THE EXPRESSIONAL CHARACTERIZATION OF MIR-222 IN MANDARIN FISH (SINIPERCA CHUATSI)

Zhu Xin, Hu Yi, Wang Kai-Zhuo, Wu Ping, Yi Tan, Chen Dun-Xue, Zhang Jun-Zhi and Chen Tao

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THE EXPRESSIONAL CHARACTERIZATION OF MIR-222 IN MANDARIN FISH (SINIPERCA CHUATSI)

Zhu Xin1, Hu Yi2, Wang Kai-Zhuo2, Wu Ping3, Yi Tan1, Chen Dun-Xue2, Zhang Jun-Zhi2and Chen Tao1

(1. College of Veterinary Medicine,Hunan Agriculture University, Changsha 410128, China; 2. College of Animal Science and Technology,Hunan Agricultural University, Changsha 410128, China;3. College of Biology, Hunan University, Changsha 410082, China)

The mandarin fish (Basilewsky) is one of the most commercially important carnivorous fish species in aquaculture. Increasing evidences have suggested that microRNAs (miRNAs) have emerged as key regulators of skeletal muscle growth, but little is known about miRNA expression profiles relate to skeletal muscle growth in teleost. The miR-222 has been reported as the key regulator during muscle differentiation. To investigate the expression pattern of miR-222 in different tissues and developmental stages in, the expression of miR-222 was detected by RT-qPCR. In the present study, we found that the miR-222 was highly expressed in muscle-related tissues, especially in fast (white) muscle of adult mandarin fish. RT-qPCR analysis revealed that the miR-222 was first detected in two cell stage embryos and highly expressed at the heart beating stage. The differential expression of miR-222 in different tissues and developmental stages indicated that miR-222 might involve in regulating muscle growth and development. We investigated the expression changes of the mature miR-222 abundance following a single satiating meal in mandarin fish juveniles after fasting for a week. MiR-222 expression was sharply up-regulated within 1h after refeeding and might be desired candidate miRNA involved in a fast-response signaling system that regulates fish skeletal muscle growth. This finding would provide a novel insight in the role of miR-222 in teleost development.

MicroRNA;; Fasting and refeeding; Skeletal Muscle

Skeletal myogenesis requires the occurrence of specific coordinated events, including exit from the cell cycle, transcription of muscle-specific proteins, fusion into polynucleated fibers and assembly of the contractile apparatus[1]. Recent studies have identified the post-transcriptional control of gene expression as a crucial level of regulation of myogenesis. MicroRNAs (miRNAs) are approximately 22-nt noncoding RNAs, which act as negative regulators of gene expression either by inhibiting mRNA translation or promoting mRNA degradation through binding to the 3￠-un-translated region (3￠-UTR) of target mRNAs[2, 3]. MiRNAs have been reported to regulate the expression of transcription factors and signaling mediators important for development and function of cardiac and skeletal muscle[3, 4]. For example, miR-222 has been found to be modulated during myogenesis and to play a role in the progression from myoblasts to myocytes via inhibiting the expression of the cell cycle inhibitor p27[1].

The mandarin fish ()is one of the most commercially important carnivorous fish species in aquaculture because of its high nutritional value and delicious taste as well as its large-scale culture in China[5—7]. In the present study, we analyzed the miR-222 expression profiling during developmental stages in mandarin fish and completely characterized the miRNA signature during the transition from catabolic to anabolic states. This study contributed to a better unders-tanding the role of miR-222 in skeletal muscle development of mandarin fish.

1 Material and Method

1.1 Individuals and tissues sampling

All mandarin fish individuals were reared at the Xingda mandarin fish hatchery, Changde, Hunan, China. Tissue samples, including brain, kidney, liver, spleen, heart, intestine, red muscle and white muscle, were collected from five adult individuals [average body weight of 500 g, 150 days of post-hatching (dph)]. The fast muscle (white muscle) was dissected from dorsal myotome of individuals at different developmental stages (20, 30, 50, 70, 90 and 150 dph). For each stage, five individuals were sampled. Samples from different embryonic developmental stages (embryos at the 2-cell stage, blastula stage, gastrula stage, neurula stage, tail-bud stage, muscular effect stage, heart beating stage and larval stage) were obtained after artificial fertilization until hatching at Hunan Aquaculture Institute, China. All tissue and embryo samples were snap-frozen in liquid nitrogen and stored at –80℃ for further processing.

1.2 Fasting-refeeding experiment and sampling

Two homogeneous groups of mandarin fish juveniles (average body weight 150 g, 90 dph) were reared respectively in two net cages (5 m × 5 m × 2 m) with fifty fish per tank. All juveniles were fed under standard conditions [with mud carp (Cuvier) as forage fish, average body weight 10 g] during 3 weeks. Juveniles were then fasted for 1 week, and fed a single meal distributed to all individuals to visual satiation. Sampling occurred at 0h (before the recovery meal), and at 1h, 3h, 6h, 12h, 24h, 48h and 96h (hours after the single meal), with seven fish sampled from random two cages at each time point.

Fast muscle was dissected from the dorsal myotome of samples. All samples were snap-frozen in liquid nitrogen and stored at –80℃ until further pro-cessing.

1.3 Real-time PCR for the miRNAs

Tissue samples were ground in liquid nitrogen. The total RNAs were extracted with the TRIzolRRea-gent (Invitrogen, USA), and then treated with RNAse-free DNAse I (Promega, USA) in the presence of RNAse inhibitor (Sigma, China Branch) followed by ethanol precipitation. The obtained RNAs were reverse transcribed with one step PrimeScript miRNA cDNA synthesis Kit (TaKaRa, Dalian, China). Each of 1 μL total RNA was added to a total volume of 20 μL reaction mix containing 10 μL reaction buffer mix, 2 μL of 0.1% BSA, 2 μL of miRNA primeScript RT enzyme mix and 5 μL nuclease-free water. The following protocol was 37℃ for 60min, 85℃ for 5s.

The expression levels of mature miRNA were quantified using real-time PCR withas the internal control in embryonic samples[8]and on account ofdo not suitable as internal control in muscle samples, we chose RPL-13 as the internal control in muscle samples[9]. The cDNA samples were used as templates for real-time PCR assays with SYBR Premix ExTMⅡ (TaKaRa, Dalian, China) and its amplification reaction was carried out using Bio-Rad CFX96 system (USA). Each of 2 μL cDNA template was added to a total volume of 25 μL reaction mix containing 12.5 μL SYBR Green mix, 1 μL of miRNA and 1 μL of miRNA universal downstream primer (as shown in Tab. 1, 10 μmol/L), 8.5 μL nuclease-free water. The following protocol used was described by Zhou,[8]. The relative expression ratio () of target miRNA was calculated by= 2–ΔΔCt[10, 11], whereis the cycle threshold. The basic equation employed was.

The miRNA expression levels were then analyzed by one-way ANOVA procedures and regression analysis of SPSS software. The differences were considered statistically significant when<0.05. Data are shown as means ± SE (= 6).

2 Result

2.1 MiR-222 expression in different tissues of adult mandarin fish

The miRNA library of mandarin fish was constructed by Chu,[12]. Their results showed that some miRNAs that may have myogenic functions were abundant in the white and red muscle libraries, including miR-222. To further verify the expression of miR-222 in different tissues, RT-PCR experiments were performed to detect miR-222 expression in white muscle, red muscle, heart, gut, liver, kidney, spleen and brain. The miR-222 expression was detected in all the sampled tissues of mandarin fish, with higher level in the white (fast) muscle. Moderate expression level was present in red (slow) muscle, gut and heart; while relative lower in liver, kidney, spleen and brain (Fig. 1).

Tab. 1 Primers used for miRNA detection

Note: The forward primers used for detection were above, the reverse primer used for detection was universal downstream primer (Uni-miR qPCR Primer, 10 μmol/L,TaKaRa)

2.2 MiR-222 expression at different embryonic developmental stages

Previous studies indicated that miRNAs were dramatically changed in different cell types and di-ff-erent developmental stages. They participate in regulating cell growth, differentiation, and program-med cell death[13, 14]. Thus, we investigated the possibility whether each embryonic developmental stage has a specific signature of miR-222. The results indicated that the expression of miR-222 had a steep increase at heart beating stage (<0.05), compared to the expression at otherembryonic developmental stage(Fig. 2). Moreover, there was a significant increase at gastrula stage (<0.05) (Fig. 2). While miR-222 was expressed lower at most of the embryonic developmental stage except gastrula and heart beating stage (Fig. 2).

-13 gene was used as the internal control. Values are the mean ± SE,= 6. Different letters in figure indicated there was ignifycant difference between different tissues (<0.05)

gene was used as the internal control. Values are the mean ± SE,= 6. Different letters in figure indicated there was significant difference between different embryonic developmental stages(<0.05)

2.3 MiR-222 expression in fast muscle of mandarin fish at different post-embryonic developmental stages

To further explore whether the miRNAs involved in the fast skeletal muscle development of the mandarin fish, we dissected fast skeletal muscle samples from different post-embryonic developmental stages (from 20 dph to 150 dph). MiR-222 was presented in all post-embryonic developmental stages. The result showed that the miR-222 expressed with a rising in the skeletal muscles from 20 to 150 days post-hatching on the whole. Furthermore, the expression of miR-222 significantly increased at 30 dph and 90 dph when compared with each previous stage (<0.05).

-13 gene was used as the internal control. Values are the mean ± SE,= 6. Different letters in figure indicated there was significant difference between different post-embryonic develop-mental stages (<0.05)

2.4 Effect of fasting and refeeding on the expre-ssion of the miR-222

To determine the effect of fasting and refeeding on miRNA expression in skeletal muscles, we analy-zed the expression of the miR-222 at various time points after refeeding. A significant up-regulation of the miR-222 was observed at 1h (hour after the single meal) (<0.05), but reached to the maximal level at 12h postprandial and then returned to initial values at 96h.

3 Discussion

Genetic studies on the components of the miRNA biogenesis pathway indicated that Dicer and miRNAs were essential for embryonic differentiation[15, 16]. The differential expression of miR-222 was showed in embryo developmental expression which indicated miR-222 might play a role in embryonic development. Among these developmental stages, heart beating stage could be a important stage when miR-222 involved in regulating embryonic development, because of its expression increasing dozens of times at this stage.Profiling of miRNA expression in different tissues indicated that miR-222 was specifically expressed in the muscle-rich tissues and organs (skeletal muscle, heart, gut), and that their expression increased with the progress of muscle development, ultimately reaching higher level in adult fish skeletal muscle. These results are consistent with previous findings in Nile tilapia (Linnaeus) and common carp (Linnaeus)[17, 18]. MiR-222 was expressed higher in skeletal muscle and differently expressed in muscle during development which indicated miR-222 was likely to involve in regulating muscle growth. Indeed miR-222 is one of the key regulators during muscle differentiation that has been found to be modulated during myogenesis, and to play a role both in the progression from myoblasts to myocytes and in the achievement of the fully differentiated phenotype by targeting p27[1]. However, whether the miR-222 influenced muscle development by targeting p27 in mandarin fish requires further investigation.

-13 gene was used as the internal control. Values are the mean ± SE,=6. Different letters in figure indicated there was significant differences between each time point (<0.05). “ab” indicated that there was no significant difference with a and b

Nutrient availability is amongst the most important environmental variable altering muscle growth[19]. Starvation and refeeding experiments have been used as the model system to study the regulation of muscle growth in fish. Short term starvation and subsequent refeeding has been reported to result in compensatory growth in larval rock carp[20]. The effects of fasting and subsequent refeeding procedure following transcript abundance over time have been studied in se-veral fish species such as Atlantic salmon (Linnaeus)[21], rainbow trout (Walbaum)[22]and Atlantic halibut (Linnaeus)[23]. MiRNAs(miR-499, -208b, -23a, -1, -133a, and -206) are changed quite rapidly (i.e. hours) in skeletal muscle following amino acid ingestion in humans[24]. However, very little information is available regarding the early transcriptional changes of miRNAs during the postprandial period. To further investigate the potential role of miR-222 involve in the stimulation of myogenesis during anabolic stimuli, we explored the postprandial regulation of miR-222 shortly after feeding a single meal in mandarin fish. The finding of this study was that miR-222 was significantly elevated at 1h and peaked at 12h after refeeding in the fast muscle of mandarin fish. Drummond,. found a rapid up-regulation of the miR-1, miR-208b, miR-23a and miR-499 following the amino acid ingestion in humans[24]which indicated that miRNAs could involve in regulating muscle growth when nutritional conditions changing. The up-regulation of miR-222 in response to nutrient supply might result in regulating its target genes to involve in promoting myogenesis, but the genetic network that is mobilised in the stimulation of myogenesis during the transition from a catabolic to anabolic state in skeletal muscle has not been exhaustively described.

In conclusion, miR-222 might be the desired candidate miRNA involved in a fast-response signaling system that regulates fish skeletal muscle growth. Further work is needed to elucidate the precise role of miRNA in the regulation of fish muscle during anabolic stimuli and to determine what specific mRNA are targeted by this novel post-transcriptional regulators.

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翹嘴鱖miR-222的表達特征

朱 鑫1胡 毅2王開卓2吳 萍3易 潭1陳敦學2張俊智2陳 韜1

(1. 湖南農業大學動物醫學院, 長沙 410128; 2. 湖南農業大學動物科技學院, 長沙 410128; 3. 湖南大學生物學院, 長沙 410082)

為了解翹嘴鱖miR-222的時空表達規律, 研究利用實時熒光定量PCR的方法檢測miR-222在翹嘴鱖不同組織、胚胎發育及胚后發育中的相對表達豐度。研究結果顯示, miR-222在肌肉相關的組織中表達較高, 特別是在成年翹嘴鱖的白肌中表達最高; 胚胎發育階段結果顯示, miR-222在胚胎發育的2細胞期就有表達, 而表達量在心動期達到最高。不同組織及不同發育階段的差異性表達結果表明, miR-222很可能參與調控鱖魚肌肉的生長發育。為研究合成代謝過程中miR-222在肌肉生長調控中的表達規律, 通過對翹嘴鱖幼魚在饑餓一周后飽食一餐的實驗處理下, 利用實時熒光定量的方法測定miR-222在骨骼肌中的相對表達變化。結果顯示, miR-222的表達量在恢復喂食后的1h顯著上升(0.05), 表明miR-222很可能是調節魚類骨骼肌生長過程中, 參與快速應答信號系統的一類miRNA。研究為miR-222在魚類發育中的調控作用提供一些理論依據。

MicroRNA; 翹嘴鱖; 饑餓和恢復喂食; 骨骼肌

10.7541/2015.42

Q344+.1 Document code: A Article ID: 1000-3207(2015)02-0315-06

date: 2014-05-08; Accepted date: 2014-12-04

National Natural Science Foundation of China (No. 31230076; 31340054); Natural Science Foundation of Hunan province (14JJ2135)

Chen Tao (1969—), Changsha Hunan; Ph.D. and Professor; major in fish physiology and molecular biology, E-mail: chentao_114@163.com

ZhuXin (1989—), male, Changsha Hunan; master; major in animal physiology and fish developmental of molecular biology,E-mail: xinzhu1219@163.com