37 publications mentioning gga-mir-155

Open access articles that are associated with the species Gallus gallus and mention the gene name mir-155. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1

Although upregulation of miR-155 appears to add complexity to regulation of gene expression in v- rel -induced malignant transformation, the downstream network of miR-155 targets, or the importance of those target genes in v- rel -induced transformation, could be an interesting area to explore.

Quite clearly, miR-155 is significantly upregulated during the time-course of v- rel transformation, with levels showing increases of fivefold (day 1), sixfold (day 4), 50-fold at day 7, 150-fold at day 9 and nearly 1500-fold at day 14, as compared with the level at day 0. Fig. 4. Upregulation of miR-155 during v- rel transformation is associated with downregulation of targets.

Quite clearly, miR-155 is significantly upregulated during the time-course of v- rel transformation, with levels showing increases of fivefold (day 1), sixfold (day 4), 50-fold at day 7, 150-fold at day 9 and nearly 1500-fold at day 14, as compared with the level at day 0. Fig. 4. Upregulation of miR-155 during v- rel transformation is associated with downregulation of targets.

Expression of v- rel increased the level of miR-155 expression by approximately 700-fold in MSB-1 cells and by about 900-fold in 265L cells, which is much higher than the miR-155 level in non-transformed CD4 [+] cells (Fig. 3b), demonstrating that ectopic expression of v- rel can induce expression of miR-155 in avian lymphoid cells.

Of the 1242 downregulated genes, 73 are predicted targets of gga-miR-155 (Fig. 4b), making it the top hit of the most enriched miRNA targets.

Analysis of the gene expression changes in the v- rel-transformed cells further demonstrated downregulation of a number of known miR-155 targets potentially affecting a number of important biological pathways.

v- rel relieves the inhibition of miR-155 expression in MSB-1 cellsWe have previously shown that miR-155 is consistently downregulated in MDV-transformed tumours and cell lines [32].

In order to analyse the behaviour of predicted gga-miR-155 targets, expression data from Affymetrix probes representing genes predicted to be targets of gga-miR-155 were extracted and analysed as a set.

The increased level of miR-155 expression after introduction of v- rel into these cells indicated that the upregulation of miR-155 is a direct effect.

Interestingly, while miR-155 was upregulated in Rev-T-transformed cell lines, it was consistently downregulated in MDV-transformed lymphocytes [52].

Together with the evidence that the potential miR-155 targets in macrophages involved in cancer stand out from those targets related to other diseases and functions, this study demonstrates the important role of miR-155 in v-rel -induced transformation.

Not only was miR-155 the most statistically enriched target within the list of significantly downregulated genes, but members of the miR-17-92 cluster are also implicated, a cluster which is known to be involved in cancer [69–72], this further emphasizing the role of oncogenic miRNAs in transformation.

The number of cancer-related genes targeted by miR-155 ranks second, implicating the importance of miR-155 as a regulator in disease pathogenesis, particularly in tumorigenesis.

During the analysis of the global changes in miRNA expression in chicken lymphocyte lines transformed by avian oncogenic viruses, we observed that miR-155 was overexpressed in v- rel-transformed chicken lymphocytes, compared with the normal spleen cells and Marek’s disease virus (MDV)-transformed cell lines [32].

An ALV-transformed B-cell line, HP45, was used as a positive control where miR-155 is upregulated due to insertional activation, and normal spleen cells, which do not express detectable levels of miR-155, were used as a negative control.

Induction of miR-155 is accompanied by downregulation of potential targets.

It has been shown more recently that EBV-encoded latent membrane protein-1 (LMP-1), a functional homologue of the tumour necrosis factor receptor family, upregulates the expression of miR-155 mainly by activating the NF-κB pathway [48].

We wanted to examine whether the downregulation of miR-155 in MDV-transformed cell lines could be rescued by expressing v- rel in these cells.

Having demonstrated the upregulation of miR-155 in Rev-T transformed cells, we examined the potential mechanisms of miR-155 overexpression by v- rel.

Demonstration of the targeting of a number of cancer-related genes in chicken macrophages overexpressing miR-155 demonstrated the importance of this miRNA as a major regulator of v- rel -induced transformation.

v- rel binds to the NF-κB sites in the Bic/miR-155 promoterHaving demonstrated the upregulation of miR-155 in Rev-T transformed cells, we examined the potential mechanisms of miR-155 overexpression by v- rel.

Although the mechanisms for this downregulation are not known, this could be due to the complementation of miR-155 functions by the high levels of the viral homologue MDV-miR-M4 expressed in these cells.

The significantly downregulated genes with miR-155 target sites in the 3′ UTR were subjected to pathway analysis using the Ingenuity Pathway Analysis tool.

Although miR-155 functions are probably rescued by the high-level expression of the MDV1-miR-M4 homologue in these cells [53], the precise molecular mechanisms of downregulation of miR-155 in MDV-transformed cells are not clear.

From microarray data on RNA of v- rel-transformed cells, 73 out of 1242 significantly downregulated genes are potential targets of miR-155.

Our studies confirm that v- rel -mediated upregulation of gga-miR-155 occurs through the direct binding to at least one of the putative NF-κB sites on the Bic/miR-155 promoter.

1, CEBPβ and/or other miR-155-regulated transcription factors in the regulation of miR-155 -inhibited genes.

As shown in Fig. 5, several potential miR-155 targets are involved in a number of diseases and cellular processes.

Fig. 5. Potential miR-155 targets are involved in a number of diseases and functions.

v- rel relieves the inhibition of miR-155 expression in MSB-1 cells.

As an NF-κB homologue [8], the most likely mechanism of miR-155 upregulation would be through the direct activation of the miR-155 promoter through the NF-κB binding sites.

Considering the complexity of target analysis in the v- rel -induced transformation system, as many miRNAs and mRNAs are affected by v- rel, we overexpressed miR-155 in chicken macrophages derived from line 0 chicken by transfection of miR-155 mimics into bone-marrow-derived macrophages.

EGFP -expressing RCAS(A)-v- rel-EGFP-infected MSB-1 and 265L cells were also sorted and examined for v- rel and miR-155 expression.

We and others have shown upregulation of miR-155 in Rev-T-transformed cell lines and CEFs [32, 33].

Additionally, miR-155 targets JARID2, a cell cycle regulator and part of a histone methyltransferase complex, to promote cell survival [33].

The promoter activity of the double mutant pBic-M1M2 construct was similar to that of the pBic-M2 construct, further confirming that the second NF-κB site in the Bic/miR-155 promoter is important for the v- rel -mediated upregulation of miR-155.

We have previously shown that miR-155 is consistently downregulated in MDV-transformed tumours and cell lines [32].

For further analysis of the global changes in miRNA profiles induced by v- rel, we used an in vitro mo del of v- rel -induced transformation of embryonic splenocytes to demonstrate the sequential upregulation of miR-155 during the transformation process.

Upregulation of miR-155 in Rev-T-transformed cell lines.

Fig. 3. Upregulation of miR-155 in MDV-transformed cell lines by v- rel.

AP-1 sites are present in chicken Bic/miR-155 promoter sequences, and the contribution of AP-1 in regulation of miR-155 expression in v- rel-transformed lymphocytes remains to be determined.

In addition to the changes in protein-coding genes, many changes in the miRNA profiles also occur in v- rel-transformed cells, and one of the miRNAs expressed at significantly higher levels in v- rel-derived tumour cell lines such as KBMC and CM758 is gga-miR-155 [33].

The known miR-155 targets Pu.

Top 20 functions (sorted by P value) of the miR-155 targets identified in primary avian macrophages transfected with miR-155 mimics.

Fig. 1. Northern blotting analysis for determining miR-155 expression.

A number of targets of miR-155 have been identified previously.

Higher expression of miR-155 is reported in a number of haematopoietic malignancies [36–40].

‘Allstars’ negative control (Qiagen) was used as control in an attempt to get a cleaner result on miR-155 targets.

RCAS -mediated transduction of v- rel did rescue the expression of miR-155 in two of the MDV-transformed cell lines, MSB-1 and 265L.

The list was further filtered for those genes predicted to be targeted by gga-miR-155.

For confirmation of the higher expression of miR-155 in v- rel-transformed cells, we examined the Rev-T-transformed cell lines AVOL-1, AVOL-2, AVOL-3 and RIR-Rev-T by Northern blot analysis.

Among the numerous miRNAs expressed in haematopoietic cells, miR-155 was shown to have the most wide-ranging effects on the biology of lymphocytes [22–25].

NF-κB site 2 in the Bic/miR-155 promoter is required for miR-155 activationHaving demonstrated the direct binding of v- rel to the NF-κB sites, we next examined the possible contribution of these elements in mediating Bic regulation.

The oncogenic effects of miR-155 are mediated through its target mRNAs.

To this end, we carried out reporter assays to examine the ability of v- rel to drive the expression of the R enilla luciferase reporter gene using constructs containing the wild-type or the mutant chicken Bic/miR-155 promoter.

The data herein are the first evidence to our knowledge showing miR-155 being regulated by an NF-κB transcription factor, the v- rel oncogene encoded by Rev-T in avian systems.

The expression levels of miR-155 were analysed using the TaqMan MicroRNA Assay System (Applied Biosystems) using 10 ng total RNA as a template for reverse transcription.

Transcriptional regulation of miR-155 by the TGF-β/Smad4 pathway using the Smad response elements in the human miR-155 promoter has also been reported [47].

v- rel binds to the NF-κB sites in the Bic/miR-155 promoter.

For this, the chicken Bic/miR-155 promoter region extending from −1829 to +3 nucleotides from the transcription start site (+1) was cloned upstream of the Renilla luciferase gene of the psiCHECK−2 vector (Promega) to replace the SV40 promoter, generating the reporter construct pBic-WT.

These data highlighted the importance of miR-155 and other miRNAs in v-rel -induced transformation.

Epstein–Barr virus (EBV) latent membrane protein-1 (LMP1) is a potent inducer of miR-155, and the NF-κB sites in the Bic/miR-155 promoter have been shown to be pivotal for this function [48, 49].

In order to establish that v- rel binds directly to the putative NF-κB sites in the Bic/miR-155 promoter, an electrophoresis mobility shift assay (EMSA) was carried out using a recombinant glutathione S-transferase (GST)–v- rel fusion protein.

1 [59], SOCS1 [60], interleukin-1 [61] and IKKε [49, 62] have been implicated in mediating functions of miR-155 in the immune system.

Southern blot hybridization of genomic DNA from AVOL-1 and AVOL-2 cells showed no evidence of genomic rearrangements in Bic loci (data not shown) discounting insertional activation of miR-155 in these cell lines.

Our results demonstrated that indeed v- rel controls miR-155 through one of the NF-κB binding sites in the Bic/miR-155 promoter.

The dynamic changes of miR-155 expression during the transformation process of splenocytes measured by qRT-PCR are shown in Fig. 4(a).

Fig. 2. Activation of miR-155 by v- rel occurs through the NF-κB pathway.

NF-κB site 2 in the Bic/miR-155 promoter is required for miR-155 activation.

Although the precise roles and molecular pathways of miR-155 in v-rel -induced transformation are not fully known, its repressive function on transcriptional factors such as Pu.

It is known that miR-155 can also be induced by a variety of immune cell stimuli such as TLR ligands, TNF-α, IFN-β and other antigens [41–45].

A conserved AP-1 element in the human Bic/miR-155 promoter was shown to be essential for some of these functions [46].

A number of recent miRNA profiling studies have shown elevated levels of miR-155 in a wide array of cancers including lymphomas [27–30].

Cloning and mutagenesis of the Bic/miR-155 promoterThe chicken Bic/miR-155 promoter region extending from −1829 to +3 nucleotides from the transcription start site (+1) was amplified by PCR from the genomic DNA prepared from CEF.

Stem-loop qRT-PCR for miR-155.

Analysis of the chicken Bic/miR-155 promoter sequence for potential transcription factor binding sites using the program tfsearch [34] identified a number of transcription factor binding sites, including two putative NF-κB sites (NF-κB1 and NF-κB2) located at positions −581 and −66, respectively (relative to the transcription start site).

The chicken Bic/miR-155 promoter region extending from −1829 to +3 nucleotides from the transcription start site (+1) was amplified by PCR from the genomic DNA prepared from CEF.

The precursor of miR-155, termed c- Bic, was first observed to cooperate with myc in chicken B-cell lymphomas induced by avian leukosis proviral integrations [26, 41].

High levels of miR-155 transcripts were readily observed in all Rev-T transformed cell lines (Fig. 1).

Cloning and mutagenesis of the Bic/miR-155 promoter.

Twenty micrograms of total RNA extracted from the cells indicated was separated on a 15 % denaturing polyacrylamide gel, blotted and hybridized with end -labelled antisense oligonucleotide probes to gga-miR-155.

2

In CEF cells, we observed that poly (I:C) stimulation had no effect on the expression of miR-21,but it could upregulate the expression of miR-155 with dose dependence (from 0.1 μ g/ml to 10 μ g/ml, Fig 4a) at 24 h after stimulation.

In contrast, high expression levels of miR-155 would inhibit TLR3 signals, and decrease the expressions of TLR3 and other important signal transduction molecules (e. g., TAB2).

We further demonstrated that miR-155 directly regulated TLR3 expression by binding to its coding region, which also regulated IFN-β production.

To confirm the regulation of miR-155 on TLR3 expression, HD11 cells were treated for 48 h with increasing amounts of the miR-155 antagomir, which can inhibit miR-155 function.

Here, we propose a viewpoint in which miR-155 could regulate TLR3 expression according to signalling thresholds rather than acting as an ‘off switch’ that intensively targets the TLR3 gene.

The role of miR-155 in the TLR3 response is very complex: miR-155 expression not only exerts negative effects on the immune response and inflammation, but also acts as a positive regulator of immunity via the modulation of cytokine expression (for review see [8, 9]).

Notably, we observed that gga-miR-155 significantly downregulated the expression of firefly luciferase fused to the TLR3 wild-type ORF, whereas mir-NC did not (Fig 2c).

Unlike these interactions, we found that miR-155 regulated the expression of TLR3 by targeting its coding region and not the 3’ UTR.

In vivo, the up-regulation of miR-155 is closely related with a variety of disease (for review see[26]).

These targets sequences indicate that miR-155 might directly target the TLR3 protein-coding region.

Here, we found that TLR3 expression was negatively regulated by miR-155, and this could be suggested that the TLR3 signal was controlled by miR-155 over -expression.

We hypothesised that if miR-155 can directly control TLR3 expression, a target site of miR-155 might exist in the TLR3 gene.

In the present study, we identified the existence of many naturally occurring miR-155 targets in the amino acid coding sequence of TLR3 and the essential role of miR-155 in the control of TLR3 expression.

Indeed, miR-155 has been as a fine-tuner of TLR signalling pathways, and regulation by miR-155 may occur at various levels of the TLR pathways via the targeting of adaptor molecules, downstream regulators and cytokines (for review see [8, 9]).

The target sites of the miR-155 sequence in the coding region of the Homo sapiens, Gallus gallus, Danio rerio, Equus caballus, Taeniopygia guttata and Cyprinus carpio TLR3 mRNAs were analysed by computational analyses and blast analyses of the miR-155 target sites.

The expression of miR-155 has been implicated in lymphoid malignancies and is mediated by several oncogenic herpes viruses[21– 23] to viral tumourigenesis, while the activation of TLR3 is involved in tumour suppression[5, 38, 39] via the recruiting of an anti-tumoural immune response.

The gga-mir-155 and mdv1-mir-M4-5p mimics, gga-mir-155 inhibitors, gga-miR-155 antagomir and agomir were synthesised by Ribobio, and the mimic and inhibitor Ncontrol, the antagomir and agomir Ncontrol were from Ribobio.

Thus, through targeting the TLR3 signalling pathway, miR-155 might exert a regulatory activity that limits the over-production of type I interferon and inflammatory cytokines during viral infection and other stimuli.

TLR3 expression is regulated by miR-155.

We analysed the most likely target sites of gga-miR-155 and mdv1-miR-M4-5p in the coding region of TLR3 mRNA (Fig 2a and 2b) by focusing on the base pairing in the seed sequence of the miRNA because the seed sequence is considered to be a critical determiners in the recognition of target mRNA by miRNAs.

This finding indicates that the complete analysis of the regulatory targets of miR-155 should be expanded to TLR3 coding regions.

Low levels of miR-155 expression would not trigger a negative regulation effect.

In vitro, the up-regulations of miR-155 following stimulation with toll-like receptor ligands (LPS, poly IC), IFN-β, IL-1β, TNF-α [27– 29] and miR-155 have been shown to be a component of the primary macrophage responses to different types of inflammatory mediators.

More recently, miR-155 has been as an important controller of TLR3 signalling via the targeting of adaptor molecules, downstream regulators and cytokines, such as TAB2, IKK-ε and RIP[8, 9].

Here, we reported that miR-155 negatively regulates TLR3 expression.

The expression of TLR3 was regulated by miR-155 and its functional orthologues.

Notably, we found miR-155 induction emerged a negative regulation on TLR3 mRNA expression (Fig 3b).

Our study demonstrated the up-regulation of miR-155 by poly (I:C) in chicken embryo fibroblast cells.

The regulation of TLR3 expression by miR-155.

The correlation between miR-155 and the responses induced by LPS are consistent with the finding that miR-155 targets transcripts that code for proteins involved in LPS/TNF-α signalling, including FADD, IKKɛ and RIPK1 [8, 9] Additionally, miR-155 has been shown to regulate STAT1, which is the key controller of the interferon (IFN) network.

Our study is the first to reveal that miR-155, and particularly the viral encoded miR-155 orthologue, can regulate TLR3 expression.

Although our findings have identified a previously unknown mechanism by which miR-155 can inhibit TLR3 signals, further studies should address the relevance of these findings in humans and mice to enhance our understanding of the regulatory functions of microRNA.

The available experimental evidence indicates that miR-155 is an important controller of TLR3 signals via the targeting of the downstream regulators, such as TAB2.

Secondary CEF cells were seeded in 6-well plate and incubated for 16~24 h. Next, cells were transfected for 48 h with gga-miR-155 mimic and inhibitor.

CEF cells were transfected with 50–200 nM of the miR-155 mimic or the negative control mimic, and TLR3 expression in the cell lysates was analysed with western blotting.

Notably, several oncogenic herpes viruses have evolved alternative strategies that either supply miR-155-like activities by encoding functional orthologous of miR-155 or induce the expression of cellular miR-155 in viral tumourigenesis [21– 23].

Two other oncogenic herpes viruses, Kaposi’s sarcoma -associated herpes virus and Marek’s disease virus, encode functional orthologues of miR-155 that are called miR-K12-11 (KSHV)[21, 33] and miR-M4 (MDV)[23, 34], respectively, and share the seed sequence of miR-155.

In contrast, IFN-β production was increased with the treatment of miR-155 inhibitor (Fig 4d).

0126012.g003 Fig 3 (a) miR-155 expression in HD11 cells treated with TLR2, 4 and 7 ligand.

Western blotting also showed that TLR3 protein expression was gradually decreased in HD11 cells along with the increased induction of miR-155 (Fig 3c).

Unfortunately, no miR-155 seed sequence was found in the 3′ untranslated regions (3′UTRs) of the TLR3 mRNA.

Additionally, the target site of mdv1-miR-M4-5p, which is the functional orthologue of miR-155 that is encoded by MDV, is in the TLR3 coding region of Gallus gallus.

Target site of miR-155 in the TLR3 coding region.

The over -expression of miR-155 has been reported in many malignant tumours, such as Hodgkin's lymphoma.

0126012.g004 Fig 4 (a) miR-155 expression in CEF cells treated with TLR3 ligand.

In the reciprocal experiment in which we transfected the cells with the firefly luciferase vector fused to the mutated TLR3 ORF, neither gga-miR-155 nor mir-NC affected the expression of firefly luciferase (Fig 2c).

In TLR ligands treated HD11 cells (an avian macrophage cell line), TLR2 and TLR4 Ligands stimulation resulted in higher expression of gga-miR-155 at 24h while had little effect on the amount of gga-miR-21 (Fig 3a and 3b).

Here, we demonstrated the existence of many naturally occurring miR-155 targets in the coding sequence (CDS) of TLR3.

miR-155 targets TLR3.

Western blotting revealed that the TLR3 protein expression was decreased in the cells transfected with the gga-miR-155 mimic or with the mdv1-miR-M4-5p mimic (Fig 2d and 2e).

Moreover, such inhibitory effects on TLR3 conferred by gga-miR-155 mimic or mdv1-miR-M4-5p mimic showed strong dose-dependence.

All these results demonstrate that miR-155 can decrease TLR3 expression.

Alternatively, herpes viruses use the functional orthologues of miR-155 to alter host cell responses and/or promote their life cycles by targeting viral transcripts, and these seem to be the predominant mechanisms of the establishment of viral latency and the induction of lymphomas.

An acceptable viewpoint is that miR-155 might function as a ‘brake’ or a molecular controller that represses the over-activation of the pro-inflammatory response without completely suppressing it.

miR-155 also plays important roles in controlling the differentiation of CD4+T cells into helper T cells[14, 15] and the development of regulatory T cells[16, 17].

Conversely, STAT1 also regulates miR-155.

Here, we found that the coding regions of TLR3 from different species (e. g., Homo sapiens and Gallus gallus) have binding sites for miR-155, which indicates that it is possible that miR-155 plays roles in the post-transcriptional regulation of TLR3 mRNA.

Recently, miR-155 has been found to play vital roles in the regulation of the immune response.

miR-155 in CD8+ T cells is critical for generating CD8+ T cell responses against viral infection and exerts this control by regulating type I interferon signalling[18– 20].

Regulation of IFN-β production by miR-155.

To test the effect of gga-miR-155 and mdv1-miR-M4 on regulating the endogenous TLR3 the CEF cells were transfected with a gga-miR-155 mimic or a mdv1-miR-M4-5p mimic, and the cells were cultured for 48 hours.

Moreover, miR-155 activates the response of CD8+ T cells to viral infection, and this activation is regulated by type I interferon signalling [18– 20].

One microRNA, miR-155 is an ancient regulator of the immune system[12] and is essential for normal immune function and antibody production[13, 14].

Activation of the TLR3 pathway by poly (I:C) has been reported to regulate IFN-β production in the chicken [46], and the induction of miR-155 by IFN-β has been demonstrated in mice [47].

The regulation of IFN-β production by miR-155.

We chose a pair of mo dels of miR-155 (gga-miR-155 and mdv1-miR-M4-5p) to confirm the direct interaction between the TLR3 mRNA coding region and miR-155.

We found that there are at least approximately 23 miR-155-5p sequences, 7 miR-155-3p sequences and 2 virus-encoded miR-155 orthologues in the miRBase v. 20.

Therefore, miR-155 might be an indicator of inflammation and an important link between cancer and inflammation.

The roles of miR-155 in the biologies and pathogeneses of several herpes viruses have been demonstrated.

Virally induced cellular microRNA miR-155 plays a key role in B-cell immortalization by Epstein-Barr virus.

Epstein-Barr virus -induced miR-155 attenuates NF-kappaB signaling and stabilizes latent virus persistence.

As described in Fig 4c, IFN-β production was decreased with the treatment of the miR-155 mimic from 50–200 nM (Fig 4c).

0126012.g001 Fig 1(a and b) The sequence logo for miR-155 was generated by Weblogo (http://weblogo.

Moreover, the miR-155 sequences in these species were highly conserved (Fig 1d).

The pGL3 firefly luciferase reporter plasmids with wild-type or mutant for the TLR3 mRNA coding regions were transiently transfected into HEK293 cells with the gga-miR-155 precursor, mdv1-mir-M4-5p or the negative control and a Renilla luciferase reporter for normalisation.

The consistent results were also observed in HD11 cells treated with increasing amounts of miR-155 agomir or antagomir (Fig 4e and 4f).

Analysis of the miR-155 seed sequence in the TLR3 coding region.

The sequence logo for miR-155 is shown in Fig 1a– 1e.

Thus, we speculated that miR-155 might control the TLR3 immune response in many manners that could be exploited by the virus via the encoding of functional orthologues of miR-155 to manipulate the host immune response.

Studies have shown that the induction of miR-155 plays a key role in B-cell immortalisation by the human herpes virus (Epstein-Barr virus) through the NF-кB pathway [31, 32].

Currently, the relationships between miR-155 and TLR3 have been extensively studied in virus infection, immune response and tumour formation.

In contrast, we treated HD11 cells with increasing amounts of miR-155 agomir significantly decreased the abundance of TLR3 (Fig 3e).

HD11 cells were seeded in 6-well plate and treated for 48 h with gga-miR-155 antagomir and agomir.

The treatment with the miR-155 antagomir resulted in increase TLR3 levels, and the concentrations of 400 nM were particularly effective (Fig 3d).

We also observed that the induction of miR-155 by poly (I:C) treatment was closely related to the IFN-β production in CEF cells (Fig 4b).

Moreover, the activation of TLR signals can also induce microRNAs (such as miR-146, miR-155 and miR-223) that can feedback the components in the TLR signalling system[9, 10].

Recent findings support the presence of a positive feedback loop involving miR-155 and STAT1.

The amino acid coding sequence of the chicken TLR3 (867–1073), mutant-type, putative gga-mir-155 binding site was cloned downstream of a firefly luciferase cassette in a pGV-272 vector.

The pri-gga-mir-155 and scrambled sequences were synthesised and cloned into pGV-268 vectors to generate the pGV-268-gga-mir-155 and the pGV268-scrambled control.

0126012.g002 Fig 2 (a and b) Predicted binding sites of gga-miR-155 and mdv1-mir-M4-5p in the coding region of the TLR3 mRNA.

3

These results indicate that similar responses are likely to be happening in the host during SE infection, that is, the down-regulation of gga-miR-101-3p may result in increased expression of IRF4 during Salmonella infection, and up-regulation of gga-miR-155 may inhibit expression of LRRC59.

The results showed that miR-155 overexpression markedly decreased the expression of IL-6 and TNF-α compared with control miRNA or miR-155 inhibitor (Figure 7A; P < 0.01), while miR-101 knockdown significantly decreased the expression of IL-6 and TNF-α compared with control miRNA inhibitor (Figure 7B; P < 0.05).

The in vitro experiment showed that gga-miR-155 directly repressed the expression of LRRC59; In addition miR-155 overexpression markedly decreased the expression of IL-6 and TNF-α compared with control miRNA or miR-155 inhibitor (P < 0.01).

For instance, few proteins (IRAK1, IRAK2, and TRAF6) within TLR signaling have been confirmed as direct targets of miR-146 (O'Neill et al., 2011); signal molecules MyD88, TAB2, SHIP1, and SOCS1 were targets of miR-155 (Eulalio et al., 2012); and cytokines IL-6 and IL-10 are targeted by Let-7 (Stae del and Darfeuille, 2013).

Two miRNAs, gga-miR-155 and gga-miR-101-3p, could directly alter the expression of target IRF4 and LRRC59 and regulate the production of pro-inflammatory cytokines, respectively.

mRNA expression of IL-6 and TNF-α in chicken HD11 6 h after LPS treatment, or 24-h post transfection with miRNA control (50 nM), miRNA inhibitor control (100 nM), miRNA-155/101 (50 nM) and miRNA-155/101 inhibitor (100 nM) then the cells were stimulated with LPS (1 μg/mL) for 6 h. Relative transcript abundances of the genes were analyzed by qPCR.

For these miRNAs, gga-miR-155 had the most abundant expression and it was significantly up-regulated in susceptible chickens (both S vs.

These results indicate that gga-mir-155 could target gene LRRC59 and then suppress the production of pro-inflammatory cytokines in response to LPS challenge.

After 36-h treatment with mimic, elevating gga-miR-155 significantly repressed the mRNA expression levels of LRRC59 compared to the miR-NC and negative controls (P < 0.05); In contrast, after 36-h treatment with gga-miR-101-3p inhibitor, the mRNA expression levels of IRF4 were significantly increased (P < 0.05) compared to the controls (Figure 6).

Based on the foregoing observations and interpretations, it is reasonable to propose that gga-miR-155 and gga-miR-101-3p contribute to SE -induced pathogenesis and regulate the production of pro-inflammatory cytokines through directly down -regulating LRRC59 and up -regulating IRF4 genes, respectively.

Previous studies have shown that miRNAs, such as miR-146a, miR-155, and Let-7 and their targets are involved in the regulation of immune response to Salmonella or lipopolysaccharide infection in mice (O'Neill et al., 2011; Schulte et al., 2011; Eulalio et al., 2012) and swine (Bao et al., 2014, 2015; Yao et al., 2016a, b).

Figure 7Gga-miR-155 and gga-miR-101-3p regulate expression of pro-inflammatory cytokine genes induced by LPS.

The pmiR-RB-Report™ (RiboBio, Guangzhou, China) including double luciferase reporter genes was used to test and validate the target sites for gga-miR-155 and gga-miR-101-3p.

The present study of splenic miRNA and mRNA profiles from chickens after Salmonella challenge has identified differential expression of several miRNAs linked to immune responses, including miR-155, miR-9, miR-30 which have been reported previously and several miRNAs, such as miR-101-3p and miR-130b-3p, which were shown here to be associated with the immune response to infection with SE.

To further validate the biological function of gga-miR-155 and gga-miR-101-3p in a chicken macrophage-like line HD11, 100 μM mimic (gga-miR-155), inhibitor (gga-miR-101-3p) and control oligos (gga-miR-NC) were transfected into HD11 cells using 12-well plates and TransIT®-2020 (Mirus Bio, Madison, WI) per the manufacturer's instructions.

Over-expressed gga-miR-155 and interference gga-miR-101-3p in chicken HD11 macrophage cells.

For example, CXCR4 is involved in cytokine-cytokine receptor interaction and was identified as a potential target of gga-miR-155 and gga-miR-9-3p.

Except for gga-miR-92-5p with a slight difference in the R group, the expression patterns of gga-miR-101-3p, gga-miR-126-3p, gga-miR-155, gga-miR-103-5p, and gga-miR-455 were comparable by both methods.

LRRC59 was predicted as a potential target of gga-miR-103-5p and gga-miR-155.

Regulation of the MIR155 host gene in physiological and pathological processes.

These data demonstrate that gga-miR-155 and gga-miR-101 could regulate the production of pro-inflammatory cytokines, IL-6 and TNF-a, which may play a negative role in response to LPS stimulation in chickens.

Interestingly, the expression of gga-mir-155 was significantly higher in the S chickens compared with R birds.

Figure 5Regulation of LRRC59 by gga-miR-155.

Since hub nodes have been found to play important roles in many networks (He and Zhang, 2006), the presence of hub miRNAs was sought and, several were identified including gga-miR-155 and gga-miR-101-3p (Figure 3).

The 3′ UTR of IRF4 and LRRC59 containing gga-miR-101-3p and gga-miR-155 binding sites were amplified from chicken genomic DNA.

Validations of miRNA-mRNA interactions using gga-miR-101-3p- IRF4 and gga-miR-155- LRRC59 mimicsThe luciferase reporter gene system was used to validate the above-stated predicted interactions.

In the current study, gga-miR-155 was significantly induced by SE infection, which was consistent with the above mammalian studies.

It has been shown that miR-155 is involved in the TLRs signaling pathway and play important roles in the innate immune response (Quinn and O'Neill, 2011; Elton et al., 2013; Li and Shi, 2013).

Several miRNAs previously reported to be involved in immune responses such as miR-155, miR-9, miR-30, miR-126, and miR-29 families were identified.

Figure 6Validations of biological function of gga-miR-155 and gga-miR-101-3p in chicken HD11 macrophages.

Validations of biological function of gga-miR-155 and gga-miR-101-3p in chicken HD11 macrophage cells.

The 3′ UTRs of IRF4 and LRRC59 were cloned into luciferase reporter plasmids to test gga-miR-101-3p and gga-miR-155 functions in vitro.

Validations of miRNA-mRNA interactions using gga-miR-101-3p- IRF4 and gga-miR-155- LRRC59 mimics.

In order to address the effect of miR-155 and miR-101 on the induction of pro-inflammatory cytokines in response to LPS, the expression levels of IL-6 and TNF-α were measured in a macrophage inflammatory response mo del.

4

Using an miRNA target prediction algorithm (Bartel, 2009), candidate cellular mRNA target sites were identified and it was subsequently confirmed that miR-M4-5p and miR-155 down-regulate the expression of a set of shared mRNA targets, including PU.

Marek’s disease virus-encoded analog of microRNA-155 activates the oncogene c-Myc by targeting LTBP1 and suppressing the TGF-beta signaling pathway.

Interestingly, it has also been demonstrated that miR-155 can suppress TGF-β signaling though targeting SMAD2 and SMAD5 in human diseases (Louafi et al., 2010; Rai et al., 2010).

These potential mRNA targets were further confirmed by DLRA, the results of which showed that targeting of the 3′UTRs of these genes via the seed regions of miR-155 family members is conserved between humans and chickens, although the data did not demonstrate a statistically significant difference in response to miR-M4-5p and/or miR-155 expression in all cases (Parnas et al., 2014).

As a viral miR-155 ortholog, the KSHV-encoded miR-K12-11 inhibits TGF-β signaling through down-regulation of SMAD5 (Liu et al., 2012).

Virus-encoded miR-155 ortholog is an important potential regulator but not essential for the development of lymphomas induced by very virulent Marek’s disease virus.

Three independent research groups identified MYB, a transcription factor involved in the regulation of hematopoiesis and tumorigenesis, as a target for miR-M4-5p, miR-K12-11, and miR-155 (Muylkens et al., 2010; Zhao et al., 2011; Parnas et al., 2014).

miR-155 is highly conserved in humans, mice, and chickens, particularly the seed region, primarily expressed in lymphocytes of the thymus and spleen, and has regulatory functions in the hematopoietic and immune systems (Rodriguez et al., 2007).

These observations suggest that miR-M4-5p may be an oncogenic miRNA, the functions of which could be partially restored by miR-155 because the two molecules regulate a common set of mRNA targets through their conserved seed region.

FIGURE 1Conserved cellular mRNA targets of GaHV2-encoded orthologs of cellular miR-155.

Both miR-K12-11 and miR-155 are associated with human lymphoma and share a common set of mRNA targets (Gottwein et al., 2007; Skalsky et al., 2007; McClure and Sullivan, 2008).

Reticuloendotheliosis virus strain T induces miR-155, which targets JARID2 and promotes cell survival.

Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek’s disease lymphomas.

Over -expression of miR-155 is related to B cell lymphomas and solid tumors (Tili et al., 2013).

Interestingly, EBV -induced cell proliferation, as well as virus latency and reactivation, are associated with elevated miR-155 expression levels (Yin et al., 2008; Linnstaedt et al., 2010; Forte and Luftig, 2011).

Importantly, it has been clearly shown that EBV -induced overexpression of cellular miR-155 is essential for transformation by EBV (Linnstaedt et al., 2010).

A functional microRNA-155 ortholog encoded by the oncogenic Marek’s disease virus.

Targeting of SMAD5 links microRNA-155 to the TGF-beta pathway and lymphomagenesis.

MicroRNA-155 is an Epstein-Barr virus -induced gene that modulates Epstein-Barr virus-regulated gene expression pathways.

MicroRNA-155 targets SMAD2 and modulates the response of macrophages to transforming growth factor-β.

In addition, Parnas et al. (2014) performed photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) analysis of RISC binding sites in the GaHV2-transformed MSB-1 cell line to identify targets of miR-M4-5p, and then compared to those of miR-155 and miR-K12-11 similarly identified in EBV-transformed lymphoblastoid cell lines (LCLs) or a KSHV-transformed PEL cell line.

It is clear that miR-155 could have an important regulatory role in many biological pathways of significance in oncogenesis, hence further investigation is needed to define the in vivo mRNA targets of this miRNA and its viral orthologs.

Collectively, these findings indicate that dysregulation of the TGF-β signaling pathway by miR-155 and its viral orthologs may be a common feature shared by oncogenic herpesviruses (Figure 2).

The potentiality of miR-K12-11 compensating for the function of miR-155 has been proved in humanized and transgenic miR-155 knockout mice; however, this is difficult to be confirmed because of the lack of a natural animal mo del for KSHV infection (Boss et al., 2011; Sin et al., 2013).

The miR-155/JARID2 axis could regulate cell apoptosis and differentiation in acute myeloid leukemia and abnormal megakaryopoiesis (Norfo et al., 2014; Palma et al., 2014).

The critical viral miR-155 orthologs, miR-M4-5p and miR-K12-11, share a set of common mRNA targets, suggesting the common viral characteristics of mimicking host miRNAs and aberrant regulation of viral miRNAs may be important in herpesvirus life cycle control, immune evasion, pathogenesis, and even tumorigenesis.

The rescue of the regulatory role of miR-M4-5p by miR-155 in vivo by viral genomic locus replacement proves that the two molecules are authentic functional orthologs.

miR-155 was initially identified as a B-cell integration cluster (bic) gene, which is activated by promoter insertion at a retroviral integration site in avian leukosis virus (ALV) -induced lymphomas (Clurman and Hayward, 1989; Rodriguez et al., 2007).

Interestingly, one GaHV2-encoded miRNA, miR-M4-5p, is a functional miR-155 ortholog (Zhao et al., 2009).

Thus, the shared seed region and common set of target mRNAs of miR-M4-5p, miR-K12-11, and miR-155 have facilitated the characterization of miRNA phenotypes in relation to viral tumorigenesis, as in MD.

miRNA-mRNA integrative analysis in primary myelofibrosis CD34 [+] cells: role of miR-155/JARID2 axis in abnormal megakaryopoiesis.

Requirement of bic/microRNA-155 for normal immune function.

Virally induced cellular microRNA miR-155 plays a key role in B-cell immortalization by Epstein-Barr Virus.

miR-155 and Its Viral Ortholog.

Interestingly, MD incidence was restored when the viral pre-miRNA, miR-M4, was replaced by gga-miR-155, although tumor induction was slow and detected later than in chickens infected by parental and revertant viruses (Zhao et al., 2011).

It is of great interest that the KSHV-encoded miR-K12-11 functions as a viral ortholog of miR-155 (Gottwein et al., 2007; Skalsky et al., 2007).

miR-155 is also involved in viral pathogenesis, particularly that of the tumor-related herpesviruses.

Kaposi’s sarcoma -associated herpesvirus encodes an ortholog of miR-155.

miR-M4-5p Functions As A Viral miR-155 Ortholog.

A Kaposi’s sarcoma -associated herpesvirus-encoded ortholog of microRNA miR-155 induces human splenic B-cell expansion in NOD/LtSz-scid IL2Rgammanull mice.

A viral microRNA functions as an orthologue of cellular miR-155.

5

The downregulation of miR-155 in MDV-transformed tumor cell lines is not a permanent defect, as these cells can be induced to express miR-155 by co-infection with retroviruses -expressing v-Rel (unpublished data).

Interestingly in KSHV -induced tumors also, there is upregulation of the miR-155 functional homolog miR-K12-11, at the expense of significant downregulation of miR-155 [39].

Demonstration of expression of miR-M4, but not gga-miR-155, in lymphoid tumors harvested from birds infected with miR-4-0-R virus (bird numbers #2166S, 2171S, 2172S and 2178K) indicated the direct correlation between the expression of miR-M4 and the induction of lymphomas (Fig. 9B–C).

One possible advantage of encoding a viral ortholog is the potential to achieve high levels of expression as seen in MDV- and KSHV -induced tumors, overriding the tighter cellular regulatory controls associated with the c- bic/miR-155 expression.

Although the types of tumors induced by recombinant viruses expressing miR-M4 or gga-miR-155 were not distinguishable (Fig. 9A), the onset of tumors in birds infected miR-4-0-155 virus was slow (Fig. 6B), possibly due to the differences in the functional context of the two miRNAs or in their processing and expression.

Although it is unclear how MDV is able to downregulate miR-155, some feedback regulatory mechanisms are the most likely mechanisms.

The lack of expression of miR-M4 and weak expression of miR-155 in the tumors induced by miR-4-0-155 virus was also confirmed by Northern blot (Fig. 9D).

The conservation of the seed sequence and the ability to regulate common sets of targets by miR-M4 and gga-miR-155 [19] prompted us to examine whether the miR-M4 functions of MDV can be replaced by gga-miR-155.

Moreover, although miR-155 and the two viral orthologs may regulate common set of target proteins through the conserved seed sequences, other potential differences in their functions, especially due to the differences in the non-seed sequences, may also exist.

Interestingly, there is consistent downregulation of gga-miR-155 in MD tumors and MDV-transformed cell lines [17], [18].

This is unlikely to be due to any spurious mutations or recombination events in the virus, as we were able to confirm the absence of miR-M4 or miR-155 expression in the tumor tissues of this bird (Fig. 9D).

Despite the low levels of expression, detection of gga-miR-155 but not miR-M4 in tumors of birds infected with miR-4-0-155 virus (bird numbers #2153L, 2153S, 2163L and 2183S) by quantitative RT-PCR and Northern blotting (Fig. 9D) suggested a direct role for gga-miR-155 in induction of these tumors.

Remarkably, miR-K12-11 and miR-M4, the miRNAs encoded by the oncogenic Kaposi's sarcoma -associated herpesvirus (KSHV) and Marek's disease virus (MDV) respectively, have been shown to be functional orthologs of miR-155.

From the demonstration of the conserved functions of miR-M4 and miR-155 in the induction of tumors, it is clear that MDV is able to restore the functions of miR-155 through the expression of high levels of the functional homolog miR-M4.

Finally, overexpression of miR-155 has been shown to be associated with lymphocyte transformation by viruses such as EBV [37] and reticuloendotheliosis virus strain T [38].

Tumors induced by the chimeric miR-4-0-155 virus are the first examples demonstrating the expression of gga-miR-155 in MD tumors.

No expression of miR-M4 but very low miR-155 was detected in the tumor that was induced in a single bird (2168L) infected with miR-M4-00 virus.

This was also evident from the expression levels of miR-M4 and miR-155 in the primary tumor samples collected from birds infected with the two viruses.

Nevertheless, the differences in the speed in the onset of tumors between MDV expressing miR-M4 and gga-miR-155 would suggest that such differences could be important.

Moreover, recent studies have demonstrated differences between miR-M4 and gga-miR-155 in targeting genes such as UL28 despite having identical seed sequences highlighting the significance of the non-seed regions of these miRNAs [22].

In order to demonstrate the expression of gga-miR-155 or miR-M4 in the tumors induced by miR-4-0-155 and miR-4-0-R viruses respectively, we carried out quantitative RT-PCR on RNA samples extracted from the lymphomas collected from infected birds at post mortem.

1, BACH1, CEBPβ, HIVEP2, BCL2L13 and PDCD6, we have previously shown that miR-M4 is a functional ortholog of gga-miR-155 [19], a miRNA known to be directly associated with several cancers [23], [36] and molecular mechanisms of cancer pathogenesis [25], [26].

Among all the miRNAs, miR-155 has been well documented for its direct role of oncogenesis in a number of species including chickens.

However, based on the direct association of miR-155 in several cancers [23], [24], including the recent findings on its roles in TGF-β pathway and lymphomagenesis [25] and modulation of mismatch repair and genomic instability [26], we hypothesised that miR-M4 has a major role in MDV oncogenicity.

Although the precise understanding of all the molecular processes would need further studies, the present findings demonstrate that miR-M4, most likely acting through the miR-155 pathway, functions as a key factor contributing to MD oncogenesis.

Oncogenic function of miR-M4- deleted virus can be rescued by gga-miR-155.

Since MDV miR-M4 in this cluster is a functional ortholog of the oncogenic miR-155 [19] implicated in a number of neoplastic disorders [23], we decided to focus on this particular miRNA.

In the present study, we explored the functional role of the miRNAs encoded within the cluster 1 of the MDV genome, including that of the miR-155 functional ortholog miR-M4, in inducing T-cell lymphomas using the well-established mo dels of MD in the natural chicken hosts of the virus.

On the other hand, EBV does not encode any functional ortholog but induces miR-155 in transformed B-cells [37], and a recent study has demonstrated its role in the induction of B-cell transformation by EBV in vitro [40].

The role of miR-M4 and the rescue of the oncogenic phenotype by miR-155 provide further evidence of the conserved biological functions of miRNA orthologs, a finding of major importance in elucidating the functions of other viral orthologs such as KSHV miR-K12-11.

Demonstration of the incidence of MD at similar levels (70%) in birds infected with miR-4-0-155 virus and the miR-4-0R revertant virus (Fig. 6B) indicated that gga-miR-155 can function as an oncogenic miRNA in the context of the MDV cluster 1 miRNAs.

For this, we generated an additional MDV construct miR-4-0-155 (Table 1), in which gga-miR-155 pre-miRNA including the loop sequence of the chicken BIC transcript [32] was introduced in the position of the viral miR-M4 pre-miRNA.

Since miR-M4 is a functional ortholog of the host-encoded gga-miR-155 [19], we wanted to examine whether gga-miR-155 can replace the oncogenic function of miR-M4 in MDV.

The demonstration of the ability of the chimeric miR-4-0-155 virus to restore the oncogenic potential to the same levels as the miR-4-0-R (Fig. 6B), further demonstrated the significance of the miR-M4/miR-155 pathway in MD lymphomagenesis.

The ability of gga-miR-155 to rescue the oncogenic potential of miR-M4- deleted virus demonstrated the conservation of oncogenic functions the two miRNAs.

The six miRNAs in cluster 1, thought to be processed from a single primary transcript upstream of the Meq locus [16] included miR-M4, the functional ortholog of miR-155 [19] and KSHV-miR-K12-11 [20], [21].

6

Apart from several cellular targets shared with miR-155, mdv1-miR-M4-5p and mdv1-miR-M4-3p have been shown to inhibit the production of the viral UL28 and UL32 proteins respectively, thus providing the first evidence of late viral gene targeting by herpesviral miRNA and making it the first avian herpesvirus miRNA known to target both viral and cellular mRNAs [33].

Interestingly, in both MDV-1 and KSHV -induced tumors, there is down-regulation of endogenous levels of miR-155 [44, 51, 53], although the mechanisms for such down-regulation is not fully understood.

MDV-1 and KSHV express two distinct miRNAs that function as an ortholog of miR-155, a conserved cellular miRNA that is highly expressed in activated lymphoid and myeloid cells and that is required for the rapid expansion of B and T cells after antigenic stimulation [32, 37, 51, 52].

The host miRNAs downregulated in the MDV-transformed cell lines include miR-155, miR-223, miR-150, miR-451, and miR-26a.

Furthermore, overexpression of miR-155 has been shown to be associated with lymphocyte transformation by EBV [58] and reticuloendotheliosis virus strain T [59].

As observed for its KSHV-encoded counterpart, several transcription factors are potentially shared targets for both miR-155 and mdv1-miR-M4-5p.

mdv1-miR-M4-5p, a member of cluster 1 miRNA and a functional ortholog of gga-miR-155 which plays a major role in lymphoid malignancies and the modulation of immune responses, is the most highly expressed viral miRNA in tumors, representing over 70% of MDV miRNA sequencing reads [37].

Zhao Y. Xu H. Yao Y. Smith L. P. Kgosana L. Green J. Petherbridge L. Baigent S. J. Nair V. Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek’s disease lymphomas PLoS Pathog.

Rai D. Kim S. W. McKeller M. R. Dahia P. L. Aguiar R. C. Targeting of SMAD5 links microRNA-155 to the TGF-beta pathway and lymphomagenesis Proc.

Bolisetty M. T. Dy G. Tam W. Beemon K. L. Reticuloendotheliosis virus strain T induces miR-155, which targets JARID2 and promotes cell survival J. Virol.

Zhao Y. Yao Y. Xu H. Lambeth L. Smith L. P. Kgosana L. Wang X. Nair V. A functional MicroRNA-155 ortholog encoded by the oncogenic Marek’s disease virus J. Virol.

There has been a number of studies showing that miR-155 has direct role of oncogenesis [54, 55] and induction of cancer [56, 57].

These observations provide additional evidence for the impact of miR-155 and its orthologs on pathways regulating lymphocyte activation, differentiation and immune tolerance [7].

Lu F. Weidmer A. Liu C. G. Volinia S. Croce C. M. Lieberman P. M. Epstein-Barr virus -induced miR-155 attenuates NF-κB signaling and stabilizes latent virus persistence J. Virol.

Introduction of chicken miR-155 into the miR-M4- deleted MDV-1 virus at least partly restored transformation ability, providing the first demonstration that viral miRNAs can play a key role in enhancing the oncogenic potential of a herpesvirus in vivo.

Skalsky R. L. Samols M. A. Plaisance K. B. Boss I. W. Riva A. Lopez M. C. Baker H. V. Renne R. Kaposi’s sarcoma -associated herpesvirus encodes an ortholog of miR-155 J. Virol.

Valeri N. Gasparini P. Fabbri M. Braconi C. Veronese A. Lovat F. Adair B. Vannini I. Fanini F. Bottoni A. Modulation of mismatch repair and genomic stability by miR-155 Proc.

1, CEBPβ, HIVEP2, BCL2L13, PDCD6, GPM6B, RREB1, c-Myb, MAP3K7IP2Mimics cellular mir-155 [32, 33, 34].

As reported above, mdv1-miR-M4-5p is a functional ortholog of cellular miR-155.

Tili E. Croce C. M. Michaille J. J. miR-155: On the crosstalk between inflammation and cancer Int.

Faraoni I. Antonetti F. R. Cardone J. Bonmassar E. miR-155 gene: A typical multifunctional microRNA Biochim.

7

However, perhaps the most striking change is in gga-miR-155, which is the most up-regulated miRNA in CD40L stimulated B cells, but it is the most down-regulated miRNA in ALV transformed DT40 cells.

NF-kappaB down-regulates expression of the B-lymphoma marker CD10 through a miR-155/PU.

Elevated expression of miRNAs such as gga-miR-155 has also been demonstrated in chicken hematopoietic cells transformed by reticuloendotheliosis virus (Bolisetty et al., 2009).

High expression of miR-155 upon CD40L stimulation is consistent the major role of this miRNA in proliferation.

The highly expressed gga-miR-155 has been extensively studied and shown to be associated with cell proliferation in a number of cancers, as well as in autoimmunity (Leng et al., 2011; Wang and Wu, 2012).

Expression levels of miRNAs obtained from the deep sequencing data were validated by carrying out quantitative RT-PCR on gga-miR-21, gga-miR-26a, gga-miR-142-3p and gga-miR-155 using RNA extracted from different cell types.

As v-Rel is an NF-κ B homolog, it is possible that the increased expression of miR-155 by CD40L is mediated through the NF-κ B pathway, although other signaling systems may also be involved.

Other viral oncogenes such as v-Rel have also been shown to drive miR-155 expression (Bolisetty et al., 2009).

Reticuloendotheliosis virus strain T induces miR-155, which targets JARID2 and promotes cell survival.

Validation of expression levels of gga-miR-21, gga-miR-26a, gga-miR142-3p, gga-miR-155 and gga-miR-223 was carried out by quantitative RT-PCR using procedures described (Yao et al., 2008).

The most striking change was in the level of gga-miR-155.

Role of miR-155 in breast cancer.

Role of microRNA-155 in autoimmunity.

One of the well characterized targets of miR-155, the transcription factor PU.

The miRNAs which showed significant increase upon CD40L-stimulation included gga-miR-21, gga-miR-155, gga-miR-146a, gga-miR-20b, gga-miR-106, gga-miR-222, and gga-miR-22.

Reduced levels of miR-155 may help in maintaining high levels of PU.

The miRNAs which showed significant increase upon CD40L-stimulation included gga-miR-21, gga-miR-155, gga-miR-146a, gga-miR-20b, gga-miR-106, gga-miR-222 and gga-miR-22 (Figure 2).

Interestingly, miR-155 was first discovered in the chicken as part of the c- bic transcript in ALV-transformed lymphomas (Clurman and Hayward, 1989).

8

Non-infected (Fold-change) miRNA [1] (fold change)MX1 myxovirus (influenza virus) resistance 1 [27]Z23168+11.46gga-miR-155(+3.55)gga-miR-206(−2.86)Interleukin 8 (IL8) [28]M16199+11.03gga-miR-32(+7.98)Interferon regulatory factor 7 (IRF7) [29]U20338+2.11gga-miR-142-5p(+2.84)Interleukin1receptor-like1, transcript variant1 [51]AB041738+1.65gga-miR-460 (only expressed in infected lungs)TNF receptor superfamily, member 19 [30]BX931334−1.85gga-miR-187(−4.35)Tipartite motif-containing 7.1 [52]BX934475−1.81NA [2]RAC serine/threonine-protein kinase3 (ATK3) [53]BX950472−1.65NAC-fringe 1 [54] U97157 −1.52 NANote: [1]miRNAs targeting on differentially expressed immune related genes; [2] No miRNAs targeting on the gene; +: Up-regulated with AIV infection; –: Down- regulated with AIV infection.

Most of the up-regulated miRNAs were predicted to target the hemaglutinin (HA) and neuraminidase (NA) mRNAs such as miR-34a and miR-155.

The up-regulation of miR-155 with poly (I:C) and IFNβ stimulation in mouse macrophages suggest an important role of miR-155 in the regulation of viral infection [37].

Based on target prediction, miR-155 could target the chicken anti-influenza gene MX1, therefore playing a role in host and AIV interactions in chickens.

The inhibition of JNK pathway blocked the expression of miR-155 in murine macrophages [37, 40].

Therefore, up-regulated miR-155 might also activate JNK pathway, and subsequently induce apoptosis to eliminate virus infected cells [37, 40].

miR-155 knock-out mice are not capable of generating defensive immune responses, developing lymphocytes, or antigen-presenting cell functions [39].

In the current study, gga-miR-155 was significantly induced by AIV infection, which was consistent with other studies [40].

The miR-155 has been reported to play important roles in both innate and adaptive immunity in mammals [7, 37, 38].

9

It has been confirmed that the hypoxia inducible miR-155 were upregulated in hypoxia and reduced its target gene HIF1α expression both in mRNA and protein levels [36].

In our results, a similar observation was found: that up-regulated miR-155 also correlated to HIF1α, which was under expressed in the pulmonary arteries of AS broilers.

Likewise, from the miRNA-mRNA association, the under expressed genes LZTFL1, JAZF1, THBS2 and RPS14 were associated with microRNAs (miR-146b-5p, miR-1684a-3p, miR-460b-3p, miR-30e-5p, miR-33-5p, miR-148a-5p, miR-32-5p, miR-155 and miR-144-3p) that were down-regulated in pulmonary arteries (Figure 4).

Considering the differential expressions of miR-155, miR-23b-3p, miR-146b-5p and miR-146b-3p found in the AS broiler pulmonary artery tissues in this study, we proposed these four miRNAs may also serve as a key regulator to pulmonary artery remo deling during AS progress.

In this study, we found that miR-155, miR-23b-3p, miR-146b-5p and miR-146b-3p upregulated in AS broilers’ pulmonary arteries tissue.

This research highlighted the potential role of specific miRNAs in disease progress [23], like miR-21, miR-204, miR-17, miR-155, miR-138 and miR-30c in human and rat mo dels of PAH [7, 8, 28, 29].

MiR-155 was among the miRNAs that were regulated in endothelial cells by shear stress forces [30, 31].

MiR-155 is expressed in multiple cell types, and it has been implicated in activating several biological processes including hematopoiesis, inflammation and immunity [32].

And in this study, we found more than one miRNA, including miR-142-5p, miR-32-5p, miR-1662, miR-155, miR-30e-5p and miR-34a-5p, that function together to target LRP1B in the pulmonary arteries of AS broilers, which was characterized by vascular cells proliferation.

Increased miR-155 levels in human Endothelial Cells (ECs) induced changes in morphology and filamentous-actin organization [33].

And it was demonstrated that miR-155 may serve as a component of a negative-feedback loop which specifically controls HIF1α resolution and finally determines transcriptional response in cells when in exposer to sustained hypoxia [36].

10

Mdv1-miR-M4 also shares some targets with mir-155, and it has been suggested that mdv1-miR-M4 target regulation plays a role in MDV -induced lymphomagenesis [29].

Sustained expression of miR-155 can stimulate production of granulocytes, and miR-155 over -expression can lead to neoplasia [26].

Thus the MDV1 ortholog of miR-155 may also be pleomorphic in that it facilitates viral growth by impeding the apoptotic response to infection and also down-regulates genes involved in cell cycle control.

An overall decline in microRNA expression has been noted in other cancers [30, 31], and we hypothesize that mdv1-miR-M4 serves to replace miR-155 in regulating functions essential to the phenotype of the transformed cell.

Mdv-miR-M4 is highly expressed in MDV induced tumors, while miR-155 is present at very low levels.

However, miR-155 expression is complicated in that its induction in macrophages by activation of the immune response with lipopolysaccharides does not result in subsequent neoplasia [27, 28].

For miR-155, loss- and gain-of-function studies have demonstrated a critical role in both immune cell development and function.

It has been proposed that kshv-miR-K11 is a functional ortholog of mir-155 and can regulate the same set of host mRNAs, and this contributes to KSHV -induced tumorigenesis [24], [25].

MDV1 encodes a microRNA (mdv1-miR-M4) that shares a seed sequence with miR-155, a microRNA important in immune function.

MDV1, like Kaposi’s sarcoma herpesvirus (KSHV), encodes a microRNA that shares a seed sequence with the host microRNA, miR-155 (kshv-miR-K11; mdv1-miR-M4).

11

In addition, the analysis of miRNA modulation allowed for the validation of two synergistic mechanisms related to miR-21 downregulation and miR-155 upregulation, as described in the Fig. 5C.

Conversely, the expression of miR-155 was significantly increased after the exposure to both gemcitabine and crizotinib monotherapies, and was additionally upregulated (up to 2.0-fold) by the combination.

On the other hand, the upregulation of miR-155 levels might be explained by a feedback mechanism caused by the inhibition of c-Met, which play an important role in tumor-fibroblasts interactions.

Remarkably, miR-155 upregulation has been associated with increased reactive oxygen species (ROS) levels 42, which have been implicated in the degradation of cytidine deaminase (CDA) - the main enzyme in gemcitabine catabolism 43.

Additionally, in the CAM tumors collected on EDD19, we determined how the treatment with gemcitabine and crizotinib affected the expression of miR-155 and miR-21 (Fig. 5B).

Furthermore, we included in our analysis two miRNAs that have been commonly associated with PDAC pathogenesis and gemcitabine chemoresistance, namely miR-155 and miR-21 46 50.

A recent study suggested indeed that PDAC cells might activate normal adjacent fibroblasts by means of secreted microvesicles containing miR-155 41.

12

We do not know, however, the normal level of miR-155 expression in target normal bursal stem cells, which is the critical factor for comparison with expression in Myc -induced neoplastic development.

Subsequently bic was found to encode a highly conserved micro RNA, miR-155, shown to be essential for normal B-cell development [33], to be lymphomagenic when over expressed in transgenic mice [34], and to be over expressed in several forms of human B-cell malignancies [35]– [37].

Therefore, and In contrast to our observations in the multistage development of bursal lymphomas, palindrome formation is unlikely to play an essential role in acute Myc transformation of DF-1 fibroblasts Palindromes Formed at bic/miR-155 Loci Are Strongly Selected during Tumorigenesis Bic is a non-coding RNA gene discovered as a site of ALV insertional mutagenesis (in addition to activating insertions at c- myc ) selected during tumor development in a large proportion of ALV -induced bursal lymphomas [19], [20].

We discovered consistent palindrome formation at a cancer -associated microRNA gene called bic/miR-155, beginning at an early stage of tumor development, and without significant further amplification of the locus.

The specific 1 to 1 correlation of palindrome formation at bic with all stages of bursal lymphomagenesis (preneoplastic TF, metastatic lymphoma and derivative established cell lines) indicates a strong selection in the development of these tumors for this otherwise uncommon change at the bic locus, and indicates a mechanistic alternative to retroviral insertional mutagenesis for alteration of bic/miR-155.

By combining GAPF with chromatin immune precipitation (ChIP) we discovered an association between palindrome formation and the occupancy of nearby Myc binding sites in chromatin, and, finally, report a strong selection for palindrome formation at an oncogenic micro RNA gene, bic/miR-155, known to cooperate with c-myc in bursal lymphomagenesis [19]– [21].

Palindromes Formed at bic/miR-155 Loci Are Strongly Selected during Tumorigenesis.

Positions of the Myc binding site (red oval), exons 1 and 2, the position of miR-155 coding sequence (black box) and relevant restriction cut sites are indicated.

As with our previous study of gene amplification in this system [11] there was no unique signature of affected genes detected with the very important exception of the Bic/miR-155 locus.

Figure 5 depicts a chart of the genomic region around bic including the positions of a Myc -binding E box (CACGTG) exons 1 and 2, the location of miR-155, key restriction endonuclease sites and the results of Southern blot hybridization analysis.

13

Their study showed that several host-encoded miRNAs, including gga-miR-155, gga-miR-150, gga-miR-451, gga-miR-26a, and gga-miR-223, were down-regulated in all MDV-transformed cell lines compared to normal splenocytes, and nine MDV-1-encoded miRNAs were up-regulated in all of the MDV-transformed cell lines compared to the MDV -negative reticuloendotheliosis virus (REV)-T-transformed cell line AVOL-1. Previous studies have mainly focused on miRNA profiles of MDV-infected CEF and MDV-transformed cell lines and expression of several individual miRNAs in MDV -induced splenic tumors.

gga-miR-155 was up-regulated in two MDV-infected samples in IDEG6 analysis, but it showed a trend toward down-regulation (p>0.05) in MDV-infected compared to non-infected samples by qPCR (Figure 2H).

Putative target genes of gga-miR-181a, gga-miR-26a, gga-miR-221, gga-miR-222, gga-miR-155, gga-miR-146b, and gga-miR-146c were predicted by TargetScan (Version 6.0) [31].

G. The expression of gga-miR-155 among TS, LL, and NS.

Only two miRNAs (gga-miR-155 and −222) showed different results from the two methods.

14

MiR-155 also controls antiviral CD8+ T cell responses by regulating interferons, and mir-155 -deficient mice had reduced viral clearance [31].

The wi dely studied miRNA, miR-155, is critical for normal B cell differentiation [29] and plays a major role in immune response and inflammation by regulating members of the tumor-necrosis factor superfamily [30].

A) Gga-mir-155, B) Gga-mir-148a and C) Gga-mir-21 display significantly increased H3K4me3 marks in the resistant line L6 [3] at the latent stage of infection.

In addition, several immune-related miRNAs, e. g. gga-miR-155 and gga-miR-10b, were associated with differential chromatin marks and could contribute to increased MD-susceptibility in line L7 [2] chickens.

Immune-related microRNAs, e. g. gga-miR-155 and gga-miR-10b, bore chromatin signatures, which suggested their contribution to MD-susceptibility.

Apart from Gga-mir-155 [48] none of the above miRNAs have been previously reported in the context of MD and their roles in determination of MD-resistance bear further analysis.

Lower H3K4me3 levels and possible reduction in transcript levels around gga-miR-155 in line L7 [2]could contribute to higher MD-susceptibility.

The varying responses in the two chicken lines particularly around gga-miR-155, gga-miR-10b and gga-miR-137, also suggested the possible contribution of these miRNAs to differential MD-resistance.

All samples exhibited strong H3K4me3 marks around gga-miR-155 at both time-points, suggesting activation, but the chromatin marks were higher in the resistant line L6 [3] particularly at 10 dpi (Figure  2A).

Similar to gga-miR-155, strong H3K4me3 marks were observed at the promoter of gga-miR-21 in all samples, with a slight reduction in line L7 [2] at 10 dpi (Figure  2A).

Close examination of the list of DMR -associated miRNAs revealed several immune-related miRNAs, e. g. gga-miR-155, gga-miR-148a (H3K4me3), gga-miR-10b and gga-miR-137 (H3K27me3).

15

Seven of the nine miRNAs, except for miR-155 and miR-9, had a relatively consistent expression in earlier developmental phases, increased significantly when puberty initiated and hold stably or showed further increment until laying the first egg.

Although the expression levels of miR-155 and miR-9 also increased significantly (P < 0.01) from 12 to 13 weeks, they then dropped significantly (P < 0.05, P < 0.01) and recovered to lower levels as early period.

Furthermore, miRNA-217, miRNA-155, miR-19b and miR-9 have target genes that are associated with puberty onset, such as FSHR, LEPR and circadian clock genes.

miR-155 is found to be a molecular switch that regulates fat metabolism [46].

According to previous reports and our sequencing results, 9 miRNAs, including miR-29c-3p, miR-375, miR-215-5p, miR-9-5p, miR-19b-3p, miR-133a-3p, let-7a, miR-217-5p and miR-155 were determined as candidates.

16

Each pre-miRNA sequence (Table  3) was designed as a single-stranded DNA oligonucleotide, ~64 nt in length and encoding, in order: sequence for 5′ overhang, a 21 nt target antisense siRNA sequence; a mouse miR-155 loop; and the 19 nt sequence deleted nucleotide No 9 and No10 of target sense siRNA sequence, with a corresponding complementary single-stranded DNA oligonucleotides.

Each sequence of target siRNA derived from the gag (p15), pol (p32), env (gp85) and LTR (U3) gene of ALV-J was embedded into mouse miR-155 backbone as a pre-miRNA hairpin oligonucleotide sequence.

The siRNA was embedded into mouse miR-155 backbone to form pre-miRNA hairpin structure, and then constructed into pcDNA6.2-GW/EmGFP-miR vector.

A number of miRNA backbones can be used, including miR-155, miR-30 and miR17-92 [23- 25].

17

Long intergenic ncRNA MEG3 (maternally expressed gene 3) could act as a tumor suppressor [44], while both the miRNA gene hsa-mir-155 and BIC RNA (MIR155HG) from which it is processed, were overexpressed in human B-cell lymphomas [45].

In some cases the designated lincRNAs have been found to be the primary transcripts and not actual lincRNA genes, for example MIR155HG (also known as BIC) and DLEU2 (deleted in lymphocytic leukemia 2 (non-protein coding), previously known as LEU2, are primary transcripts of their resident miRNA genes hsa-mir-155 and hsa-mir-15a/16-1, respectively.

18

Hepatitis C virus -induced up-regulation of microRNA-155 promotes hepatocarcinogenesis by activating Wnt signaling.

In HCV- or HIV-infected cells, the up-regulation of miR-155 and miR-21 represses the NF-κB signaling pathway (Houzet et al., 2008; Marquez et al., 2010; Zhang Y. et al., 2012).

19

Similarly to Kaposi’s sarcoma -associated herpesvirus (KSHV)-encoded miR-K12-11 [21], the most highly expressed miR-M4-5p encoded by GaHV2 has been characterized as a viral analogue of cellular miR-155 and specifically inhibits the translation of viral proteins involved in virus DNA cleavage/packaging [22, 23].

Since miR-155 is a host oncogene associated with several cancers [24, 25], miR-M4-5p has also been hypothesized to play a critical role in GaHV2 oncogenesis.

Earlier studies of the oncogenic GaHV2 have demonstrated that most of the Meq-clustered miRNAs, especially miR-M4-5p, a virus-encoded miR-155 analogue, play critical roles in MD lymphomagenesis [22, 23, 26–30].

20

[50] The downregulated expression of miR-203 from myoblast proliferation to differentiation is similar to that observed for miR-155, miR-125, miR-669a and miR-669q, 2, 51, 52 which all act to repress skeletal muscle differentiation.

21

Zhao S. Zhang J. Hou X. Zan L. Wang N. Tang Z. Li K. OLFML3 expression is decreased during prenatal muscle development and regulated by microRNA-155 in pigs Int.

22

MiR-21, miR-146a/b and miR-155 were obviously up-regulated in rat’s mononuclear cells after Salmonella infection [14, 15].

23

The physiological functions of miR-1744, miR-155, miR-6548, miR-1684a, miR-1665 and miR-6557 are still not known, but their significant expression changes from BO to AO indicate that they are likely to function in chicken rapid gonad development onset.

24

Bolisetty M. T. Dy G. Tam W. Beemon K. L. Reticuloendotheliosis virus strain T induces miR-155, which targets JARID2 and promotes cell survivalJ.

Yao Y. Vasoya D. Kgosana L. Smith L. P. Gao Y. Wang X. Watson M. Nair V. Activation of gga-miR-155 by reticuloendotheliosis virus T strain and its contribution to transformationJ.

25

It has been shown that a viral analog of cellular miR-155 in MDV, which is critical for oncogenicity of MDV, down-regulate TGF-beta signalling [41].

26

Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek's disease lymphomas.

27

Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting.

28

For example, overexpression of miR-155 in mice reduces liver and serum lipid, triglycerides (TG), high density lipoproteins and free fatty acids [27].

29

Reticuloendotheliosis virus strain T induces miR-155, which targets JARID2 and promotes cell survival.

30

However, miR-155 showed very low abundance in both lungs and tracheae and no significant differential expression was observed in the present study.

31

miR-M4, an ortholog of the oncogenic miR-155, was shown to have a direct effect on inducing MDV -induced T cell lymphoma, as viruses deleted in miR-M4 or having mutations in the seed region failed to induce lymphoma [67].

32

00556-12 22647701 8. Zhao Y. Xu H. Yao Y. Smith L. P. Kgosana L. Green J. Petherbridge L. Baigent S. J. Nair V. Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek’s disease lymphomasPLoS Pathog.

33

Zhao Y. Xu H. Yao Y. Smith L. P. Kgosana L. Green J. Petherbridge L. Baigent S. J. Nair V. Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek’s disease lymphomas PLoS Pathog.

34

In addition to miR30 -based designs, mouse miR155 -based design has also been used to knockdown multiple genes [19].

35

[40] It was revealed that miR-146, [41] miR-155, [42] and miR-203 [43] regulate arthritic inflammatory response.

36

miR-155 integrations were also frequently seen with MYB in tumors (S4 Table).

37

Integrations into a number of proto-oncogenes, including MYC, MYB, TERT, mir-155, MET and EGFR, have been seen in past screens [1, 5– 9].