InteractiveFly: GeneBrief

mir-8 stem loop: Biological Overview | References

BIOLOGICAL OVERVIEW

How body size is determined is a long-standing question in biology, yet its regulatory mechanisms remain largely unknown. This study finds that a conserved microRNA miR-8 and its target, U-shaped (USH), regulate body size in Drosophila. miR-8 null flies are smaller in size and defective in insulin signaling in fat body that is the fly counterpart of liver and adipose tissue. Fat body-specific expression and clonal analyses reveal that miR-8 activates PI3K, thereby promoting fat cell growth cell-autonomously and enhancing organismal growth non-cell-autonomously. Comparative analyses identify USH and its human homolog, FOG2, as the targets of fly miR-8 and human miR-200, respectively. USH/FOG2 inhibits PI3K activity, suppressing cell growth in both flies and humans. FOG2 directly binds to p85α, the regulatory subunit of PI3K, and interferes with the formation of a PI3K complex. This study identifies two novel regulators of insulin signaling, miR-8/miR-200 and USH/FOG2, and suggests their roles in adolescent growth, aging, and cancer (Hyun, 2009).

Animal body size is a biological parameter subject to considerable stabilizing selection; animals of abnormal size are strongly selected against as less fit for survival. Thus, the way in which body size is determined and regulated is a fundamental biological question. Recent studies using insect model systems have begun to provide some clues by showing that insulin signaling plays an important part in modulating body growth. The binding of insulin (insulin-like peptides in Drosophila) to its receptor (InR) triggers a phosphorylation cascade involving the insulin receptor substrate (IRS; chico in Drosophila), phosphoinositide-3 kinase (PI3K), and Akt/PKB. An active PI3K complex consists of a catalytic subunit (p110; dp110 in Drosophila) and a regulatory subunit (p85α; dp60 in Drosophila). Phosphorylated Akt (p-Akt) phosphorylates many proteins -- including forkhead box O transcription factor (FOXO) -- which are involved in cell death, cell proliferation, metabolism, and life span control. Once activated, the kinase cascade enhances cell growth and proliferation (Hyun, 2009).

Organismal growth is achieved not only by cell-autonomous regulation but also by non-cell-autonomous control through circulating growth hormones. Recent studies in insects indicate that several endocrine organs, such as the prothoracic gland and fat body, govern organismal growth by coordinating developmental and nutritional conditions. However, detailed mechanisms of how body size is determined and modulated remain largely unknown (Hyun, 2009).

microRNAs (miRNAs) are noncoding RNAs of ~22 nt that act as posttranscriptional repressors by base-pairing to the 3' untranslated region (UTR) of their cognate mRNAs. The physiological functions of individual miRNAs remain largely unknown. Studies of miRNA function rely heavily on computational algorithms that predict target genes. In spite of their utility, however, these target prediction programs generate many false-positive results, because regulation in vivo depends on target message availability and complementary sequence accessibility. To overcome the difficulties in identifying real targets, various experimental approaches have been developed, including microarrays, proteomic analyses, and biochemical purification of the miRNA-mRNA complex. Genetic approaches using model organisms can also be useful tools for studying the biological roles of miRNAs at both the organismal and molecular levels. Despite these advances, however, it is still a daunting task to understand the biological function of a given miRNA and to identify its physiologically relevant targets (Hyun, 2009).

This study found using Drosophila as a model system that conserved miRNA miR-8 positively regulates body size by targeting a fly gene called u-shaped (ush) in fat body cells. It was further discovered that this function of miR-8 and USH is conserved in mammals and that the human homolog of USH, FOG2, acts by directly binding to the regulatory subunit of PI3K (Hyun, 2009).

Small body phenotype of the mir-8 mutant

: In a screen for miRNAs that modulate cell proliferation, it was observed that human miR-200 miRNAs (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) promote cell growth when transfected into several human cell lines (Park, 2009). Members of the miR-200 family are upregulated in certain cancers, including ovarian cancer, consistent with the observation of the proliferation promoting effects (Iorio, 2007; Nam, 2008). Because the miR-200 family is highly conserved in bilaterian animals, with miR-8 being the sole homolog in Drosophila, D. melanogaster was used in order to uncover the biological function of the miR-200 family (Hyun, 2009).

The phenotype of the miR-8 null fly was first analyzed using mir-8^δ2. It has been shown that mir-8 mutation results in increased apoptosis in the brain and frequent occurrence of malformed legs and wings (in about one-third of the mutants) (Karres, 2007). Interestingly, in addition to these phenotypes, it as found that miR-8 null flies are significantly smaller in size and mass than their wild-type counterparts (Hyun, 2009).

The determination of the final body size in insects during the larval stage is analogous to that which occurs during the human juvenile period. It is generally known that reduced body size in insects is caused by either slow larval growth, precocious early pupariation that shortens the larval growth period, or both. It was observed that, at 100 hr after egg laying (AEL), miR-8 null larvae exhibit a significantly smaller body volume than do wild-type larva. The onset of pupariation in miR-8 null flies was not significantly different from that in wild-type flies, and adult emergence was slightly delayed (~12 hr). Thus, the smaller body size of miR-8 null flies is likely to be caused by slower growth during the larval period rather than by precocious pupariation. Insufficient food intake has been reported to accompany either precocious or delayed pupariation, depending on the onset of reduced feeding. However, the levels of Drosophila insulin-like peptides (Dilps), which are known to be reduced in starvation conditions, were not downregulated in miR-8 null larvae. Given the unaffected onset time of pupariation and the levels of Dilps in this animal, the small body size of miR-8 null flies is unlikely due to reduced feeding (Hyun, 2009).

Next, it was asked whether the small body phenotype was caused by a reduction in cell size, cell number, or both. Cell size and number were measured and it was found that cell number was reduced in the wing in miR-8 null flies, whereas cell size was not significantly different from that of wild-type. Thus, assuming that similar regulation takes place in other body parts, the reduced growth in the peripheral tissues of the miR-8 null flies may be ascribed to decreased cell number rather than reduced cell size (Hyun, 2009).

To understand why miR-8 null animals grow slowly, the activities of the proteins involved in insulin signaling were examined in the miR-8 null flies. The level of activated Akt was measured by Western blotting using a p-Akt-specific antibody. The p-Akt level was reduced in the mutant flies, suggesting that Akt signaling is impaired in the absence of miR-8. Activated p-Akt is known to inactivate FOXO via phosphorylation. Phosphorylation prevents nuclear localization of FOXO, which, in turn, results in the reduction of transcription of FOXO target genes. Consistent with the reduced level of p-Akt, the FOXO target gene, 4EBP, was increased in mir-8 mutant larvae, indicating that insulin signaling is indeed significantly reduced in the miR-8 null animal (Hyun, 2009).

Fat body-specific expression of miR-8 rescues the small body phenotype

The spatial expression pattern of miR-8 was examined in larvae using a mir-8 enhancer trap GAL4 line (hereafter referred to as mir-8 gal4), which, when combined with UAS-GFP, expresses GFP under the control of the mir-8 enhancer (Karres, 2007). In addition to the signals in the brain, wing discs, and leg discs that were previously observed by Karres, strong signals were noticed in the fat body. The level of miR-8 was determined by Northern blotting using total RNA prepared from different larval organs, which showed that miR-8 is indeed highly abundant in the fat body (Hyun, 2009).

Recent studies suggested that Drosophila fat body may be an important organ in the control of energy metabolism and growth. Therefore, it was reasoned that if miR-8 in the larval fat body is critical for body size control, exclusive expression of miR-8 in the fat body alone should alleviate the whole body size defect observed in the mir-8 mutants. To test this idea, transgenic flies were generated to specifically reintroduce miR-8 into the fat bodies of mir-8 mutant larvae using a fat body-specific GAL4 driver, Cg gal4 (CgG4). Remarkably, miR-8 expression in the fat body alone rescued the phenotype to near wild-type levels in both body weight and body size, suggesting that miR-8 in the fat body is important for systemic body growth. Another interesting observation was that the miRNAs from the human miR-200c cluster, which includes miR-200c and miR-141, could also yield a comparable rescue effect. Human miR-200 family miRNAs, which are located in two chromosomal clusters, have extensive homology to miR-8. The fact that miRNAs of the human miR-200c cluster effectively compensate for the loss of miR-8 suggests that these human miRNAs can be processed by the Drosophila miRNA processing machinery and that they share a conserved biological function. Because CgG4 is expressed in the anterior lymph gland as well as in the fat body, an additional GAL4 driver, ppl gal4 (pplG4), was used that is active mainly in the fat body and slightly in the salivary gland (Zinke, 1999). Similar rescue effects were observed with pplG4, in support of the fat body-specific function of miR-8 (Hyun, 2009).

USH is a critical target of miR-8 in the regulation of body growth

To further understand the molecular function of miR-8, miR-8 target genes responsible for body size control were identified. Because miR-8 is a highly conserved miRNA and because the human homologs promote cell proliferation in human cells, it was assumed that the targets involved in the cell proliferation phenotype may be conserved throughout evolution. To discover such conserved targets,the candidate target genes of both fly miR-8 and human miR-200 miRNAs were listed using five different target prediction programs. The two groups of putative targets, one from flies and the other from humans, were then compared with each other to identify homologous gene pairs. The Drosophila gene u-shaped (ush) and its human homolog Friend of GATA 2 (FOG2, also known as ZFPM2) were the most frequently predicted gene pair conserved in both species. From the target candidate list, 15 genes, including ush/FOG2, were selected that are known as tumor suppressors or negative regulators of cell proliferation in at least one species. These target genes were validated with luciferase reporter plasmids. Briefly, each target's 3' UTR, which contains the miRNA target sites, was inserted downstream of a luciferase gene. The reporter activities were measured following cotransfection of miRNAs and the reporter plasmids into Drosophila S2 or human HeLa cells. After extensive reporter assays, seven gene pairs were identified that respond both to miR-8 and to miR-200 family miRNAs in fly and human cells, respectively (Hyun, 2009).

To examine which targets among the candidates are physiologically relevant to the phenotype observed, the candidate genes were knocked down in the fat body of miR-8 null flies and it was asked whether the knockdown could rescue the small body phenotype. Using the UAS-RNA interference (RNAi) lines obtained from the Vienna RNAi Library Centre, dsRNAs of five candidate genes were expressed in the fat body of mir-8 mutants using CgG4. Lap1 knockdown was unsuccessful and, thus, did not rescue the mir-8 mutant phenotype. Among the RNAi lines tested, the one against ush rescued the dwarf phenotype most dramatically. RNAi of ush in wild-type background did not significantly increase body weight, ruling out the possibility that the effects of ush knockdown and mir-8 mutation are additive (Hyun, 2009).

Because a previous study showed that miR-8 targets atrophin (atro) to prevent neurodegeneration (Karres, 2007), whether atro is also involved in body size regulation was tested. Knockdown of atro in the fat body, however, failed to rescue the small body phenotype of miR-8 null flies. Thus, the reported function of miR-8 in the prevention of neurodegeneration (Karres, 2007) may be separate from its function in body growth, not only spatially but also at the molecular level. To exclude possible off-target effects of ush RNAi, the ush¹⁵¹³ hypomorph, which expresses a reduced level of ush as the result of a mutation in the promoter region, was used. Consistent with the results of the ush RNAi, ush¹⁵¹³ heterozygotes have larger adult bodies than do the control flies. This result indicates that USH may indeed suppress body growth (Hyun, 2009).

Next, whether the level of USH was elevated in miR-8 null animals was examined. The endogenous ush mRNA level was determined by qRT-PCR analysis of the RNAs from whole larva or larval fat body. The ush mRNA is, indeed, significantly upregulated in the fat body of miR-8 null larvae (δ2.0 fold), suggesting that miR-8 suppresses ush in the fat body. Upregulation of ush mRNA in whole larval RNA was less prominent (~1.3 fold). Thus, ush may be more strongly suppressed in the fat body than in other body parts. Notably, USH protein levels are more dramatically affected than the mRNA levels, indicating that miR-8 represses USH production by both mRNA destabilization and translational inhibition. Furthermore, a point mutation of the miR-8 target site in the 3' UTR of ush abolished the suppression of the 3' UTR reporter, indicating that the suppression is mediated through the direct binding of miR-8 to the predicted target site. Putative target sites for miR-8 are found in all Drosophila species examined, including distant species such as D. virilis and D. grimshawi. Together, these results demonstrate that ush is an authentic target of miR-8 (Hyun, 2009).

miR-8 and USH regulate PI3K

Several reports have suggested that insulin signaling in the larval fat body controls organismal growth in a non-cell-autonomous manner (Britton, 2002; Colombani, 2005). In support of this idea, it was observed that suppression of insulin signaling in the larval fat body yielded smaller flies and fewer cells in the wing. This phenotype is similar to that of miR-8 null flies. To further investigate whether insulin signaling is indeed defective in the fat body of miR-8 null animals, the tGPH reporter, a GFP protein fused to a PH domain, was used. Using this technique, the activity of PI3K can be measured by monitoring the membrane-associated GFP signal, because PH domains bind to the membrane-anchored phosphatidylinositol-3,4,5-triphosphate (PIP₃) produced by PI3K. It was found that PI3K activity was downregulated in mir-8 mutant fat bodies and that FOXO was more strongly localized to the nucleus of mutant fat bodies than the controls. Moreover, the level of p-Akt was significantly reduced in the fat body, whereas the phosphorylated JNK level did not change. Thus, insulin signaling is specifically disrupted in the fat bodies of miR-8 null larvae (Hyun, 2009).

To more precisely analyze miR-8's function in fat cells, flip-out GAL4 overexpressing clones of miR-8 were generated in the fat body of mir-8 heterozygote. In the mosaic fat cells overexpressing miR-8, the tGPH signals was augmented in the membrane, indicating that miR-8 promotes PI3K activity in a cell-autonomous manner. Cell size also increased with miR-8 overexpression (Hyun, 2009).

Next mitotic null clones were generated to observe the loss of function phenotype. Cells of the miR-8 null clone were smaller than the adjacent cells in the twin spot -- the cells harboring wild-type copies of miR-8. This suggests that miR-8 promotes fat cell growth in a cell-autonomous manner, as expected if miR-8 enhances insulin signaling in the fat body. Fewer (or no) null clone cells were often observed next to the twin spot cells when the mitotic clones were induced at embryonic stage or newly hatched larval stage. This suggests the frequent failure of proliferation and survival of miR-8 null cells during larval development. It is noted that null clones of miR-8 were generated in the wing or eye disc but little growth defect was found in these organs. Therefore, the effect of miR-8 on cell growth is dependent on tissue type, which may be explained by the fact that USH is present in the fat body but not in wing precursor cells or the eye disc (Hyun, 2009).

To determine whether USH negatively regulates insulin signaling, mosaic clones of fat cells overexpressing USH were generated. USH-overexpressing cells were smaller in size and showed significantly lower tGPH signals in the membrane and higher FOXO signals in the nucleus than did the neighboring wild-type cells. Also mosaic fat cells expressing dsRNA against ush were created to observe the knockdown phenotype. The tGPH signal was significantly enhanced in the mosaic cells depleted of USH. In mosaic ush mutant cells, the nuclear FOXO signals decreased. Together, these observations indicate that USH inhibits insulin signaling upstream of or in parallel with PI3K in a cell-autonomous manner (Hyun, 2009).

Whether reduced insulin signaling caused by the absence of miR-8 could be rescued by knockdown of USH was further examined. Excessive insulin signaling is known to reduce the levels of insulin receptor (Inr) and cytohesin Steppke (step) through negative feedback by FOXO. These two targets of FOXO were upregulated in the fat body of miR-8 null larvae, whereas reintroduction of miR-8 dramatically reduced their expression. Notably, ush RNAi also restores the mRNA levels of the FOXO target genes Inr and step in mir-8 mutant fat bodies. Thus, the defect of insulin signaling in the fat body of miR-8 null larvae is at least partially attributable to elevated ush levels (Hyun, 2009).

MicroRNA miR-8 regulates multiple growth factor hormones produced from Drosophila fat cells

Metabolic organs such as the liver and adipose tissue produce several peptide hormones that influence metabolic homeostasis. Fat bodies, the Drosophila counterpart of liver and adipose tissues, have been thought to analogously secrete several hormones that affect organismal physiology, but their identity and regulation remain poorly understood. Previous studies have indicated that microRNA miR-8, functions in the fat body to non-autonomously regulate organismal growth, suggesting that fat body-derived humoral factors are regulated by imiR-8. This study found that several putative peptide hormones known to have mitogenic effects are regulated by imiR-8 in the fat body. Most members of the imaginal disc growth factors and two members of the adenosine deaminase-related growth factors are up-regulated in the absence of imiR-8. Drosophila insulin-like peptide 6 (Dilp6) and Imaginal morphogenesis protein-late 2 (Imp-L2), a binding partner of Dilp, are also up-regulated in the fat body of miR-8 null mutant larvae. The fat body-specific reintroduction of miR-8 into the miR-8 null mutants revealed six peptides that showed fat-body organ-autonomous regulation by miR-8. Amongst them, only Imp-L2 was found to be regulated by U-shaped, the miR-8 target for body growth. However, a rescue experiment by knockdown of Imp-L2 indicated that Imp-L2 alone does not account for miR-8's control over the insect's growth. These findings suggest that multiple peptide hormones regulated by miR-8 in the fat body may collectively contribute to Drosophila growth (Lee, 2014).

FOG2, a target of miR-200, regulates PI3K in humans

The ush mRNA has one predicted binding site for miR-8, whereas the mammalian ortholog of ush, FOG2, has at least three predicted sites for miR-200 family miRNAs. When all the putative sites in the 3' UTR reporter of FOG2 are mutated, the reporter became refractory to the miR-200 family miRNAs. These predicted sites in the 3' UTR of FOG2 are, therefore, responsible for miR-200-mediated FOG2 regulation. It is noted that miR-200a and miR-141 that have one nucleotide mismatch in the seed sequence suppressed the UTR reporter, albeit less effectively than the other members did, suggesting that the noncanonical target sites may also lead to repression (Hyun, 2009).

FOG2 is expressed in the heart, brain, testes, liver, lung, and skeletal muscle. Despite its relatively broad expression in adult tissues, little is known about the function of FOG2 beyond its role in embryonic heart development. The miR-200 miRNAs have also been reported to be expressed in various adult organs, including pituitary gland, thyroid, pancreatic islet, testes, prostate, ovary, breast, and liver. A correlation was sought between the expression of FOG2 protein and miR-200c cluster miRNAs in human cell lines derived from different organs. There is generally a negative correlation between miR-200 miRNAs and FOG2 in a given tissue type, consistent with a suppressive role for miR-200 in FOG2 regulation (Hyun, 2009).

Attempts were made to confirm the repression of FOG2 by miR-200 miRNAs. Transfection of miR-200 miRNAs significantly reduced FOG2 protein levels in hepatocellular carcinoma Huh7 cells that express relatively low but detectable levels of miR-200 and FOG2. In addition, the inhibition of miR-200 miRNAs by 2'-O-methyl oligonuclelotides antisense to miR-200 increased FOG2 protein levels in pancreatic cancer AsPC1 cells that express relatively high levels of miR-200 miRNAs. These data demonstrate that FOG2 is an authentic target of endogenous miR-200 miRNAs (Hyun, 2009).

Next, whether human miR-200 miRNAs have a conserved role in the modulation of insulin signaling was investigated, as in the case of fly miR-8 miRNA. Transfection of miR-200 increases p-Akt levels, whereas miR-200 inhibitors reduce p-Akt levels. Moreover, knockdown of FOG2 increased p-Akt levels, mimicking the effect of miR-200. Also, the effect of FOG2 on PI3K activity by immunocomplex kinase assay was tested using an antibody against p85α. When Hep3B cells were transfected with a FOG2-expression plasmid, IGF-1 (Insulin like growth factor-1) failed to induce PI3K activity, indicating that FOG2 suppresses PI3K. Consistent with this result, Akt was not phosphorylated in IGF-1-treated cells when FOG2 was ectopically introduced. The effect was further analyzed of miR-200 miRNAs on the downstream transducers of Akt. Because activated Akt represses FOXO activity, a luciferase reporter plasmid (pFK1tk-luc) containing eight FOXO-binding sites was used to determine the level of FOXO activity in cultured cells. The activity of this FOXO reporter in Hep3B cells was significantly repressed by transfection of miR-200 miRNAs and by FOG2 knockdown. Furthermore, treating the cells with miR-200 inhibitors elevated FOXO activity. Consistently, when FOG2 was overexpressed, FOXO activity was upregulated. In contrast to PI3K pathway components, the level of phosphorylated Erk was not significantly impaired. In addition, inhibitors of JNK or Mek1/2 did not affect miR-200-mediated FOXO regulation, whereas PI3K inhibitor abrogated this FOXO regulation by miR-200. Thus, these data suggest that miR-200 specifically modulates PI3K-Akt-FOXO signaling (Hyun, 2009).

Because stimulation of PI3K and Akt is known to facilitate cell proliferation and antagonize apoptosis, cellular viability was measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in Hep3B cells. Introduction of miR-200 miRNAs increased cell viability, whereas miRNA inhibitors produced the opposite effect (Hyun, 2009).

To investigate the action mechanism of FOG2, Western blotting was performed against p85α, p110, and IRS-1 following p85α immunoprecipitation. Notably, when FOG2 was expressed, reduced amounts of p110 and IRS-1 were coprecipitated with p85α. Thus, FOG2 may act as a negative regulator of PI3K by interfering with the formation of a IRS-1/p85α/p110 complex (Hyun, 2009).

FOG2 directly binds to p85α to inhibit PI3K

Although FOG2 is thought to be a nuclear transcriptional coregulator, several studies have reported that FOG2 also localizes to the cytoplasm. To confirm this finding, subcellular fractionation and Western blotting were performed. FOG2 was, in fact, observed predominantly in the cytoplasm rather than in the nucleus in HeLa and PANC1 cells. Immunostaining also showed cytoplasmic localization of FOG2 in HepG2 cells, suggesting a cytoplasmic role of FOG2 (Hyun, 2009).

Given that FOG2 suppresses PI3K and colocalizes with p85α, it is suspected that FOG2 may interact with PI3K. Notably, a significant amount of p85α, the regulatory subunit of PI3K, was coprecipitated with anti-FOG2 antibody. Interaction between FOG2 and p85α was also observed when the FOG2 was ectopically expressed in a FLAG-tagged form (Hyun, 2009).

To map the interaction domain of FOG2, several truncated mutants of FOG2 were generated. The mutants containing a FLAG-tag in the N termini were coexpressed with V5-tagged p85α and were analyzed by immunoprecipitation using anti-FLAG antibody. The results indicate that the middle region of FOG2 (507-789 aa) mediates the interaction with p85α. It was then asked whether the middle region is sufficient to inhibit PI3K activity when it is ectopically expressed in HepG2 cells. The middle region suppressed PI3K, whereas neither the N-terminal part nor the C-terminal part had a significant effect on PI3K activity (Hyun, 2009).

To test whether FOG2 binds to p85α directly, the FOG2 protein was expressed and purified from bacteria and was used in an in vitro binding assay, along with purified recombinant p85α protein fused to GST. The recombinant FOG2 protein containing the middle region of FOG2 (413-789 aa) specifically bound to recombinant p85α (Hyun, 2009).

Finally, it was asked whether FOG2 can directly inhibit p85α by performing an in vitro PI3K assay using recombinant FOG2. Addition of the recombinant FOG2 protein containing the middle region (FOG2[413-789]) to the immunoprecipitated PI3K complex significantly inhibited the PI3K activity. This finding suggests that direct binding of FOG2 to p85α leads to the inhibition of PI3K activity. Notably, it was also found that Drosophila USH physically interacts with Drosophila p60 (dp60, the fly ortholog of p85α) when dp60 is coexpressed with USH in human HEK293T cells. Therefore, the action mechanism of USH/FOG2 may be conserved across the phyla (Hyun, 2009).

This study has revealed two novel regulatory components of insulin signaling: miR-8/miR-200 and USH/FOG2. miR-8/200 negatively regulates USH/FOG2 through direct base-pairing to the 3' UTR of the ush/FOG2 mRNA. USH/FOG2, in turn, inhibits the formation of an active PI3K complex via direct interaction with dp60/p85α, the regulatory subunit of PI3K. In fly fat bodies, miR-8 suppresses ush, which causes cell-autonomous increase of fat cell growth. The roles of miR-8 and USH are conserved in mammals; miR-200 miRNAs target FOG2 to upregulate insulin signaling and cell proliferation in human cells. Given that the PI3K-Akt-FOXO pathway plays central roles in many developmental processes and that defects of this pathway have been associated with cancer, diabetes, neuropathology, and aging, further investigation of the miR-8/200 family and USH/FOG2 may contribute to the understanding and amelioration of such human diseases (Hyun, 2009).

In Drosophila, miR-8 posttranscriptionally represses USH, thereby activating insulin signaling, which results in cell-autonomous growth of fat body cells. This process also causes nonautonomous organismal growth, likely through the induction of humoral factors. In human liver cells, miR-200 posttranscriptionally represses FOG2, which directly binds to p85α and blocks the formation of an active PI3K complex. As such, the repression of FOG2 by miR-200 stimulates insulin signaling and cell proliferation (Hyun, 2009).

The results support and extend the emerging theory that the fat body is a central organ coordinating metabolic condition and global growth of the organism. It is proposed that miR-8 regulates the growth of peripheral tissues in a non-cell-autonomous manner by modulating the secretion of the humoral factors that are under the control of insulin signaling (see A model for the functions of miR-8/miR-200 and USH/FOG2). Future investigation is needed to identify the humoral factors that mediate the communication between the fat body and other tissues. Because the larval fat body is considered the Drosophila counterpart of mammalian liver and adipose tissues, it will be interesting to study whether miR-200 and FOG2 play a similar role in liver and adipose tissues to control body growth during the human juvenile period (Hyun, 2009).

Previous studies suggest that USH/FOG2 may function as either transcriptional coactivators or corepressors by partnering with various GATA transcription factors. However, FOG2 is localized to the cytoplasm in some tissues. FOG1, the other human homolog of Drosophila USH, was also reported to remain in the cytoplasm of skin stem cells that lack GATA-3 and was shown to be sequestered in the cytoplasm by a cytoplasmic protein TACC3. USH/FOG2 have been studied mainly in hematopoiesis and heart development in both flies and mammals. However, it was recently shown that USH suppresses cell proliferation in Drosophila hemocytes. It is also noteworthy that FOG2 is frequently downregulated in human cancers of the thyroid, lung, and prostate, which suggests a role of FOG2 as a tumor suppressor. This study is the first report that FOG2 acts as a negative modulator of the PI3K-Akt pathway via direct binding to p85α. It remains to be determined whether the newly discovered molecular function of USH/FOG2 is related to the previously described phenotypes of ush/FOG2 (Hyun, 2009).

This study also offers a comprehensive way of discovering the physiological function of conserved miRNAs. By systematically mapping the protein homologs of miRNA targets and by validating them experimentally, seven gene pairs were identified as conserved targets of the miR-8/200 family. Also fly genetics and human cell biology were used to identify ush/FOG2 as the target gene that is responsible for one particular phenotype. Of note, six other genes (Lap1/ERBB2IP, CG8445/BAP1, dbo/KLHL20, Lar/PTPRD, Ced-12/ELMO2, and CG12333/WDR37) may also be authentic targets of miR-8/200, although they need to be further verified by additional methods. These six genes may function in different organs and/or at different developmental stages. Karres (2007) has reported that miR-8 prevents neurodegeneration by targeting atro (Karres, 2007). This study observed that atro knockdown does not rescue the small body phenotype of mir-8 mutants and that ush knockdown cannot reverse the wing and leg defects attributed to atro. Thus, a single miRNA may have several distinct functions in different cell types, likely depending on the availability of specific targets or downstream effectors. In a recent study, miR-8 gain of function was shown to affect the WNT pathway, although this finding was not sufficiently supported by the phenotype resulting from miR-8 loss of function (Kennell, 2008). The miR-200 family has also been shown to interfere with epithelial to mesenchymal transitions in humans (Gregory, 2008) to enhance cancer cell colonization in distant tissues (Dykxhoorn, 2009) and to regulate olfactory neurogenesis and osmotic stress in zebrafish (Choi, 2008; Flynt, 2009). It remains to be determined whether these previously described functions of the miR-8/200 microRNAs are systemically interconnected in a single organism and how widely each of these functions is conserved among animals expressing miR-8/200 microRNAs (Hyun, 2009).

The microRNA miR-8 is a conserved negative regulator of Wnt signaling

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wg pathway, a genetic screen was performed in Drosophila. The microRNA miR-8 was identified as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. These data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway (Kennell, 2008).

Ectopic activation of Wg signaling in the developing eye using the GMR-Gal4 driver causes a dramatic reduction in eye size. To identify regulators of the Wg pathway, a genetic screen was performed to identify genes that, when misexpressed, suppress this small-eye phenotype. Wg was coexpressed with random genes that were placed under the control of bidirectional Gal4-dependent (UAS) promoters by GSV transposable element insertions. Two GSV transposon insertions (GSV^1305-2 and GSV²¹⁹⁶), known to suppress the GMR/Wg phenotype, were located upstream of the microRNA miR-8. Both GSV insertions also suppressed the phenotype resulting from ectopic expression of Arm*, a stable form of Arm, in the developing eye. To verify that the phenotype of these insertions was because of expression of miR-8 and not to expression of surrounding genes, transgenic flies were generated expressing miR-8 under the control of a Gal4-dependent promoter (UAS-miR-8). Expression of miR-8 suppressed the small-eye phenotype caused by ectopic expression of Wg or Arm*. These data suggest that miR-8 can inhibit ectopic Wg signaling in the developing fly eye (Kennell, 2008).

These studies suggest that miR-8 is a potent antagonist of Wg signaling in vivo and in cell culture. Inhibition of Wg signaling by miR-8 may be because of targeting of the pathway at multiple levels, from Wg secretion to the reception of the signal in the nucleus by TCF. Finally, these studies of miR-8 extended across species to examine the effects of miR-8 family members in a cell culture model of mouse mesenchymal stem cell differentiation. The studies suggest that miR-8 family members promote adipogenesis by inhibiting endogenous Wnt signaling. Negative regulation of Wnt signaling by these microRNAs is consistent with a study reporting that all five miR-8 family members are highly expressed in the epidermis of the skin but are excluded from the hair follicle, a site of Wnt activity. Together, these data suggest that miR-8 may play an evolutionarily conserved role in negatively regulating Wnt signaling (Kennell, 2008).

This study found that miR-8 inhibits TCF protein levels without affecting TCF mRNA, suggesting that TCF is a direct target of miR-8. However, the 3'UTRs of the reported TCF isoforms were not regulated by miR-8, suggesting that miR-8 may directly target TCF mRNA independently of its 3'UTR or through an indirect mechanism. An intriguing possibility is that miR-8 may directly target an unidentified gene that is required for TCF protein stability. Studies have shown that TCF/LEF transcription factor activity or subcellular localization is regulated by posttranslational modifications, such as sumoylation, phosphorylation, and acetylation; however, none of these studies have reported an effect of these modifications on overall TCF protein expression. Interestingly, Sox17 has been reported to negatively regulate both TCF-4 and β-catenin protein levels in human colorectal cell lines by a mechanism that appears to require the proteasome; however, a positive regulator of TCF protein stability has not yet been reported, and this study found that knockdown of the miR-8 target CG32767 did not affect TCF protein levels (Kennell, 2008).

Another microRNA, miR-315, was recently reported to be a positive regulator of the Wg pathway (Silver, 2007). miR-315 was identified in a cell culture-based screen for effects on Wg dependent reporter gene activity. In contrast to the current findings, that study did not identify miR-8 as a pathway regulator in its screen. These discrepant findings may be because of the use of different cell lines (Kc167 vs clone8) and overall approaches (in vivo vs cell culture-based screen). This study did not test the effects of miR-8 expression in clone8 cells, although robust inhibition of the Wg pathway in the wing imaginal disk, the original source of the cell line, was noted (Kennell, 2008).

miR-8 loss of function mutant flies were reported recently (Kerres, 2007). The authors described a subtle mutant phenotype with defective leg and wing extension and behavioral defects, in part, because of an increased expression of Atrophin. Although the miR-8 mutants do not demonstrate an obvious Wg-related phenotype, Wg signaling does play an important role in Drosophila leg and wing development, and increased expression of other direct or indirect targets of miR-8 (e.g., TCF, wls, CG32767) also may contribute to the mutant phenotype in the leg and elsewhere (Kennell, 2008).

The subtle phenotype of the miR-8 mutant is not surprising, given similar reports for other microRNA loss of function mutants. The differences in scale of the loss-of-function versus the gain-of-function phenotype that is reported in this study may be, in part, because of redundancy, because all three targets that were identified contain predicted binding sites for multiple microRNAs. Mutation of multiple microRNAs may be required to produce dramatic effects on some signaling and developmental pathways. In addition, negative and positive regulators of the Wg pathway, such as nkd and fz3, are dispensable in certain tissues, including the wing imaginal disk. This may be indicative of redundant negative and positive regulator activities in these tissues. Redundancy may protect the Wg pathway in certain tissues from aberrations because of alterations in regulator gene expression. Overall, the studies of miR-8 suggest that the role of miR-8 is not as a developmental switch, but instead miR-8 may act as a modulator of multiple pathways in vivo by precisely tuning target gene expression in concert with other microRNAs and genes (Kennell, 2008).

Conserved miR-8/miR-200 defines a glial niche that controls neuroepithelial expansion and neuroblast transition

Neuroepithelial cell proliferation must be carefully balanced with the transition to neuroblast (neural stem cell) to control neurogenesis. This study shows that loss of the Drosophila microRNA mir-8 (the homolog of vertebrate miR-200 family) results in both excess proliferation and ectopic neuroblast transition. Unexpectedly, mir-8 is expressed in a subpopulation of optic-lobe-associated cortex glia that extend processes that ensheath the neuroepithelium, suggesting that glia cells communicate with the neuroepithelium. Evidence is provided that miR-8-positive glia express Spitz, a transforming growth factor α (TGF-α)-like ligand that triggers epidermal growth factor receptor (EGFR) activation to promote neuroepithelial proliferation and neuroblast formation. Further, these experiments suggest that miR-8 ensures both a correct glial architecture and the spatiotemporal control of Spitz protein synthesis via direct binding to Spitz 3' UTR. Together, these results establish glial-derived cues as key regulatory elements in the control of neuroepithelial cell proliferation and the neuroblast transition (Morante, 2013).

This analysis reveals that the production by an optic-lobe-associated cortex glia of the EGFR ligand Spitz is critical for coordination of neuroepithelial proliferation and the spatiotemporal emergence of neuroblasts. External signaling from the surrounding microenvironment is a common mechanism for the regulation of stem cell number and behavior in mature tissues, but the need of a niche microenvironment during early neurogenesis was unknown (Morante, 2013).

Flies deficient for the microRNA mir-8 exhibit brain degeneration and behavioral defects. Strikingly, the microRNA is expressed in cortex glial cells lying underneath the blood-brain-barrier (subperineurial) glial layer. These glia are of large size and produce long protrusions that ensheath the developing optic lobe neuroepithelium and can be distinguished by their selective expression of the EGFR ligand spitz as well as its modulators aos and rho (Morante, 2013).

Genetic manipulation of mir-8, spi, or aos, in this glial cell population unveiled cell nonautonomous influences of these glia on the development of the underlying neuroepithelium. Similar glial signaling to neuroblasts in the larval Drosophila brain has been demonstrated for a class of neuroblasts that remain quiescent until nutrient-responsive satellite and cortical glia reactivate their proliferation. This study identifies another population of glia cells that sustain growth of neuroepithelial cells and the neuroepithelial-neuroblast transition via a mir-8-Spitz axis (Morante, 2013).

Glial-mediated regulation of the neuroepithelium is reminiscent to the roles of mammalian astrocytes that are known to potently stimulate neurogenesis in cell culture and a component of endogenous neural stem cell niche in adult mammalian neurogenesis. Additionally, EGFR is also implicated in glial cell proliferation in Drosophila and human glioma often exhibits elevated EGFR signaling (Morante, 2013).

The notion that mir-8-Spitz-positive cortex glia constitute an anatomically and functionally distinct population of surface-associated glia cells is strongly supported by the finding that RNAi knockdown of spi in subperineurial glia using moody- Gal4 has no effect on neuroepithelial development. Spitz protein is converted to its active form by the Rhomboid protease, which is also expressed by miR-8 glia. The extracellular factor Aos limits Spitz spreading and signaling level that may influence the effects of Spitz-EGFR in the responding neuroepithelium (e.g., sustaining proliferation and preventing premature or ectopic neuroblast formation). The posttranscriptional silencing of spi mRNA by the miR-8 binding to a sequence in its 3' UTR provides another layer of regulation to fine-tune the timing, localization and/or amount of Spitz protein translation. Moreover, the distinctive architecture of miR-8-Spitz-positive cortex glia appears to be regulated by endoreplication regulators dup/ Cdt1 and the microRNA miR-8. Importantly, expression of a spitz transgene lacking its 3' UTR (and hence unable of regulating by miR-8) fully rescued the undergrowth defect caused by mir-8 overexpression in the glia cells. Therefore, it is suggested that cortex glia employ a coordinated strategy that is mediated by miR-8 to ensure that: (1) the glia establish a correct architecture to provide a continuous layer of cortex glia cells that extend long processes to the neuroepithelium; and (2) correct local (or temporal) control of Spitz protein synthesis. Given the expression of rho and aos is directly induced by EGFR signaling in other context, a feed-back signaling via EGFR may also occur in miR-8-Spitz positive glial cells, thereby contributing to the fine-tuning of Spitz protein activation and secretion (Morante, 2013).

A niche typically refers to a confined anatomical location where adult stem cells reside and provides the signals required to sustain stem cell function and number. Niches are usually composed of supporting cells that make physical contact with the stem cells and act locally. The optic-lobe-associated miR-8-Spitz-positive cortex glia appear to represent a niche that contributes signals for the growth and morphogenesis of the neuroepithelium and that constitutes a functionally distinct population of that of the bloodbrain- barrier glial cells (Morante, 2013).

Extrinsic signaling in the coordination of neuroepithelial proliferation in the developing mammalian forebrain of Foxc1 mutant mice has also been suggested. In these mice, the meninges are reduced or absent, resulting in an expansion of the neuroepithelium due to the predominance of symmetric divisions. The meninges are a source of the retinoic acid required for the transition of neuroepithelial cells into radial glia and neurons (Siegenthaler, 2009). Furthermore, meningeal cells secrete and organize the pial basement membrane, a thin sheet of extracellular matrix that covers the brain and that is enriched in a variety of growth factors. Rupture of the basement membrane in the developing brain causes type II lissencephaly, generating ectopic precursor clusters and cortical heterotopias due to impaired attachment of the radial glia to the basement membrane, resulting in a general laminar disorganization. These defects are reminiscent to those of disrupted surface glia cells described in this study (Morante, 2013).

In summary these findings suggest that neuroepithelial proliferation and the onset of neuroblasts in the developing optic lobe neuroepithelium are largely influenced by extrinsic cues via a miR-8-dependent mechanism in the overlying glia. The reprogramming of neuroepithelial cells into neural stem cells (neuroblasts) is associated with dramatic morphological and molecular changes, including the loss of epithelial determinants DE-Cadherin, Crumbs and PatJ, and enhanced expression of the Snail-family zinc-finger transcriptional repressor Worniu. These changes are strikingly reminiscent of the events that drive the epithelial-to-mesenchymal transition (EMT), which confers a stem-like character in mammalian epithelial cells and in cancer cells and which are regulated by the miR-200 family. Indeed, downregulation of human mir-200 genes in epithelial normal and cancer cells promotes EMT and the acquisition of 'stemness' effects that are presumed to be cell-autonomous. These findings demonstrate that miR-8 can exert its effects non-cell- autonomously, opening the possibility that microRNA of the miR-200 family may play similar roles in stem cell fate niches and/or microenvironmental regulation of metastasis (Morante, 2013).

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. The localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, this study found that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage it was found that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins (Lu, 2014).

Search PubMed for articles about Drosophila Mir-8

Britton, J. S., et al. (2002). Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2: 239-249. PubMed ID: 11832249

Choi, P. S., et al. (2008). Members of the miRNA-200 family regulate olfactory neurogenesis. Neuron 57: 41-55. PubMed ID: 18184563

Colombani, J., et al. (2005). Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science 310: 667-670. PubMed ID: 16179433

Dykxhoorn, D. M., et al. (2009), miR-200 enhances mouse breast cancer cell colonization to form distant metastases. PLoS One 4: e7181. PubMed ID: 19787069

Flynt, A. S., et al. (2009). miR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos. J. Cell Biol. 185: 115-127. PubMed ID: 19332888

Gregory, P. A., Bracken, C. P., Bert, A. G., and Goodall, G. J. (2008). MicroRNAs as regulators of epithelial-mesenchymal transition. Cell Cycle 7: 3112-3118. PubMed ID: 18927505

Hyun, S., et al. (2009). Conserved MicroRNA miR-8/miR-200 and its target USH/FOG2 control growth by regulating PI3K. Cell 139(6): 1096-108. PubMed ID: 20005803

Iorio, M. V., et al. (2007). MicroRNA signatures in human ovarian cancer. Cancer Res. 67: 8699-8707. PubMed ID: 17875710

Karres, J. S., Hilgers, V., Carrera, I., Treisman, J. and Cohen, S. M. (2007). The conserved microRNA miR-8 tunes atrophin levels to prevent neurodegeneration in Drosophila. Cell 131: 136-145. PubMed ID: 17923093

Kennell, J. A., Gerin, I., MacDougald, O. A. and Cadigan, K. M. (2008). The microRNA miR-8 is a conserved negative regulator of Wnt signaling. Proc. Natl. Acad. Sci. 105(40): 15417-22. PubMed ID: 18824696

Lee, G. J., Jun, J. W. and Hyun, S. (2014). MicroRNA miR-8 regulates multiple growth factor hormones produced from Drosophila fat cells. Insect Mol Biol. PubMed ID: 25492518

Lu, C. S., Zhai, B., Mauss, A., Landgraf, M., Gygi, S. and Van Vactor, D. (2014). MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development. Philos Trans R Soc Lond B Biol Sci 369(1652). PubMed ID: 25135978

Morante, J., Vallejo, D. M., Desplan, C. and Dominguez, M. (2013). Conserved miR-8/miR-200 defines a glial niche that controls neuroepithelial expansion and neuroblast transition. Dev Cell. PubMed ID: 24139822

Nam, E. J., et al. (2008). MicroRNA expression profiles in serous ovarian carcinoma. Clin. Cancer Res. 14: 2690-2695. PubMed ID: 18451233

Park, S. Y., et al. (2009). miR-29 miRNAs activate p53 by targeting p85 alpha and CDC42. Nat. Struct. Mol. Biol. 16: 23-29. PubMed ID: 19079265

Siegenthaler, J. A., Ashique, A. M., Zarbalis, K., Patterson, K. P., Hecht, J. H., Kane, M. A., Folias, A. E., Choe, Y., May, S. R., Kume, T., Napoli, J. L., Peterson, A. S. and Pleasure, S. J. (2009). Retinoic acid from the meninges regulates cortical neuron generation. Cell 139: 597-609. PubMed ID: 19879845

Silver, S. J., et al. (2007). Functional screening identifies miR-315 as a potent activator of Wingless signaling. Proc. Natl. Acad. Sci. 104: 18151-18156. PubMed ID: 17989227

Zinke, I., et al. (1999). Suppression of food intake and growth by amino acids in Drosophila: the role of pumpless, a fat body expressed gene with homology to vertebrate glycine cleavage system. Development 126(23): 5275-84. PubMed ID: 10556053

date revised: 7 October 2021

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