A novel microRNA and its pfk target control growth length in the freshwater shrimp Neocaridina heteropoda

The expansion of aquaculture is important from both economic and food security perspectives. In general, aquaculture of shrimps is challenging because of their large size (up to about 30 cm) and long growth cycle, making them unsuitable for laboratory research. A short growth cycle and facile breeding make the shrimp species Neocaridina heteropoda (family Decapoda, class Atyidae) suitable for developmental, ecotoxicological and reproductive studies (FAO, 1961). Neocaridina heteropoda is therefore a useful model Decapoda species for basic theoretical research on shrimp physiology and aquaculture.

As interest in genes and phenotypes has grown, the focus has transferred to the molecules that regulate DNA expression in organisms. In the past decade, we have begun to realize the importance of RNA molecules of ∼20−30 nucleotides (nt) in length in eukaryotic gene expression and functional regulation. These RNAs are mainly divided into two types: small or microRNAs (miRNAs) and small interfering RNAs (siRNAs) (Carthew and Sontheimer, 2009). miRNAs are non-coding RNAs ∼22 nt in length that affect target gene expression by promoting the degradation of target mRNAs or hindering the translation of target genes into proteins. In the cytoplasm, mature miRNAs participate in the formation of the RNA-induced silencing complex (RISC) that binds to target RNAs via recognition regions that include the coding region, 3′-untranslated region (UTR) and 5′-UTR of mRNAs. When Argonaute (AGO) proteins possessing lytic (i.e. slicer) activity are associated with the RISC, they induce cleavage degradation of target mRNAs (Bartel, 2004; Brennecke et al., 2005; Doench and Sharp, 2004).

miRNAs play an important role in various biological processes in crustaceans. For example, in Eriocheir sinensis, miR-217 can enhance White Spot Syndrome Virus (WSSV) infection by regulating the Tube gene, thereby regulating innate immunity and interactions with the external environment (Huang et al., 2017). miR-100 may promote the anti-Vibrio immune response of Penaeus vannamei by regulating cell apoptosis, phagocytosis and phenoloxidase (PO) activity, and to some extent affects the progress of WSSV infection (Wang and Zhu, 2017). In addition, two miRNAs (miR-9041 and miR-9850) in the gill of Macrobrachium rosenbergii can promote the replication of WSSV by regulating the signal transducer and activator of transcription (STAT) gene (Huang et al., 2016). Some crustaceans must endure long periods of hypoxia to survive, and miRNA-based regulation aids survival in hypoxic environments. For example, the northern crayfish (Orconectes virilis) often endures constant hypoxia and hypoxia stress, and miR-33-5p, miR-125-5p and miR-190-5 have been shown to directly or indirectly target the alpha subunit of hypoxia-inducible factor 1 (HIF1), a major regulator of oxygen stress (English et al., 2018). Recent studies have shown that miRNAs play a key role in the responses of crustaceans to heavy metals. miRNAs may increase the tolerance of Daphnia to cadmium by inhibiting cell growth and proliferation through GTPase and keratin pathways, thereby converting cell energy allocation into detoxification (Chen et al., 2015, 2016). miRNAs also play an important role in the development of crustaceans. Song et al. (2014) identified a large number of miRNA transcripts from the ovaries of E. sinensis, including miR-2 and miR-133, which are differentially expressed during meiotic maturation of oocytes and bind to the 3′-UTR of the cyclin B gene to inhibit its translation (Song et al., 2014). miR-34 in the eyestalk of the mud crab Scylla paramamosa regulates reproduction by inhibiting the expression of the molt-inhibiting hormone (MIH), crustacean hyperglycemic hormone (CHH), ecdysone receptor (EcR) and farnesoic acid O-methyl transferase (FAMeT) genes (English et al., 2018). However, we know little about the role of miRNAs in crustacean muscle growth.

The growth of crustaceans has been studied for a long time, and glycolysis pathways are known to be closely related to growth. Glycolysis can support embryonic muscle growth by promoting myoblast fusion, and mRNA transcripts of phosphofructokinase (pfk), triosephosphate isomerase (Tpi), glyceraldehyde 3-phosphate dehydrogenase (Gapdh1), phosphoglycerate kinase (Pgk), phosphoglyceromutase (Pglym) and pyruvate kinase (PyK) accumulate specifically and synchronously in developing muscles during the embryonic stage, suggesting that glycolysis-stimulated biomass production is a core myogenic program that operates in both invertebrate and vertebrate embryos, and promotes the formation of syncytial muscles (Tixier et al., 2013). Expression of PFK in muscle of callipyge goats is higher than that in normal goats, especially in goats aged 20 days when hypertrophy begins. It is speculated that high expression of PFK may generate energy for muscle protein synthesis (Fleming-Waddell et al., 2007). In one study, transcriptome profiles were determined for the hepatopancreas of E. sinensis during ecdysis (molting), postmolt, intermolt and premolt stages, and numerous genes related to glycolysis pathways were highly expressed during the intermolt stage, including ADP-dependent glucokinase (adpgk), phosphoglucose isomerase (pgi), phosphofructokinase (pfk), triosephosphate isomerase (tpi1b), phosphoglycerate kinase (pgk) and phosphoglyceratemutase (pgam2) (Huang et al., 2015). In one study on the Tribolium castaneum chitin biosynthetic pathway, gene knockdown of trehalase decreased the transcription of genes related to chitin synthesis and energy generation pathways, including those encoding hexokinase (HK), glucose 6-phosphate isomerase (GPI), fructose 6-phosphate transaminase (GFAT) and PFK; it also caused dysecdysis and high mortality (Tang et al., 2016). Taken together, these studies indicate that the glycolysis pathway is closely related to muscle growth and molting in crustaceans, and as the key rate-limiting enzyme in glycolysis, PFK plays an important role.

Our previous miRNA and transcriptome analyses revealed that novel miR-26 is differentially expressed in abdominal muscle of female shrimps during different growth rate stages (Li et al., 2020). We also investigated pfk, a key target gene of novel miR-26, using bioinformatics analysis, and revealed differences in expression levels during different growth rate stages (Li et al., 2020). Here, we explored the mechanism by which miRNAs regulate the growth of N. heteropoda through PFK.

Neocaridina heteropoda Liang 2002 were purchased from a local aquatic product market in the city of Ningbo, China, and acclimated at the aquaculture center of Tianjin Normal University. Shrimps were maintained in aerated water at 25°C and pH 7.

microRNA.org (http://www.microrna.org/microrna/home.do) was used to predict target genes of novel miR-26, and an RNA hybridization program (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/) was then employed to predict the minimum hybridization free energy between target genes and novel miR-26.

Total RNA was extracted from abdominal muscles using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer's protocol, and then reverse-transcribed by PrimeScript RT Master Mix (TaKaRa, Shiga, Japan) for subsequent analysis. Real-time RT-PCR assays were carried out using AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) with 18S rRNA as the internal reference gene. The nucleotide sequence of pfk cDNA was submitted to GenBank under accession number MK685666. All primers used in this study are listed in Table S1.

A mirVana miRNA Isolation Kit (Invitrogen, Carlsbad, CA, USA) was used to obtain miRNAs from abdominal muscles according to the manufacturer's protocol. A Bulge-Loop miRNA qRT-PCR Starter Kit (RiboBIO, Guangzhou, China) was then used to synthesize cDNAs from miRNA templates and to amplify novel miR-26 and reference U6 snRNA (small nuclear RNA) molecules. RT-PCR assays were performed on a Roche Light Cycler 480II system (Roche, Basel, Switzerland).

HeLa cells (ATCC, CCL-2™, passage number 10) were maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Invitrogen).

miRNA agomirs, antagomirs and their negative controls were purchased from RiboBIO (Guangzhou, China). Wild-type and mutant DNA fragments of the 3′-UTR of the PFK gene were synthesized by GENEWIZ (Beijing, China).

To construct recombinant vectors, the corresponding target region in the 3′-UTR of pfk mRNA was inserted downstream of the luciferase reporter gene in the pmirGLO vector (Promega, Madison, WI, USA). As a control, we used the pfk pmirGLO construct containing a mutant binding site (T substituted for C, A substituted for G). HeLa cells (10⁵–10⁶) were maintained in 12-well plates, transfected with recombinant vectors and miRNA agomirs using Lipo2000 (Thermo Fisher Scientific), and cultured until covering 60−70% of the dish. At 24 h after transfection, pyrolysis buffer was used for cell lysis, and luciferase activity was measured on an Infinite M200 instrument (Tecan, Mannedorf, Switzerland). All experiments were repeated at least three times.

Muscle tissue was ground with a sterilized pestle and mortar, and proteins were extracted using tissue protein extraction reagent (Thermo Fisher Scientific) containing phenylmethylsulfonyl fluoride (PMSF). Proteins were separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) using a dedicated transfer apparatus (Bio-Rad, Hercules, CA, USA). The membrane was incubated with PFK-specific (1:1000, custom made) or β-tubulin-specific (1:1000; Proteintech, Rosemont, IL, USA) antibody. Western blot assays were performed using ECL Plus (Millipore).

All shrimps used in this study were females of the same size in the intermolt stage. Double-stranded (ds)PFK and dsGFP were injected into abdominal muscle (between the third and fourth abdominal segments) using a micro-injection system (WPI, Sarasota, FL, USA). Each shrimp was injected with 1 μg dsRNA dissolved in 500 nl sterilized RNAse-free water, and supplementary injections were made every 72 h. External skeletons were counted every day, and shrimps were photographed and subjected to length measurement after 30 days.

Agomirs and antagomirs of novel miR-26 and ecdysone 20E were injected into shrimp muscle (between the third and fourth abdominal segment) using a micro-injection system (WPI). Each shrimp was injected with 800 nl, and the concentrations of agomirs, antagomirs and ecdysone 20E were 20 μmol l⁻¹, 20 μmol l⁻¹ and 2000 ng ml⁻¹, respectively. Molted skin was counted every day, and animals were photographed and subjected to length measurement after 30 days. At 0.5, 12, 24 and 36 h after injection, levels of pfk mRNA were measured in test and control groups, and levels of PFK protein were measured at 12 and 24 h.

The content of fructose 1,6-bisphosphate was analyzed using a dedicated fructose 1,6-diphosphate research kit (Jianglai, Shanghai, China). Muscle tissue was rinsed using pre-cooled phosphate-buffered saline (PBS; 0.01 mol l⁻¹, pH 7.4) to remove residual blood. Muscle samples were weighed and cut into small fragments and homogenized in PBS (100 mg muscle in 500 μl PBS) on ice by repeated freezing and thawing. Then, the homogenate was centrifuged at 5000 g for 5−10 min and the supernatant was used for detection.

Wells were divided into standard and test groups; standard wells were treated with 50 μl standard solution at different concentrations (0, 2.5, 5, 10, 20 and 40 μg ml⁻¹), and 50 μl samples were added to test wells (but not blank wells). Test wells (but not standard or blank wells) were treated with 50 μl of biotin-labeled antibody and incubated at 37°C for 30 min. After incubation, well solutions were discarded and 350 μl wash buffer was added and incubated for 1 min. Then, wash buffer was discarded and plates were wiped dry. The wash step was repeated five times. All wells except blanks were treated with 100 μl horseradish peroxidase-conjugated antibody and incubated at 37°C for 30 min. Solution A or B was added separately to wells and incubated at 37°C for 15 min in the dark, and the absorbance at 450 nm (OD₄₅₀) was measured. All experiments were conducted independently at least three times, and SPSS (version 11.5; Chicago, IL, USA) was used for statistical analysis (P<0.05 was considered statistically significant).

According to the results of miRBase analysis (Li et al., 2020), pfk appears to be a potential target of novel miR-26 in N. heteropoda, as there are potential novel miR-26 binding sites in the 3′ UTR of pfk (Fig. 1A). The minimum free energy of hybridization between the pfk sequence and novel miR-26 was predicted using RNAhybrid software, and the results further supported binding between novel miR-26 and pfk (Fig. 1B). To verify whether novel miR-26 can interact with the 3′-UTR region of pfk in vivo, we constructed a pmirGLO vector containing the potential novel miR-26 binding site for reporter gene detection. Dual-luciferase activity was determined 24 h after transfection and incubation with novel miR-26 agomirs. The results showed that dual-luciferase activity in the experimental group was decreased by 40−50% compared with that in the control group, and the inhibition of luciferase activity increased with increasing concentration of novel miR-26 analog (Fig. 1C). Together, the above results confirmed that pfk is a direct target gene of novel miR-26.

As novel miR-26 can directly interact with pfk, we investigated the effects on PFK of artificially increasing or decreasing miR-26 in shrimps. To this end, we injected agomirs and antagomirs of novel miR-26 into shrimps and measured PFK protein levels by western blotting. The results showed that the relative PFK protein content in the experimental group decreased gradually at 24, 48 and 72 h after injection of novel miR-26 agomir (Fig. 2A). By comparison, at 24, 48 and 72 h after injection of novel miR-26 antagomir, the PFK protein content in the experimental group was, respectively, 1.72-, 1.21- and 1.24-fold higher than in the control group (Fig. 2B). Thus, overexpression of novel miR-26 decreased the abundance of PFK protein, while inhibition of novel miR-26 increased PFK protein levels.

We injected the abdomen muscle of shrimps with GFP/PFK dsRNA (dsGFP, dsPFK) every 3 days, measured body length at different time points, and counted the daily number of molted skins of each group. At 25 days after injection, the body length of shrimps in the test group was significantly shorter than that in the control group (Fig. 3A,C). The cumulative molting rate reached 100% at 7 days after injection of dsGFP, while the cumulative molting rate after injection of dsPFK took 12 days to reach 100% (Fig. 3B), suggesting that PFK may promote the process of ecdysis. To further verify the regulatory effects of miRNAs on PFK, we also conducted injection experiments using novel miR-26 antagomirs and agomirs. We found that at 30 days after injection of the antagomir, the average body length of shrimps in the test group was significantly increased compared with that of controls (Fig. 3D,F). Additionally, the cumulative molting rate after injection of novel miR-26 antagomir was more obviously different between groups. After 24 days, the cumulative molting rate reached 554% in the test group and 470% in the control group (Fig. 3E). By comparison, at 30 days after injection of agomir, the average body length of shrimps in the test group was significantly decreased compared with controls (Fig. 3G,I). There were no apparent differences in cumulative molting rate between test and control groups within 9 days of injection, but there was clear differentiation between the two groups at subsequent time points, and differences were greatest after 17 days. The cumulative molting rate reached 268% in the experimental group and 334% in the control group (Fig. 3H).

The results described above suggest that miRNAs and PFK are intimately involved in the molting process. To directly verify their relationship with molting, female shrimps at the same molting stage and with similar body length were injected with ecdysone 20E or solvent alone (control). Relative expression of the pfk gene was then detected after 0.5, 12, 24 and 48 h at the mRNA level by qPCR using 18S rRNA as a reference, and at the protein level by western blotting using β-tubulin as a reference protein. The results showed that expression of PFK was up-regulated at 0.5 h after ecdysone 20E injection, and up-regulation lasted for 12 h. The pfk gene was down-regulated 24 h after injection and slightly up-regulated at 48 h (Fig. 4A). In terms of PFK protein levels, the relative expression ratio of control and test groups was 0.71:1.00 at 12 h and 1.00:0.64 at 24 h (Fig. 4B).

We also investigated the expression of novel miR-26 at these four time points and found that it was negatively correlated with expression of the pfk gene. Novel miR-26 expression was strongly inhibited at 0.5 h and inhibition lasted until 12 h, before returning to normal (Fig. 4C). Interestingly, changes in gene and miRNA expression coincided with ecdysis; molting of shrimps occurred successively in the test group between 12 and 24 h, but not in the control group (Fig. 4D).

The most important role of PFK in cells is to act as a rate-limiting enzyme in glycolysis. As miRNAs regulate PFK and thereby affect body length growth in shrimps, we investigated whether this is related to the classical glycolysis function of PFK in glycometabolism. We therefore measured the expression of all enzymes in the glycolysis pathway following injection of ecdysone 20E (Fig. 5). The results showed that only three enzymes, Glut1, PFK and Pgk, were up-regulated 12 h after ecdysone 20E injection, while others were down-regulated compared with the control group. At 24 h after injection, when most shrimps had molted, expression of most genes was down-regulated compared with levels before molting (12 h), except for those encoding Pk and Tpi. At 48 h, expression of most genes had recovered slightly compared with levels at 24 h.

Overall, glycolysis pathways appear to be inactive under stimulation during ecdysis. Thus, miRNAs decrease the inhibition of PFK, resulting in increased PFK expression, which may elevate the abundance of intermediate metabolites such as fructose 1,6-bisphosphate (FDP). We therefore measured FDP content at 0.5, 12, 24 and 48 h after ecdysone 20E injection, and the highest FDP content was 13.7 µg ml⁻¹ at 0.5 h, while the lowest content was 8.5 µg ml⁻¹ at 24 h, consistent with the high expression of PFK at these two time points.

We next investigated the purpose of accumulating a large amount of FDP before molting. Based on one of the most important physiological functions of FDP, excluding glycolysis, we measured expression of the eIF2 β-subunit. We found that expression peaked at 12 h (before molting) and recovered at 24 h (after molting), consistent with the observed changes in PFK expression and FDP content. Overall, these results indicate that novel miR-26 negatively regulates the expression of pfk, and this impacts the function of eIF2 and the protein translation system, thereby mediating signal transduction via FDP.

In our previous work, novel miR-26 was identified by high-throughput sequencing of muscle samples from N. heteropoda at different growth stages (Li et al., 2020). We found that novel miR-26 was highly expressed during slow growth stages and expressed at low levels during faster growth stages, implicating it as a potential regulatory factor inhibiting growth in shrimps.

pfk was identified as a candidate target gene of novel miR-26. PFK is a rate-limiting enzyme in glycolysis that catalyzes the transfer of phosphoric acid from ATP to fructose 6-phosphate. After knocking down the pfk gene, the molting rate was decreased. Interfering with PFK expression may therefore prolong the molting cycle of shrimps and slow down the growth of individuals. When we injected the agomir and antagomir of novel miR-26 into the abdominal muscles, the molting cycle was prolonged and shortened, respectively, and the growth of individuals was correspondingly slowed down and accelerated. There were significant differences in body size. However, changes in molting rate and body length caused by agomir injection were not as obvious as those caused by antagomir injection. This may be due to the fact that endogenous miRNAs are in a near-saturated state for regulating pfk; hence, the effect of exogenous agomir is not obvious in this instance. As molting and growth are closely correlated, we believe that novel miR-26 may regulate the growth of individual shrimps by negatively regulating pfk expression. To prove that this was not a special phenomenon specific only to N. heteropoda, we performed the same experiment on another shrimp, M. rosenbergii. The relative expression of pfk in abdominal muscle of M. rosenbergii was measured at different growth rates, and the results showed that pfk expression was positively correlated with growth rate (Fig. S1). This consistency with the results for N. heteropoda further confirmed our speculation that PFK is closely correlated with growth, possibly in all crustaceans. This hypothesis will be validated in the future.

Molting in crustaceans is a highly complex process, and ecdysone 20E is the most important hormone promoting this process (Kim et al., 2010). At 12 h after ecdysone 20E injection, all shrimps had molted completely. Expression of pfk gene increased before molting and decreased after molting, indicating that PFK may respond to molting signals. There was a strong negative correlation between novel miR-26 and pfk expression at different time points. We therefore speculate that ecdysone 20E may regulate the expression of PFK by affecting the expression of novel miR-26, but the relationship is not obvious from the perspective of PFK alone. We subsequently examined the expression levels of other genes in the glycolysis pathway following ecdysone 20E injection and found that glut1 gene expression was up-regulated before molting, suggesting that more glucose was transported into cells, but only pfk was up-regulated among the three major rate-limiting glycolytic enzymes Hex, Pk and PFK, while other enzymes were down-regulated compared with the control group. This indicates that the glycolysis pathway was inhibited following ecdysone 20E injection. However, we believe that up-regulation of PFK and down-regulation of aldolase (Ald) may lead to the accumulation of FDP. We found that the FDP content increased when PFK was increased and decreased when PFK was decreased. FDP reportedly activates eIF2B, an essential molecule for generating sufficient eIF2-GTP for translation initiation and protein synthesis (Rabinovitz, 1996). We detected the expression of eIF2β at four time points after ecdysone 20E injection, and the relative expression of eIF2β was increased only at 12 h, indicating that protein synthesis was most vigorous at this time point. Conversely, up-regulation of Pgk and inhibition of downstream pgam and enolase (Eno) expression at this time point may result in accumulation of 3-phosphoglycerate, the precursor of serine, which not only plays a role in protein synthesis but also provides a carbon unit involved in maintaining nucleotide biosynthesis (Possemato et al., 2011; Samanta and Semenza, 2016). Serine also plays a role in lipid and fatty acid metabolism, reproduction and muscle growth, and it is needed to maintain a healthy immune system (Brauer et al., 2019; Brown et al., 2016; Li and Zhang, 2016; Oslund et al., 2017). This may explain why expression of Pgk, pgam and Eno was altered in this way to prepare for the following molting process.

In conclusion, we identified a novel miRNA in N. heteropoda that regulates the expression of pfk in muscle and participates in growth and molting. Furthermore, under stimulation by ecdysone 20E, glycolysis in shrimp muscle was inhibited overall, although some enzymes were up-regulated. In response to ecdysone 20E signals, novel miR-26 appears to indirectly regulate eIF2 by targeting pfk and the accumulation of the intermediate metabolite FDP. The novel miR-26 ultimately affects pre-ecdysis protein synthesis. Our results provide a new perspective for understanding molting and growth mechanisms in crustaceans, as well as a reference for identifying future gene editing targets.