore, complex interactions involved in the populations the ability to adapt to changing environmental regulation of gene expression have been well-established. At conditions [13]. Population genetic structure is influenced least up to 60% of protein-coding genes expressed in various by the effects of gene flow, natural selection, genetic drift, species, and down-regulated more than 1,000 miRNAs [7]. and mutation [14]. The olive flounder Paralichthys olivaceus is one of the most According to the several functional studies, miRNA and important fish species that widely cultured in Korea, Japan, their targets interaction in olive flounder indicated invol- Received July 31, 2017; Revised August 8, 2017; Accepted August 15, 2017 *Corresponding author: Tel: +82-51-510-2259, Fax: +82-51-581-2962, E-mail: [email protected] Copyright © 2017 by the Korea Genome Organization CC It is identical to the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/). Genomics & Informatics Vol. 15, No. 3, 2017 vement in development, apoptosis, and metabolism [6, 15]. These RNAs were treated with RNase-Free DNase I (New One of the representative studies is about the miR-17, and it England Biolabs, Beverly, MA, USA) to remove genomic is about metamorphic development of olive flounder that DNA contamination. RNAs were separated by 1% agarose overexpressed miR-17 in FEC cells and suppressed Cdc42 gel electrophoresis for analysis of the 28S and 18S RNA expression due to miR-17 binds to 3´ UTR of Cdc42 gene. bands. The RNA concentration was quantified to 500 ng The declined metamorphic development means growth level using a ND-1000 UV-Vis spectrophotometer (Nano Drop, of olive flounder also diminished [16]. Recently, studies Wilmington, DE, USA). ReverTra Ace qPCR RT Master Mix have shown that 21 let-miR-7 precursors control metamor- kit (TOYOBO, Osaka, Japan) was used for cDNA synthesis. phosis stages from the larval to juvenile form in P. o l i v a c e u s . Prior to use, a 1 in 50 volume of gDNA remover was added These let-7 miRNAs were shown in different positions of P. to 4× DN Master Mix and then each quantitated 500 ng RNA olivaceus genome and significantly expressed in adult tissues samples 6 μL denatured at 65°C for 5 min. Then, 2 μL of 4× of olive flounder, but not expressed in embryonic DN Master Mix was added to RNA in order to increase development tissues, indicating that let-7 miRNAs could be DNase I reaction and incubated at 37oC for 5 min. After that, associated with tissue development and metabolism process 2 μL of 5× RT Master Mix was added to 4× DN Master Mix. [17]. There is limited evidence between the miR-21-3p Reverse transcription response was performed by following expression pattern and the quantitative expression levels of steps; incubation at 37oC for 15 min, 50oC for 5 min, heating the target gene in various olive flounder tissues. Previous 98oC for 5 min and stored the solutions at ‒20oC. studies reported miR-21-3p function in different fields. For Quantitative real-time RT-PCR analyses instance, some miRNAs have been found to function as oncogenic miRNAs, this can be observed in the study where Quantified RNA samples were used for miR-21-3p miR-21-3p was found to be up-regulated in solid and expression and its target gene; HOXC9 3´ UTR expression hematological cancer tissues thus leading to the conclusion analysis. HB miR Multi Assay Kit System I (Heim Biotek, that miR-21-3p repress the expression of tumor suppressors Seongnam, Korea) was used to miRNA analysis that miR-21-3p like PTEN phosphatase and actin-binding protein tropo- reverse transcription (RT) and real-time PCR. First, 500 ng myosin I [18-20]. In addition, the set of miR-21 and its target RNA reaction volume per each tissue need to 11 μL con- mRNA, TGFBR2, were predicted as high binding affinity in taining RNase free water. Then for synthesis of cDNA—HB_I seven types of solid tumor namely; lung, breast, colorectal, RT Reaction Kit and its reagents was used in compliance pancreatic, prostate, stomach, and bladder that indicated with manufacturer’s instructions and reverse transcription strong molecular interaction between miR-21 and TGFBR2 polymerase chain reaction (RT-PCR) amplification was per- [21, 22]. The hsa-miR-21-3p was known as bipolar disorder formed in a thermal cycler (Eppendorf, Hamburg, Germany) (BP) dominant miRNA by verifying only increase of on terms of 37oC for 60 min followed by incubation at 95oC miR-21-3p in BP on fibroblast culture [23]. The miR-21-3p for 5 min for one cycle and then held at 4oC. Synthesized also upregulated expression in hearts of lipopolysaccha- cDNA samples in various tissues olive flounder were stored ride-treated mice [24]. Due to down regulation of SH3 at ‒20oC. Second, HB_I Real-time PCR Master Mix Kit from domain-containing protein 2 (SORBS2) is one of target the HB miR Multi Assay Kit System I was followed by genes of miR-21-3p and putative target of SORBS2 controls manufacturer’s protocols. All cDNA samples were amplified septic infection-associated cardiac dysfunction [24]. in triplicate to ensure reproducibility with a Rotor-Gene Q In the present study, the evolutionary conservation system (Qiagen, Hilden, Germany). Real-time PCR was patterns of miR-21-3p in various species and miR-21-3p initial denaturation for 15 min at 95oC, 45 thermal cycles of target genes were analyzed using bioinformatic tools. Also, 95oC for 10 s and 60oC for 40 s and standard melting condi- expression patterns of miR-21-3p were analyzed in various tions of ramp ranging from 55oC to 99oC with 1oC rise on tissues of olive flounder in relation to the target PPIL2 gene each step. As a standard control, miRNA U6 was used as using qRT-PCR. reference. Relative expression ratio of the target miR-21-3p (5’-CGACAACAGUCUGAAGGCUGUC-3’) to miRNA U6 Methods were analyzed the comparative threshold method (2-∆∆ Ct). Total RNA isolation and cDNA syntheses In case of target gene expression analysis, 7 μL of ddH2O, Total RNA was extracted from ten tissues of olive flounder 10 μL of QuantiTect SYBR Green PCR Master Mix (Qiagen, (spleen, gill, muscle, kidney, intestine, stomach, liver, fin, Valencia, CA, USA), 1 μL of each forward (5´-TCGTGCA- brain, and testis) using TRIzol reagent (Invitrogen, Carlsbad, TCTACCTCGACAG-3´) and reverse (5´-CCTCCTCCT- CA, USA) depending on the manufacturer’s instructions.
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