Down-Regulated gga-miR-223 Inhibits Proliferation and Induces Apoptosis of MG-infected DF-1 Cells by Targeting FOXO3

Mycoplasma gallisepticum (MG) is a major poultry pathogen that can induce Chronic Respiratory Disease (CRD) in chickens, causing serious economic losses in poultry industry worldwide. Increasing evidence suggests that microRNAs (miRNAs) act as a vital role in resisting microbial pathogenesis and maintaining cellular mechanism. Our previous miRNAs sequencing data showed that gga-miR-223 expression level signicantly decreased in MG-infected chicken lungs. The aim of this study was to reveal the role of gga-miR-223 in MG-induced CRD progression. We found that gga-miR-223 was remarkably down regulated and forkhead box O3 (FOXO3) was upregulated in both MG-infected chicken embryos lungs and the chicken embryonic broblast cell line (DF-1) by qPCR. FOXO3 was veried as the target gene of gga-miR-223 through bioinformatics analysis and dual-luciferase reporter assay. Further studies showed that overexpressed gga-miR-223 could promote cell proliferation, cell cycle, and inhibit cell apoptosis by notably promoting the expression of cell cycle marker genes cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 6 (CDK6) and Cyclin D1 (CCND1) and inhibiting the expression of apoptosis markers Bcl-2-like 11(BIM), FAS ligand (FASLG) and TNF-related apoptosis-inducing ligand (TRAIL). As expected, FOXO3 knockdown group got similar results. Overexpression of gga-miR-223 observably promoted cell multiplication, cell cycle progression, and inhibited apoptosis of MG-infected DF-1 cells, while inhibited gga-miR-223 had the opposite effect. downregulated


Abstract Background
Mycoplasma gallisepticum (MG) is a major poultry pathogen that can induce Chronic Respiratory Disease (CRD) in chickens, causing serious economic losses in poultry industry worldwide. Increasing evidence suggests that microRNAs (miRNAs) act as a vital role in resisting microbial pathogenesis and maintaining cellular mechanism. Our previous miRNAs sequencing data showed that gga-miR-223 expression level signi cantly decreased in MG-infected chicken lungs. The aim of this study was to reveal the role of gga-miR-223 in MG-induced CRD progression.

Results
We found that gga-miR-223 was remarkably down regulated and forkhead box O3 (FOXO3) was upregulated in both MG-infected chicken embryos lungs and the chicken embryonic broblast cell line (DF-1) by qPCR. FOXO3 was veri ed as the target gene of gga-miR-223 through bioinformatics analysis and dual-luciferase reporter assay. Further studies showed that overexpressed gga-miR-223 could promote cell proliferation, cell cycle, and inhibit cell apoptosis by notably promoting the expression of cell cycle marker genes cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 6 (CDK6) and Cyclin D1 (CCND1) and inhibiting the expression of apoptosis markers Bcl-2-like 11(BIM), FAS ligand (FASLG) and TNF-related apoptosis-inducing ligand (TRAIL). As expected, FOXO3 knockdown group got similar results.
Overexpression of gga-miR-223 observably promoted cell multiplication, cell cycle progression, and inhibited apoptosis of MG-infected DF-1 cells, while inhibited gga-miR-223 had the opposite effect.

Conclusions
Taken together, upon MG-infection, downregulated gga-miR-223 could decrease proliferation, cycle progression, and increase apoptosis through directly targeting FOXO3 to exert an aggravating MGinfectious effect.

Background
Chronic Respiratory Disease (CRD) is one of the most common chronic diseases in the global poultry industry. Mycoplasma is a kind of cell wall-free, self-reproduction, and intracellular parasitics. Mycoplasma is widespread all over the world and can invade a wide range of species such as human, dog, rabbit, chicken and all domestic animals, resulting in multiple diseases [1]. Mycoplasma gallisepticum (MG) is one of the most important pathogens in avian mycoplasma infection and frequently causes CRD in chickens. The host mounts a vigorous in ammatory response to MG, resulting in respiratory distress in chickens. Although vaccination and antibiotics can be used to control the infection, it is impossible to completely clear MG from infected chickens. MG-caused avian chronic respiratory disease has an increased epidemic, leading to tremendous economic losses in poultry industry [2]. MG-HS, isolated from a chicken farm in Hubei Province, was demonstrated to be a virulent strain of MG through a series of physiological and biochemical reactions [3]. Nowadays, increasing attention is paid to Mycoplasma-induced CRD, which remains much to be explored [4][5][6].
Additionally, it has been reported that miRNAs are involved in many major poultry diseases, such as avian in uenza, infection bursal disease, and avian leucosis [12][13][14][15]. Our previous research has revealed that some miRNAs (gga-miR-99a, gga-miR-16-5p, gga-miR-451 and gga-miR-101-3p) signi cantly suppressed the proliferation of MG-infected DF-1 cells by promoted apoptosis [16][17][18][19]. However, it is largely unclear how miRNAs regulate MG infection. MiR-223 is highly conservative among different species and plays an important role in immune cell development, in ammation and other biological processes [11,20]. MiR-223 could inhibit nicotinamide phosphoribosyl transferase (NAMPT) to regulate the proliferation and senescence of endothelial progenitor cells (EPCs) [21]; miR-223 inhibited cell proliferation and enhanced cell apoptosis in acute myeloid leukemia (AML) cell lines [22]. Numerous studies have revealed that miR-223 functions as a regulatory factor to assist the proliferation and invasion of diverse cancers, such as liver cancer [23], lung cancer [24] and breast cancer [25]. Even though current studies have shown that miR-223 plays an important role in immunity and various pathological responses [26], it still remains unclear how miR-223 is involved in the regulation of MG infection.
We found that gga-miR-223 was signi cantly down-regulated in MG-infected chicken embryo lungs by Solexa deep sequencing [27]. In the present study, we further validated that gga-miR-223 was remarkably down-regulated in MG-infected chicken embryo lungs and MG-infected DF-1 cells. FOXO3 was demonstrated as the direct target gene of gga-miR-223. Down-regulated of gga-miR-223 signi cantly inhibited the MG-infected DF-1 cells proliferation and promoted apoptosis by regulating CDK1, CDK6 CCND1 BIM, FASLG and TRAIL. This study suggested that down-regulated 223 inhibits cell proliferation and promotes apoptosis by targeting FOXO3 to facilitate MG infection.

Reagents and cell culture
The chicken DF-1 cells were purchased from Huiying (Shanghai, China). All cells were cultured in Dulbecco's modi ed Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Shanghai, China) in a carbon dioxide cell incubator with 5% CO 2 at 37 ℃. study report [28]. A color-changing unit (CCU) assay was applied to determine MG-HS viable number in suspension.

Infection Experiments
To study the MG infection mechanism, we established an experimental group and a blank group and started infection experiment. Meanwhile, DF-1 cells were cultured in six-well plates without any antibiotics. When cells covered 50-60% con uence, the experimental group was infected with MG-HS (1×10 10  with SYBR-green (Tiangen, Beijing, China). Each sample was repeated least three times.

DNA Primers and RNA Oligonucleotides
In this experiment, total sequences of DNA primers that was adopted are presented in Table 1. In addition, All RNA oligonucleotides designed and synthesized by GenePharm (Shanghai, China). Bioinformatic analysis, TargetScan (http://www.targetscan.org/) and the other is miRDB (http://www.mirdb.org/miRDB/), were used to nd which gene was the most potential target. The gene prediction scores and conservation degree as well as experimental goals were taken into account.
Besides, it meant quali ed that the prediction score was 60 and perfect that the prediction score was 100. RNA secondary structure between gga-miR-223 and the 3'-UTR of its target gene was analyzed by RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/).

Construction of 3'-UTR-luciferase Plasmid and Dual-luciferase Reporter Assay
For dual-luciferase reporter assay, we had designed wide-type (WT) and mutant 3'-UTR primers of FOXO3 and obtained corresponding DNA fragment which covered the predicted gga-miR-223 binding site.
DF-1 cells were cultured in 24-well plates at 2 × 10 5 cells or so per well for the luciferase assay. When cells reached 80-90% con uence, using Lipofectamine 3000 (Invitrogen Life Technologies, USA) each cotransfected cells with wildtype or mutant reporter plasmid (200 ng) and 10 pmol of the indicated RNA oligonucleotides, including gga-miR-223 and gga-miR-223-NC. DF-1 Cells were harvested after transfection for 48 hours. Next, the luciferase activity in each group was detected by using automatic microplate reader (Bio-Rad, Hercules, CA, USA) in accordance with the dual luciferase reporter gene detection kit instructions (Promega, Madison, WI, USA) according to the manufacturer's protocol.
Overexpression and Inhibition of gga-miR-223 To explore the effects of overexpressed and inhibitory gga-miR-223 on FOXO3, we conducted a series of experiments to detect FOXO3 mRNA relative expression under different circumstances where gga-miR-223 was overexpressed or inhibited. DF-1 cells were cultured in 6-well plates. Once reached 80-90% con uence, each group cells would be transfected separately with gga-miR-223, gga-miR-223-NC, gga-miR-223-Inh and gga-miR-223-Inh-NC. 48h after transfection, TRNzol was used to harvest total cells. Subsequently, we had extracted total RNA and performed reverse transcription experiment. Next, the expression levels of gga-miR-223 and FOXO3 in each group were detected by qPCR. Meanwhile, 5S-RNA and GAPDH served as an international control.

Cell Proliferation and Apoptosis Assays
The Cell Counting Kit-8(CCK-8) Kit was used for growth curve experiments. Cell suspension was prepared from logarithmic growth phase cells and the DF-1 cells were then diluted and inoculated on a 96-well plate at a density of 2x10 4 cells per well. At 39 °C with 5% CO 2 DF-1 cells were cultured to stick to the wall. Next, MG-HS was utilized to infect DF-1 cells for 2 hours. Each group cells were separately transfected gga-miR-223 mimics, NC, inhibitor and inhibitor NC using Lipofectamine TM 3000 and each group had 6 biological replicates. Meanwhile, a blank group without any RNA oligonucleotides was also set up. At 24h, 48h, and 72 h post-transfection, 10 ul of CCK-8 solution were added to each well of the cells to be tested and all cells continued to incubate in a cell incubator for 1-4 hours. Subsequently, absorbance at a 450 nm wavelength was measured on a microplate reader (Bio-Rad, USA).
Made from logarithmic growth phase DF-1 cells, Cell suspension was infected MG-HS for 2 hours and then separately transfected RNA oligonucleotides according to the instruction of Lipofectamine TM 3000. At 48h post-transfection, cold-ice phosphate-buffered saline (PBS) was utilized to wash cells once, harvested cells with the centrifuge (2000 r/min, 5 min) and resuspended in binding buffer containing 5μl Annexin V-FITC at a density of 1 -5x105 cells/mL. DF-1 Cells were then incubated with 5 μl propidium lodide (PI) for 15 min in the dark. Finally, the apoptosis of DF-1 cell was analyzed by uorescenceactivated cell sorting (BD Biosciences). Each group was repeated three times.

Statistical Analysis
In this study, all data were presented as the mean values ± SDs of at least three independent experiments. The one-way ANOVA with the Duncan's multiple-range test and Student's t-test were to evaluate statistically signi cant differences. P < 0.05 was considered signi cant. Signi cant differences were performed as *p < 0.05 and **p < 0.01.

Results
Gga-miR-223 is Remarkably Suppressed in MG-Infected DF-1 Cellsand chicken embryonic lung tissue In the previous study, we found that gga-miR-223 was signi cantly down-regulated in chicken embryonic lungs with MG infection by miRNAs deep sequencing [29]. The expression level of miR-223 in chicken embryo lung tissues and cells were further veri ed by qPCR. The results showed that compared with normal lung tissue, gga-miR-223 was down-regulated (p<0.05) after 4 and 6 days of infection, and on the 8th day of infection, the expression level of gga-miR-223 was highly down-regulated (p<0.01) ( Figure. 1A).
Meanwhile, gga-miR-223 was remarkably suppressed in DF-1 cells with MG infection compared with control group (Figure. 1B), which was consistent with the sequencing results. Therefore, these results imply that gga-miR-223 may be involved in MG infection. (B) DF-1 cells were cultured in 6-wells plate and treated MG. After 24h infection, the total RNA was isolated from infected cells via TRNzol Universal and the gga-miR-223 expression was detected by RT-qPCR. 5s-RNA was used as the internal quantitative control gene. All measurements shown were the means ± SD from three independent experiments, each with three replicates. ( *, P < 0.05 ;**, P < 0.01) miR-223 is able to bind to the 3′-UTR of FOXO3 mRNA It is well known that miRNAs exert a role in decreasing and inhibiting transcriptional gene level by binding target miRNAs, the expression of their made a difference by degrading corresponding target genes.
TargetScan (http://www.targetscan.org) and miRDB (http://www.mirdb.org/miRDB/index.html) were utilized to predict the potential target genes of gga-miR-223. FOXO3 was chosen the best appropriate target gene within 107 potential targets with the help of Gene Scoring system, and the highly conserved target sequence from position 42 to 49bp in different species. ( Figure.  To further verify whether gga-miR-223 could combine with FOXO3 3'-UTR, we performed the dualluciferase reporter assay by transfecting the luciferase reporter vectors containing either the wild-type or the mutant 3cifer of FOXO3 into DF-1 cells. The transfection of gga-miR-223 mimics signi cantly decreased the luciferase activity of the reporter containing wild-type 3'-UTR compared with the control, but there was no signi cant effect of the reporter containing mutant 3'UTR ( Figure 2C, D). These results showed that gga-miR-223 inhibited FOXO3 expression by directly binding to its complementary sequence in the 3'-UTR of FOXO3. FOXO3 expression is negatively regulated by gga-miR-223 In order to further explore the function of gga-miR-223, we examined whether gga-miR-223 could regulate the expression of FOXO3 in DF-1 cells. The DF-1 cells transfected with gga-miR-223 mimics were marked as miR-223; cells transfected with a non-speci c RNA (marked as miR-223-NC); cells transfected with gga-miR-223 inhibitor (marked as miR-223-Inh). We found that, whether at the mRNA level or at the protein level, the over-expression of gga-miR-223 signi cantly suppressed the expression of FOXO3 in DF-1 cells, whereas miR-223-Inh had the opposite function ( Figure 3A, 3B). These results indicate that FOXO3 is a direct target of gga-miR-223 in DF-1 cells and its expression is negatively regulated by gga-miR-223.  compared with miR-223-NC (MG+) group, miR-free (MG +) group and miR-223 inhibitor (MG+), the number of cells in miR-223-mimics (MG+) group was signi cantly increased (p<0.05) ( Figure 5); but the number of cells in miR-223-inhibitor (MG+) group was markedly lower than that in other groups (p < 0.01); after 72 hours, although the number of cells in the miR-223-mimics (MG+) group was not different from that in the blank (MG-) group, it was signally higher than the other groups (p<0.05) ( Figure 5). Date indicates that the presence of gga-miR-223 can resist the cell reduction caused by MG infection, thereby promoting the proliferation of DF-1 cells infected with MG.   The cell apoptosis of DF-1 cells was promoted by inhibition of gga-miR-223. Bars that do not share the same letters are signi cantly different (p < 0.05) from each other. All data from the triplicate experiments carried out independently were adopted as mean value ± SD.
Gga-miR-223 promotes cycle gene expression and inhibits apoptosis gene expression We have known that gga-miR-223 could regulate the cycle of MG-infected cells, promote their proliferation and inhibit their apoptosis. In order to further explore the details of the role of gga-miR-223, the expressions of genes which related to cell cycle and apoptosis were measured after transfection of synthetic RNA oligodeoxynucleotides in DF-1 cells. RT-qPCR detection showed that the relative expression of CDK1, CDK6 and CCND1 in miR-223-mimics group was dramatically higher than that in miR-223inhibitor group and miR-223-NC group, while the relative expression of CDK1, CDK6 and CCND1 in miR-223-inhibitor group was signi cantly lower than that in miR-223-NC group (p < 0.05) ( Figure 8A). These results indicate that the low expression of gga-mir-223 inhibits the cell cycle progression by inhibiting the expression of CDK1, CDK6 and CCND1.
The RT-qPCR detection of apoptotic genes showed that the relative expression of BIM, FASLG and TRAIL in miR-223-mimics group was notably decreasing than that in miR-223-inhibitor group and miR-223-NC group (p < 0.05), while the relative expression of FASLG, BIM and TRAIL in miR-223-inhibitor group was prominently increasing than that in miR-223-NC group (p < 0.05) ( Figure 8B). The data proved that lowexpressed gga-miR-223 promoted apoptosis by promoting the expression of BIM, FASLG and TRAIL. Knockdown of FOXO3 promoted cell cycle genes expression and inhibited apoptosis genes expression We have known that gga-miR-223 could promote cell proliferation by promoting the expression of cycle genes and inhibiting the expression of apoptotic genes in DF-1 cells. FOXO3, the direct target gene of gga-miR-223, its knockdown was used to further investigate the role of gga-miR-223 in DF-1 cells. The si-FOXO3 and siRNA-NC were transfected into DF-1 cells, and the relative expression of FOXO3 mRNA and protein were detected by RT-qPCR and Western-blotting. The results of qPCR and Western-blotting were consistent, that is, the expression of FOXO3 in FOXO3 knockdown group was much lower than that in siRNA-NC group (P < 0.05) ( Figure 9A, 9B). It showed that the FOXO3 gene was successfully knocked down in DF-1 cell.
Based on the knockdown of FOXO3 gene, we measured the expression of cycle marker genes and apoptosis marker genes in DF-1 cells. As expected, the relative expressions of CDK1, CDK6 and CCND1 in si-FOXO3 group were observably higher than that in siRNA-NC group (p<0.05), while the relative expression of FASLG and TRAIL were markedly lower than that in siRNA-NC group (p<0.01). And compared with siRNA-NC group, the relative expression of BIM in the si-FOXO3 group was extremely signi cantly reduced (p<0.01) ( Figure 9C). Therefore, these results indicate that the up-regulated gga-miR-223 negatively regulated the expression of FOXO3, thereby inhibiting the expression of cycle genes and promoting the expression of apoptotic genes, to achieve the effect of affecting cell proliferation, cycle and apoptosis.

Discussion
The CRD induced by MG, a worldwide avian pathogen, has caused huge economic loss, making it a hot topic in poultry research [31,32]. MG can activate in ammatory response in the respiratory tract of chickens [33]. Notably, it is extremely contagious and easy to induce prevalence of other diseases such as in uenza A virus [34]. Therefore, how to solve the problem of MG infection is particularly important. Previously, a large number of researches indicated that pathogenic microorganisms could induce the differential expression of some host miRNAs, regulating the expression of numerous target genes by binding to them and affecting the corresponding cellular behaviors and metabolism processes [35,36].
Hence, it will be crucial to increase our understanding of MG infection mechanisms by studying the potential role of miRNAs involved in MG infection.
Based on the previous studies, we found that MG could signi cantly regulate the expression of multiple miRNAs in the lung tissue of chicken embryo [27], suggesting that these differentially expressed miRNA might be effective markers for the diagnosis and prognosis and potential therapeutic interventions of MG-induced CRD. Previous high-throughput sequencing data showed that gga-miR-223 was signally down-regulated in MG-infected chicken embryo lung [37]. Importantly, accumulating data has suggested that miR-223 plays an important role in various diseases, especially respiratory disease [20,38,39]. MiR-223 plays a key role in regulating the immune in ammatory response to neutrophil asthma by inhibiting the NLRP3/ IL-1β [40]; pharmacological blockade of the miR-223-NLRP3-IL-1β signaling axis alleviates acute respiratory distress syndrome [41]. In addition, the expression level of miR-223 has dramatically altered by other bacterial infections, For instance, gastric in ammation caused by Helicobacter pylori infection through induction of miR-223-3P expression in a Helicobacter pylori CagA-dependent manner [42]; the expression of miR-223 in the breast tissue of dairy cows with Streptococcus agalactis mastitis increased three-fold, which may be involved in the signal pathway of Streptococcus agalactis-induced mastitis [40]. It indicates that miR-223 is strongly related to microbial infection. Consistent with these results, we discovered that gga-miR-223 was notably down-regulated in chicken embryonic lung tissue and DF-1 cells infected with MG ( Figure 1), which indicated that gga-miR-223 was likely to be involved in the regulation of MG infection mechanism.
It is documented that miRNAs usually exert their regulatory effect the negative regulation of target genes at post-transcriptional level. There is innumerable evidence that miR-223 regulates cell physiological process by targeting FOXO3 [43,44]. Here, we have also identi ed FOXO3 as a target gene of gga-miR-223 by bioinformatic analysis and dual-luciferase assay ( Figure 2). FOXO3 is a member of forkhead O (FOXO) transcription factors family, which has crucial roles in cell proliferation, cell cycle and apoptosis [45].
Many studies have shown that the up-regulated expression of FOXO3 in cells is associated with many diseases. FOXO3 is up-regulated in the cytoplasm of in ammatory colonic epithelial cells, leading to intestinal in ammation [46]; and up-regulated FOXO3 plays a key role in the migration of esophageal cancer cells [47]. Coincidentally, here, the expression of FOXO3 in MG-infected lung tissues and DF-1 cells also increased signi cantly (Figure 4), which is contrary to gga-miR-223, indicating that DF-1 cells infected with MG promote the expression of FOXO3 by downregulating gga-miR-223, thus presenting cellular physiological and biochemical changes.
In the process of MG infection, exogenous DNA and components from mycoplasma cause DNA damage to host cells and affect cell function, leading to apoptosis and inhibiting cell proliferation and cell cycle [48,49]. MiR-223 has an effect on the proliferation and apoptosis of colorectal cancer cells by regulating FOXO3/BIM [50]. Some studies have shown that FOXO3 inhibits the proliferation of chicken liver cancer cell line (LMH) and induces its apoptosis [51]. It also has demonstrated that miR-223 directly suppresses FOXO3, and FOXO3 plays a critical role as a mediator of the biological effects of miR-223 in macrophage apoptosis [52]. Our results suggested that the presence of gga-miR-223 could resist the cell reduction caused by MG infection ( Figure 5); overexpression of gga-miR-223 could effectively promote cell proliferation ( Figure 6); and inhibit apoptosis rate of MG-infected cells with gga-miR-223 mimics was remarkably decreased (Figure 7).
CDKs are the core of cell cycle regulation, and Cyclins have a positive regulatory effect on CDKs ; BIM, FASLG and TRAIL are classic downstream genes of apoptosis [53], which suggest that abnormal expression of them contributes cell cycle arrest or apoptosis. In liver cancer cell, the translocated FOXO3 can directly promote the transcription of cyclin-dependent kinase inhibitors (CDKIs) p21Cip1 and p27Kip1, and on other hand can inhibit the expression of Skp2, leading to cell arrest in the GO/G1-phase [51]. FOXO3 can induce apoptosis by promoting TRAIL and inhibiting Bcl-2 expression. What's more, the phosphorylation of FOXO3 can be inhibited by casein, which reduces the expression of FOXM1 and its downstream gene CDK1, remarkably inhibiting the cell viability and cell growth. FOXO3 induced by mannitol can observably reduce the expression of CCND1 and CDK6, arresting cells in G1 phase, and inhibiting breast cancer cell cycle and activity. We found that, in this manuscript, cycle genes (CDK1, CDK6, CCND1) were inhibited and apoptosis genes (BIM, FASLG, TRAIL) were promoted in cells which treated with gga-miR-223 mimics or si-FOXO3 (Figure 8, 9). In general, it is now becoming clear that MG can regulate gga-miR-223 expression to affect the expression of FOXO3, and the in uence cell proliferation and apoptosis. Therefore, we have su cient reasons to speculate that MG regulates FOXO3 gene by changing the expression of gga-miR-223 to achieve regulation of cell proliferation and apoptosis.

Conclusions
In conclusion, when responding to MG infection, down-regulation of gga-miR-223 can increase the mRNA expression of cycle genes or decrease the mRNA expression of apoptosis genes, inhibit cell proliferation and promote cell apoptosis by targeting FOXO3. Therefore, the regulation of gga-miR-223 may provide a new therapeutic target for the CRD induced by MG in poultry.
More importantly, because of the high conservation of miR-223 and FOXO3 among different species ( Figure.  Availability of data and materials All data and analysis in the manuscript, if necessary, can be obtained from the corresponding author on reasonable request.

Ethics approval and consent to participate
These studies were approved by Huazhong Agriculture University Animal Research Ethics Committee (Wuhan, Hubei, China).

Consent for publication
Not applicable.