Drosophila Myc restores immune homeostasis of Imd pathway via activating miR-277 to inhibit imd/Tab2

Summary

Drosophila Myc (dMyc), as a broad-spectrum transcription factor, can regulate the expression of a large number of genes to control diverse cellular processes, such as cell cycle progression, cell growth, proliferation and apoptosis. However, it remains largely unknown about whether dMyc can be involved in Drosophila innate immune response. Here, we have identified dMyc to be a negative regulator of Drosophila Imd pathway via the loss- and gain-of-function screening. We demonstrate that dMyc inhibits Drosophila Imd immune response via directly activating miR-277 transcription, which further inhibit the expression of imd and Tab2-Ra/b. Importantly, dMyc can improve the survival of flies upon infection, suggesting inhibiting Drosophila Imd pathway by dMyc is vital to restore immune homeostasis that is essential for survival. Taken together, our study not only reports a new dMyc-miR-277-imd/Tab2 axis involved in the negative regulation of Drosophila Imd pathway, and provides a new insight into the complex regulatory mechanism of Drosophila innate immune homeostasis maintenance.

Keywords:

Drosophila melanogaster – Immune response – Homeostasis – MicroRNAs – Escherichia coli infections – Luciferase – Transcriptional control – Regulator genes

Introduction

Innate immune system plays critical roles in host defensing foreign pathogenic microorganisms [1]. Drosophila melanogaster is an important model for studying innate immune response in animals. Drosophila involves both cellular and humoral mechanisms to produce diverse antimicrobial peptides (AMPs) to resist the invasion of foreign pathogens via innate immune responses [2, 3]. Transcriptional expressions of Drosophila AMPs are primarily controlled by Toll and the immune deficiency (Imd) signaling pathways [4, 5]. Drosophila mainly utilizes the Imd signaling pathway to resist Gram-negative bacteria infection [6]. Currently, although the activation mechanisms of innate immune response have been well-established, the study on restoration mechanism of innate immune homeostasis remains a major challenge [7].

The intensity and duration of Drosophila immune response can be positively or negatively regulated at multiple layers [8]. For example, STING and sick can activate Drosophila Imd innate immune response via upregulating the expression of the NF-κB transcription factor Relish [9, 10]. The E3-ligase inhibitor of apoptosis 2 (Iap2) could activate Dredd expression to positively regulate the Imd immune response [11, 12]. Furthermore, Imd-mediated immune response can be negatively regulated by some immune suppressors, such as WntD, Die, PGRP-LF, pirk, dUSP36, CYLD, Dnr1, dRYBP and Caspar [13–23], which can prevent the excessive activation of Drosophila Imd pathway to maintain innate immune homeostasis. In addition, many miRNAs have been reported to participate in fine-tuning Drosophila Imd immune response positively or negatively. Studies have shown that Drosophila miR-8 and miRNA let-7 could negatively regulate the Imd pathway [24, 25], whereas miR-34 could positively regulate the Imd pathway [8]. Our previous works have also demonstrated that both miR-9a and miR-981 could negatively regulate Drosophila Imd-dependent immune response via directly targeting the AMP gene Diptericin (Dpt) [26]. Although several regulators involved in Drosophila innate immune responses have been identified, the further study of the restoration mechanism of Drosophila innate immune homeostasis is still needed.

Myc serves as a broad-spectrum transcription factor to control the expression of a large number of genes for diverse cellular processes, including cell cycle progression, cell growth, proliferation and apoptosis [27–31]. Myc family includes three member of c-Myc, N-Myc, and L-Myc in human [27]. It’s well documented that Myc can function as a proto-oncogene in human cancers [32, 33]. Drosophila has only one single Myc gene, referred to as dMyc or diminutive (dm), which is homologous to human c-Myc [34]. Studies have revealed that dMyc could involve in ribosome biogenesis [35], protein synthesis [36, 37], cell-autonomous apoptosis [38, 39], and cell competition [40, 41]. Although Myc can regulate the expression of some miRNAs to participate in immune response in human [42–48], it’s largely unknown whether and how dMyc can regulate miRNA expression to control innate immune response in Drosophila.

In this work, we firstly report that dMyc can inhibit the immune response of Drosophila Imd pathway by genetic screening. Secondly, we confirm that dMyc directly activate the transcription of miR-277 using Chromatin immunoprecipitation (ChIP)-qPCR analysis and promoter reporter activity system. Thirdly, we find that miR-277 can negatively regulate Drosophila Imd signaling response via targeting and downregulating the expression of imd and Tab2-Ra/b, but not Tab2-Rc. We further verify that dMyc could negatively regulate Drosophila Imd signaling pathway via activating miR-277 transcription to inhibit imd/Tab2 expression in vivo using dMyc and miR-277 SP co-highexpressed flies. Finally, we provide evidences to show that dMyc could restore immune homeostasis of Drosophila Imd pathway to promote the survival of flies upon infection.

Result

dMyc is a negative regulator of Drosophila Imd pathway

Drosophila defends against Gram-negative bacteria infection through Imd pathway to produce Diptericin (Dpt) and other anti-microorganism peptides such as Drosocin, Attacin and Cecropin A1. In this work, via screening the fly strains from the Bloomington Drosophila library, we found Gal80^ts-UAS-dmyc flies significantly decrease the expressions of Dpt, Drosocin, Attacin and Cecropin A1 after infection with gram-negative bacteria Escherichia coli (E. coli), indicating dMyc regulates the Imd pathway (S1 Fig). Therefore, we here chose the Dpt as the representative for further exploring the regulatory role of dMyc in immune response of Drosophila Imd pathway. Our results demonstrate that the expression level of Dpt in the dMyc high-expressed flies with E. coli infection is, respectively, significantly lower than wild-type flies at all five time points (3, 6, 12, 24, 48h) (Fig 1A). The expression level of dMyc exists significant differences within different dmyc mutant fly strains (Fig 1B), and the order of the expression level of dMyc is dMyc high-expressed flies > dMyc and dMyc-RNAi co-highexpressed flies > wild-type flies > dMyc-RNAi high-expressed flies. Remarkably, the expression level of Dpt has no significant difference in these above dmyc mutant flies without infection (Fig 1C), but after infected with E. coli, the expression level of Dpt in dMyc-RNAi high-expressed flies is significantly higher than dMyc high-expressed flies and the control flies, respectively (Fig 1D). Especially, the expression level of Dpt in the dMyc and dMyc-RNAi co-highexpressed flies could be nearly restored to the normal expression level of the control flies (Fig 1D). Taken together, our findings strongly suggest that dMyc is a novel important negative regulator of Drosophila Imd pathway.

dMyc regulates the expression of immune related miRNAs

To investigate whether and how dMyc negatively regulates Imd pathway via regulating miRNA expression, we used in silico analysis to identify potentially immune related dMyc-miRNA-gene axis (Fig 2A). We collected 63 genes involved in Drosophila Imd pathway from the literature data, and downloaded the maturation and upstream promoter sequences of Drosophila miRNAs from miRBase (http://www.mirbase.org/) and NCBI (https://www.ncbi.nlm.nih.gov/). The relationships between the 63 genes and miRNAs were next predicted by TargetScan and miRanda. Then miRNAs regulated by dMyc were predicted using the PROMO website and TransmiR 2.0 database.

We found 12 candidate dMyc-regulated miRNAs, which have been identified as differentially expressed miRNAs (DEmiRNAs) between flies with E. coli infection and the control from our previous small RNA-seq data [26] (Fig 2A, 2B and 2C). We next constructed these 12 DEmiRNAs high-expressed fly strains. We found that the expression level of Dpt is significant lower than the control after E. coli infection in miR-10, miR-1012, miR-277, miR-2b-2 and miR-996 high-expressed flies, respectively (Figs 2D and S2). We also investigated the expression relevance between dMyc and these 5 miRNAs, finding only miR-277’s expression level in dMyc high-expressed flies is gone up 1.15 times than the control, but not miR-2b-2, miR-1012, miR-10 and miR-996 (Fig 2E). In contrast, the miR-277’s expression level in dMyc knocked down flies is significant lower than the control (Fig 2F). In addition, two ChIP-seq data for dMyc from ENCODE database (https://www.encodeproject.org/) indicated that dMyc could bind to the upstream promoter sequences of Drosophila miR-277 gene (S3 Fig). Taken together, our results suggest that dMyc might regulate miR-277 expression to negatively control Imd signaling pathway.

dMyc directly activates the transcription of miR-277

To further study how dMyc regulates miR-277, we performed an analysis for the upstream promoter sequences of miR-277 and found that miR-277 gene contains 2 transcriptional start sites (TSSs) [49] (Fig 3A). The upstream sequences of these two TSSs have the promoter activity, and the promoter activity of TSS1 upstream sequence is stronger than the TSS2 (Fig 3B). Lipopolysaccharide (LPS) stimulation could enhance these two promoter activities to increase miR-277 expression (Fig 3B). Furthermore, dMyc could enhance the luciferase activities of these two promoter regions, and the promoter activity of TSS1 upstream sequence is consistently stronger than that of TSS2 (Fig 3C). ChIP-qPCR assay showed that dMyc is enriched at three candidate regions in the promoter sequences, and the neighbor region of TSS1 (ChIP1) is most enriched (Fig 3D). Taken together, our results suggest that dMyc activates miR-277 transcription via directly binding to its promoter.

miR-277 inhibits the expression of imd and Tab2-Ra/b

To further determine how miR-277 regulates the Imd pathway, first, we examined the expression level of Dpt in miR-277 high-expressed flies compared to the control group flies upon infection. The result showed that the expression level of Dpt in miR-277 high-expressed flies is significantly down-regulated at 3, 6 and 12 h post-infection compared with the control (Fig 4A). Moreover, the miR-277 rescue assay showed that this miR-277 and miR-277 sponge co-highexpressed flies could restore the expression level of miR-277 to the normal level (Fig 4B). Without infection, the expression level of Dpt has no significant difference between miR-277 mutant flies and the control group flies (Fig 4C). Remarkably, the expression level of Dpt could be recovered to the comparable level of the control group in miR-277 and miR-277 sponge co-highexpressed flies after E. coli infection (Fig 4D). Taken together, these results confirm that miR-277 negatively regulates the Imd pathway.

To further explore how miR-277 inhibits the Imd pathway, we predicted the potential target genes of miR-277 using targetScan and miRanda. We found that miR-277 could target to the 3’UTR of imd and Tab2 Ra/b transcripts (Fig 5A and 5B). Imd and Tab2 Ra/b are key components of the Imd pathway in Drosophila [6, 50]. As expected, the expression levels of Dpt in both imd-RNAi and Tab2-RNAi mutant flies are significantly lower than the control upon E. coli infection (S4A and S4B Fig). Consistently, compared with the control, the expression levels of both imd and Tab2 are also significantly down-regulated in flies with high-expressed miR-277 at 3 h, 6 h and 12 h post infection, respectively (Fig 5C and 5D). In addition, the expression levels of imd and Tab2 in miR-277 and miR-277 sponge co-highexpressed flies could be restored to the level of the control group (Fig 5E and 5F). Our findings suggest that miR-277 could inhibit the expression of both imd and Tab2 in vivo.

To further evaluate the direct targeting relationship between miR-277 and imd as well as Tab2, we carried out the Luciferase Reporter Assay in Drosophila S2 Cell. The results showed that compared with the pAc empty vector, miR-277 could significantly reduce the activity of the luciferase reporter containing the 3'UTR of imd and Tab2 Ra/b, but not Tab2 Rc (Fig 5G and 5H). Furthermore, we performed the target site mutation in the 3'UTR of imd and Tab2 Ra/b, finding that the reporter activity of imd and Tab2 Ra/b could be nearly restored to the normal level in these cells with co-transfected miR-277 expression vector and 3'UTR mutant reporters of imd and Tab2 Ra/b (Fig 5G and 5H). Taken together, our results suggest that miR-277 directly targets the 3’UTR of imd and Tab2 Ra/b.

dMyc negatively regulates Drosophila Imd pathway via activating miR-277 to inhibit imd/Tab2

To further ascertain whether dMyc can regulate the Drosophila immune response via activating the transcription of miR-277 in vivo, we constructed the dMyc and miR-277 SP co-highexpressed mutation flies. We found that the expression level of miR-277 in the dMyc and miR-277 SP co-highexpressed flies is significantly lower than the dMyc highexpressed flies, and is nearly restored to the control level (S5A and S5B Fig). Without infection, the expression level of Dpt shows no significant difference in aforesaid mutant flies, and the expression level of Dpt in this dMyc and miR-277 SP co-highexpressed flies is significantly higher than the dMyc highexpressed flies upon infection (Fig 6A and 6B). In addition, the expression level of Dpt in the dMyc and miR-277 SP co-highexpressed flies at 6 h post infection could be restored to 55% of the control level, and is nearly 3 times than the dMyc highexpressed flies (Fig 6B). The expression levels of imd and Tab2, respectively, are also restored to 83% and 58% of the control level, and are 2.1 and 1.8 fold than the dMyc highexpressed flies, respectively (Fig 6C and 6D). Taken together, our data suggest that dMyc negatively regulates Drosophila Imd immune response via activating miR-277 transcription to inhibit imd/Tab2 expression.

dMyc controls Drosophila Imd immune homeostasis

dMyc could negatively regulate Drosophila Imd immune response, indicating dMyc could be involved in the maintenance of Imd immune homeostasis. To test this point, we further monitored the dynamic expressions of Dpt, dMyc, miR-277, imd and Tab2 in this wild-type flies at 0, 3, 6, 12, 24 and 48 h after E. coli infection. Our results showed that the expression level of Dpt is increasing before 3 h and reaching its peak expression level at 12 h, then gradually decreased to the basal level post-infection (Fig 7A). In contrast, the expression level of dMyc is decreased before 6 h, implying that the expression of dMyc is inhibited for avoiding the inadequate of immune response in the early stage of E. coli infection (Fig 7B). The expression level of dMyc is markedly up-regulated at 12 h post-infection, and subsequently is restored to the pre-infection level (Fig 7B). Moreover, the expression pattern of miR-277 is very similar with that of dMyc, whereas the expression patterns of both imd and Tab2 are opposites of that of dMyc and miR-277 (Fig 7C and 7D). Taken together, we propose that dMyc could play a key role in restoring Drosophila Imd immune homeostasis post infection.

Immune homeostasis post infection is essential to organisms. We hypothesized that dMyc could protect Drosophila from damage caused by over-activation of immune response. To test this, we further investigated the survival rate of dMyc high-expressed flies and the control (Gal80^ts; Tub-Gal4/+) flies without infection and with PBS as well as the Gram-negative bacteria Enterobacter cloacae (E. cloacae) infection, respectively (Fig 8). We found that the survival rate of dMyc high-expressed flies is significantly lower than the control group both in the absence of infection and after infection with PBS. However, the survival rate of dMyc high-expressed flies is significantly higher than the control group after infection with E. cloacae. These results seem to support the important role of dMyc in negatively regulating the Imd pathway for immune homeostasis, which is essential for fly survival.

Taken all results together, we proposed a molecular mechanism by which dMyc plays an important role in Drosophila Imd immune homeostasis (Fig 9). On the one hand, down-expressed dMyc could down-regulate miR-277 expression to ensure the elevated expression of imd and Tab2 at the early stage of E. coli infection to promote the expression of Dpt against pathogenic bacteria. On the other hand, to prevent the overactivation of Imd immune response, over-expressed AMP Dpt induces dMyc expression to activate the expression of miR-277 for down-regulating the expression of imd and Tab2 to reduce Dpt expression, which restores Imd immune response to homeostasis to protect Drosophila from damage caused by overactivation of immune response, and improve the survival of Drosophila.

Discussion

The Drosophila innate immune system plays critical roles in defending invading pathogens. Depression and overactivation of innate immune responses are both harmful for Drosophila. Thus, the Drosophila innate immune system must gain an unknown mechanism to resist pathogen challenges without overactivation of innate immunity. Although studies have revealed that the Drosophila innate immune response could be controlled by a series of negative or positive regulators at transcriptional and post-transcriptional levels [8, 24–26, 51, 52], the mechanism of maintaining immune homeostasis is largely unknown. In this study, we reveal a new dMyc-miR-277-imd/Tab2 axis to play an important role in negatively regulating Imd pathway, and provide a mechanistic insight into immune homeostasis in Drosophila.

We found that high-expressed dMyc led to decrease of Dpt expression, conversely knock-down dMyc resulted in increase of Dpt expression (Fig 1B), indicating that dMyc act as a negative regulator of Drosophila Imd pathway. Previous studies have revealed that human Mycs play key roles in activating both innate and adaptive immune cells to defense invading pathogens [1, 53, 54]. Functions of Myc are evolutionarily conserved between fruit fly and vertebrate [55]. The fruit flies and vertebrate proteins can substitute for each other to the extent [56]. Thus, our study provides an important insight into illuminate the conservative immune regulation function of Myc between Drosophila and human.

Human Mycs, as important transcriptional factors, not only can control the expressions of a large number of protein-coding genes, but can regulate the expressions of many miRNAs [57, 58]. In this work, we further identified miR-277 as a target gene of dMyc (Fig 3), and found miR-277 colud negatively regulate Dpt expression by targeting imd and Tab2 in Drosophila Imd immune responses (Fig 6). Previous studies have indicated that imd and Tab2 are specifically required for the immune activation of Drosophila Imd signaling pathway [6, 59]. Thus, our results suggest that dMyc might negatively regulate Drosophila Imd immune response via activating miR-277 transcription to inhibit imd/Tab2 expression. Previous reports have indicated that miR-277 not only can control branched-chain amino acid catabolism, affect lifespan [60] and wing imaginal discs development of Drosophila [61], but modulate the Neurodegeneration Caused by Fragile X Premutation rCGG Repeats [62]. Whereas, our present results demonstrate that miR-277 is a new negative regulator involved in Drosophila Imd signaling pathway. Especially, the Tab2 is an alternative splicing gene, which contains three transcripts, i.e. Tab2 Ra, Tab2 Rb and Tab2 Rc, of which Tab2 Ra and Tab2 Rb’s 3’UTR is identical. Here, our results showed that miR-277 can target to the 3’UTR of Tab2 Ra and Tab2 Rb transcripts, but not Tab2 Rc (Fig 5). This result suggests that miRNA might play a critical role in selectively regulating the expression of alternative splicing gene in the post-transcriptional level.

Innate immune is a rapid and short immune response process, and inactivation or overactivation of innate immune responses can result in the normal tissue damage [63–66]. We reported the tightly coordinated expression of dmyc, miR-277, imd,Tab2 and Dpt, suggesting that dMyc as a novel negative regulator primarily prevents the over-activation of Drosophila Imd immune response at this middle and later stage of E. coli infection, and helps Drosophila restore to a new immune homeostasis.

Recently, overexpression of dMyc has been reported to be able to significantly diminish Drosophila adult longevity, which might is due to over-expressed dMyc greatly resulting in genome instability [67]. In addition, studies have indicated that down-regulating expression level of c-Myc can significantly increase mice longevity due to heterozygosity [27, 68]. However, in our present study, we found that the overall survival rate of dMyc high-expressed adult male flies is similar with the control group at the early stage (0~5h) after E. cloacae infection (Fig 8). Whereas, the survival rate of dMyc high-expressed adult male flies was significantly higher than the control group after 5 h post infection (Fig 8). Taken together, our findings suggest that dMyc contributes to the survival of flies likely via preventing over-activation of innate immune responses to avoid excessive damage of many tissues.

Conclusions

In this work, we identify dMyc as a novel negative regulator of Drosophila Imd pathway. Mechanically, dMyc positively activate miR-277 transcription, to target the 3’UTR of imd and Tab2-Ra/b to inhibit their expression, leading a new immune homeostasis. Our present results provide a new comprehensive understanding on the complex regulatory mechanism of maintaining innate immune homeostasis in Drosophila.

Materials and methods

Fly stocks

Flies were obtained from the Bloomington Drosophila Stock Center: UAS-dmyc (#7118); UAS-miR-277 (#36559); UAS-miR-277^sponge (#61408). These Fly lines carrying UAS-RNA interference (RNAi) constructs were obtained from the Tsinghua Fly Center: dmyc^RNAi (#47953); imd^RNAi (#31706); Tab2^RNAi (#24667). As well as previously purchased tubulin-Gal80^ts;TM2/TM6B (#7019) and tubulin-Gal4/TM3, Sb¹, Ser¹ (#5138), all flies were raised on maize malt molasses food in a light-dark (12-hr cycle) incubator at 25°C and 60% humidity. Flies were shifted to 30°C 24 h prior to and then during infection for UAS-protein/UAS-miR-277^sponge/UAS-protein ^RNAi overexpression experiments.

Bioinformatic analysis

These mature sequences of Drosophila miRNAs were downloaded from miRBase (http://www.mirbase.org/). 3’UTRs of 63 Imd pathway-related genes and promoter sequences of Drosophila miRNAs were extracted from the fruit fly genome in FlyBase (http://flybase.org/) and NCBI (https://www.ncbi.nlm.nih.gov/). These relationships between mature miRNAs and 3’UTR of genes were predicted using two miRNA target prediction programs with default parameters, i.e. TargetScan (www.targetscan.org/fly_12/) [69] and miRanda v3.3a tool downloaded from microRNA.org-Targets and Expression [70, 71]. Whilst these sites of transcription factor (dMyc) binding at these promoter sequences of miRNAs were predicted through PROMO website [72, 73] and TransmiR 2.0 database (http://www.cuilab.cn/transmir).

Infection and survival experiments of adult flies

Three to four-day-old adult male flies were used for septic injury experiments. Control and high-expressed or knockdown gene/miRNA flies were infected by E. coli, which is a widely used bacterial strain that can activate the Imd-mediated immune response to induce the expression of Diptericin. Infection experiments were performed by pricking the thorax of the flies with a pulled glass capillary carrying E. coli inoculant using a Nanoject apparatus (Nanoliter 2010, WPI). Next, flies were collected at specified time-points for subsequent experiments. Survival to infection is the most holistic approach to assess these defects in immune response [74]. For the survival experiment, flies were infected with a concentrated culture of E. cloacae by pricking as above, and then the survival situation of flies was detailedly recorded for 5 days.

RNA extraction and RT-qPCR

Total RNAs were isolated from these treated adult flies using TRIzol Reagent (Invitrogen) following the instructions. For RT-PCR, A first-strand cDNA synthesis kit (Vazyme, China) was used to prepare the cDNA. These stem-loop primers were synthesized for reverse transcription to generate the specific stem-loop cDNA of miRNA. Quantitative PCR reactions were performed using AceQ SYBR Green Master Mix (Vazyme, China) on the ABI StepOne Plus Real-Time PCR System (Applied Biosystems, USA). The expression levels of mRNA and miRNA were normalized to the control rp49 and U6 snRNA, respectively. All experiments were in triplicate. The relative 2^-△△CT method was used for data analysis [75]. All primers used in this analysis were listed in S1 Table.

Cell culture and immune stimulations

Drosophila S2 cells were maintained at 28°C in Schneider’s medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen). For immune stimulation, cells were incubated with 10μg/ml commercial LPS from E. coli 055:B5 (Sigma, St. Louis, MO), which is characteristic components of the cell wall of Gram-negative bacteria, for 6 h [76, 77].

Chromatin immunoprecipitation (ChIP)

For ChIP experiment, Cells were fixed by cross-linking with a final concentration of 1% formaldehyde solution for 10 min at room temperature and then quenched with 125 mM glycine for 5 min. After washing with cold PBS containing a protease inhibitor cocktail and PMSF twice, these cells were lysed with cell lysis buffer and nuclear lysis buffer. The clarified lysate was subject to sonication. The chromatin was then sheared to fragments of 200–500 bp. The chromatin was used for ChIP incubating with Dynabeads protein G (Thermo Fisher Scientific) coated with either an anti-dMyc antibody (P4C4-B10; DSHB) or mouse IgG control antibody overnight at 4°C on a rotating platform. After repeated washes using a magnetic rack (Thermo Fisher Scientific), dMyc-bound genomic DNA was eluted from Dynabeads, and then the cross-links were reversed at 65°C for 4h (or overnight). DNA fragments then were purified with AxyPrep PCR Cleanup Kit (Axygen). qRT-PCR analysis was performed using the DNA from the Input and ChIP experiments with primers listed in S2 Table. At least three independent experiments were carried out for the miR-277 promoters, as well as for Fibrillarin gene served as a positive control [78].

Luciferase reporter construction and luciferase assay

This pri-mir-277 has two transcription initiation sites (TSSs) as reported [49], so we further divided the promoter region of pri-mir-277 into two parts for luciferase promoter analysis. Promoter sequences of miR-277 and CDS of dMyc were amplified by PCR from Drosophila genomic DNA. The DNA fragments were then isolated and inserted respectively into the restriction enzyme digested the promoterless pGL3 Basic and pAc5.1 Vector using T4 DNA ligase. pAc5.1 luciferase reporter constructs carrying the 3’-UTR of either imd or Tab2 with wild-type or mutated sequences of their respective miR-277 target sites were utilized to analyze the effects of the miR-277. All constructs were confirmed by sequencing. All PCR primers for the reporter constructs were listed in S3 Table. Drosophila S2 cells were transfected with each reporter construct for 48 h followed by assessment of luciferase activity. Luciferase activity was then measured with Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions and normalized to the Renilla luciferase activity for each transfected well. Each assay was performed in triplicate.

Statistical analysis

All experimental data in this work were collected from three independent biological replicates. All statistical analyses were presented as means ± SEM. Significant differences between the values under different experimental conditions were subjected to two-tailed Student’s t-test. Statistical analysis of fly survival experiments was performed using the log-rank (Mantel-Cox) test. For all tests, P value < 0.05 was considered as statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001; and ns, no significance vs. the control groups.

Supporting information

S1 Fig [a]
The expression level of multiple s in the dMyc high-expressing flies and the control flies.

S2 Fig [tif]
The expression level of in 7 miRNA highexpressed fly strains.

S3 Fig [tif]
The bind sites of dMyc on the upstream of gene.

S4 Fig [a]
The expression level of in this imd-RNAi and Tab2-RNAi highexpressed fly strains.

S5 Fig [a]
The expression level of and miR-277 in dMyc and miR-277 sponge co-highexpressed fly strains.

S1 Table [docx]
Primers used for quantitative RT-PCR

S2 Table [docx]
Primers used for ChIP-qPCR

S3 Table [docx]
Primers used for transgene vector construction