Caenorhabditis elegans: An important tool for dissecting microRNA functions

Caenorhabditis elegans (C. elegans), a member of the phylum Nematoda, carries the evolutionarily conserved genes comparing to mammals. Due to its short lifespan and completely sequenced genome, C. elegans becomes a potentially powerful model for mechanistic studies in human diseases. In this mini review, we will outline the current understandings on C. elegans as a model organism for microRNA (miRNA)-related research in the pathogenesis of human diseases. Correspondence to: Yang Jin, MD, Ph.D., Division of Pulmonary and Critical Care, Department of Medicine, Boston University Medical Campus, 72 East Concord St., Boston, MA 02118, USA, E-mail: yjin1@bu.edu


Introduction
In the 1960s, Dr. Sydney Brenner first proposed using C. elegans as a model organism for the investigations focused on the neural development in animals [1]. Two decades later, the lineage of all 959 cells in the adult C. elegans were identified in Dr. John Sulston's lab [1]. Furthermore, the first C. elegans gene was cloned, and the physical map was constructed [2]. One important milestone in biological research is that C. elegans is also the first multi-cellular organism with complete genome sequenced [3].
C. elegans is a round worm, a member of the phylum Nematoda, and lives in the aqueous layer between the soils [1]. Its adult body only is approximate 1 mm in full length and 70 µm in diameter [4]. Bacteria (usually E. Coli strains in laboratory) serve as the food for C. elegans [5]. Interestingly, the bodies of C. elegans are transparent, providing convenience for investigators to observe the worm organs such as the intestine, gonads, etc under the microscope with no needs of staining. Furthermore, under a high-power phase-contrast microscope, C. elegans can be observed at a single cell resolution [6]. Therefore, C. elegans is a good model to observe cell division, differentiation and death. The life cycle of C. elegans is short. It only takes 3-4 days to reach the adult stage [7]. The embryonic development of C. elegans is much quicker comparing to other animals. Only after approximate 12 hours at 25°C, one can observe the evolution of each single cell through the entire development [8]. The culture of C. elegans is also convenient. Generally, agar plates using the Petri dishes serve as a good condition for them [9]. Thus, hundreds of plates for genetic mutation screening can be easily prepared in one lab. Conveniently, the long-term storage of C. elegans only requires -80°C freezer. The L1 or L2 stage of worms can recover in the room temperature. Moreover, C. elegans can be immersed in a solution containing the designated nucleic acids to acquire exogenously delivered gene modifiers, instead of requiring tedious transfection procedures. Also, the RNA interference can be performed by feeding the C. elegans with bacteria expressing siRNA [9].
MicroRNAs (miRNAs) are a group of highly conserved noncoding RNAs and approximately 22 nucleotides in length [10]. Most of primary miRNAs (pri-miRNAs) are transcribed in the nucleus. After transcription, pri-miRNAs are then processed by microprocessor complex. Microprocessor complex binds to the stem-loop structure of pri-miRNAs and cleaves the primary transcripts to generate a hairpin-shaped RNA molecule known as precursor miRNAs (pre-miRNAs) [10]. These double-stranded pre-miRNAs are composed of 70-100 nucleotides each and subsequently transported from nucleus to the cytoplasm. Dicer is required for the mature of pre-miRNAs in the cytoplasm. The mature miRNA duplex is recognized by the RNA induced silencing complex (RISC) containing Dicer and AGO2 (argonaute RISC catalytic component 2), which are essential to miRNA-induced silencing [10]. Only one strand of miRNA duplex can be incorporated into the RISC to form miRISC, while the other strand, named miRNA* is mostly degraded. The miRNA loaded RISC binds to the target mRNAs and silence these gene expression through either degradation of mRNA or inhibition of translation at posttranscriptional level [10].
In the process of discovering miRNAs, C. elegans played an important role as a model organism. In 1993, Lee et al. found that the gene lin-4 is not encoded for the protein, but encoded a small RNA [11]. They predicted that the lin-4 binds to the lin- 14

Cell death
C. elegans is an easy model to study survival and death. C. elegans has also been used as space animals for several times. Gao et al. found 17 miRNAs which are involved in the space radiation-caused death [16].

Aging
The different miRNAs can control the same target gene. On the other hand, one miRNA can regulate a variety of target genes [18]. C. elegans can be used as a good model for this type of studies, given its relatively easy-signaling pathways. MiR-34 was the first identified miRNA up-regulating in C. elegans [19]. MiR-34 can inhibit autophagy by targeting atg-9a [20]. Lencastre et al. identified that 17 miRNAs are altered during aging processes. Moreover, they prove that miR-71, miR-238, and miR-246 have the function of increasing longevity, demonstrated using mutant worms. On the other hand, miR-239 can decrease life span [21]. MiR-71 interacts with the DNA damage response signaling pathways via CDC-25.1 and CHK-1 mediated pathways. The expression of CDC-25.1 in aged miR-71 knock-out worms is increased by 8-folds [21]. Despite that several screenings have suggested that the aging process relates to miRNAs, the mechanisms underlying the miRNA-mediated regulation of longevity remain to be further determined [21,22].

Metabolism
Dietary restriction (DR) is the most effective way to reduce agerelated phenotypes and to extend lifespan, it can also promote longevity and protect against age-associated disease across species. C. elegans is a great model to examine the molecular mechanisms by which miRNAs coordinate food intake with health-promoting metabolism [23]. Vora et al. found that mir-80 is a major regulator for the DR state [24]. MiR-80 knock-out worms maintain cardiac and skeletal muscle-like function at older age, reduce accumulation of lipofuscin, and extend lifespan. These functions of miR-80 is very similar to the physiological features of DR [24]. With food limitation, decreased miR-80 levels upregulate CBP-1 protein levels to engage metabolic loops that promote DR [24]. Also, miR-71 and miR-228 mediate dietary-restriction-induced longevity [25]. Furthermore, PHA-4 and SKN-1 are negatively regulated by miR-228. On the other side, miR-71 represses PHA-4 [25].

Innate immunity
The let-7 family is initially found relating to the innate immunity of C. elegans [26]. The let-7 family mutant worms are more resistant to pathogen infections by downregulating of heterochronic genes and the p38 MAPK pathways [26]. The developmental timing signal or ATF-7 regulate hbl-1 to control seam cell proliferation via regulating miR-48, miR-84 and miR-241 [26].

Development
C. elegans is an easy model to study the development, given that it only takes 2-3 days for this round worm to grow from eggs to adults. Karp et al. identified 14 miRNAs which are related the development of C. elegans. Most development miRNAs are altered during the stages from L2 to L2d or L2 molt to dauer [27]. The second found miRNA, let-7, has an important role in embryonic developing [28]. Let-7 regulates the transition process from L4 stage to adult stage. Without let-7, worms can't grow into mature stage due to uncontrolled downregulation of hbl-1, lin-4 and daf-12 [12,29]. Further studies found that let-7 family members miR-48, miR-84, and miR-241 all regulate developmental timing from the stage L2 transit into L3. The hbl-1 is also a target similar to let-7 [29]. MiR-51 family contains miR-51, miR-52, miR-53, miR-54, miR-55, and miR-56. This family has also been proved involved in the early development stage. Mutant worms of these miRNAs have the phenotypes of unattached penetrant pharynx [30,31]. Additionally, CDH-3, the mammalian fat cadherin homolog, is the target of the miR-51 family miRNAs [30].

Stress response
The miR-360 knock-out worms were also proved to increase the function of glycyrrhizic acid [32]. And the same group prove that graphene oxide can regulate miR-360 to decrease DNA damageapoptosis signaling by targeting CEP-1 [33,34]. Also miR-355 could regulate MWCNTs toxicity by target daf-2, the gene encodes for the insulin-like growth factor 1 [35] (Table 1).