gga-miR-146c Activates TLR6/MyD88/NF-κB Pathway through Targeting MMP16 to Prevent Mycoplasma Gallisepticum (HS Strain) Infection in Chickens

Mycoplasma gallisepticum (MG), a pathogen that infects chickens and some other birds, triggers chronic respiratory disease (CRD) in chickens, which is characterized by inflammation. The investigation of microbial pathogenesis would contribute to the deep understanding of infection control. Since microribonucleic acids (miRNAs) play a key role in this process, gga-mir-146c, an upregulated miRNA upon MG infection, was selected according to our previous RNA-sequencing data. In this paper, we predicted and validated that MMP16 is one of gga-miR-146c target genes. Results show that MMP16 is the target of gga-miR-146c and gga-miR-146c can downregulate MMP16 expression within limits. gga-miR-146c upregulation significantly increased the expression of TLR6, NF-κB p65, MyD88, and TNF-α, whereas the gga-miR-146c inhibitor led to an opposite result. gga-miR-146c upregulation effectively decreased apoptosis and stimulated DF-1 cells proliferation upon MG infection. On the contrary, gga-miR-146c inhibitor promoted apoptosis and repressed the proliferation. Collectively, our results suggest that gga-miR-146c upregulation upon MG infection represses MMP16 expression, activating TLR6/MyD88/NF-κB pathway, promoting cell proliferation by inhibiting cell apoptosis, and, finally, enhancing cell cycle progression to defend against host MG infection.


Introduction
Mycoplasma, a prokaryotic parasite, has negative impacts on human health and livestock farming [1][2][3]. Of those, Mycoplasma gallisepticum (MG) could cause severe chronic respiratory disease (CRD) in chickens and sinusitis in turkeys, represented by severely inflamed tracheas, lungs, and air sacs [4,5]. Controlling the impact of the disease on a global level is done by vaccination and medication. Unfortunately, they cannot eliminate all pathogens of infected chickens, and some vaccines may revert to virulence [6][7][8]. MG infection results in considerable losses consisting of reducied weight gain and egg production, as well as increased embryo morbidity [9].
Microribonucleic acids (miRNAs) act as critical regulators of gene expression by binding to the 3 -untranslated regions (3 -UTRs) with sequence complementarity and decreasing the stability or translation efficiency of target mRNAs [10]. Increasing evidence suggests that miRNAs can extensively regulate various physiological and pathological processes and serve as important regulators of the defense and inflammatory responses to the host, including pathogenesis of many avian diseases [11,12]. Recent researches have indicated this. For instance, miR-181 and miR-29c might act as a Marek's disease tumor suppressor by targeting MYBL1 and inhibitor of accelerated avian influenza virus replication,

Dual-Luciferase Reporter Assay
In order to construct the reporter plasmid, MMP16 3 -UTR covering the seed sequence binding site was amplified by RT-PCR. The cDNA template was extracted from chicken embryo lung tissues, extracting the normal luciferase reporter plasmid, then mutating three core sequences through PCR. The amplified products included the sites of the enzyme cut Xho I/Not I. The primer sequences were showed in Table S1.
DF-1 cells were seeded on 24-well plates, and 2 × 10 5 cells per well were used for the luciferase assay. Next, 10 pmol miR-146c, miR-146c-NC, and 200 ng reporter plasmid were transfected into cells together by Lipofectamine 3000 (Invitrogen). The cells were gathered to measure dual-luciferase activity 48 h transfection later by using Lumat LB 9507 Ultra Sensitive Tube Luminometer (Titertek Berthold, Nanjing, China). The luciferase activity of each sample was normalized by the firefly luciferase activity and the Renilla luciferase activity. All experiments were carried out three independent repeats.

RNA Extraction and RT-qPCR
The total RNA was extracted with Trizol Reagent (Invitrogen). The purification of RNA was carried out using RNeasy microcolumns on the basis of the manufacturer's protocol (Qiagen, Valencia, CA, USA). TransStart Top Green qPCR SuperMix (TRANSGEN, Beijing, China) was used to mix with total RNA, then each RNA sample was tested by RT-qPCR on CFX96 or CFX384 TouchTM (Bio-Rad, Hercules, CA, USA). The relative expression of gga-miR-146c, TLR6, MMP16, NF-κB p65, MyD88, and TNF-α were calculated by Ct (2 −∆∆Ct ) method. To analyze the data, 7500 software v.2.0.1 (Applied Biosystems, Foster City, CA, USA) was used. The internal control of gga-miR-146c was 5S-RNA, and the internal control of TLR6, MMP16, NF-κB p65, TNF-α, and MyD88 was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Table S1 showed the primers. The experiment was carried out three independent times. There were 6 × 10 3 DF-1 cells and 100 µL DMEM containing 10% (v/v) FBS in each well on six-well plates. Then plates were putted into an incubator overnight (39 • C, 5% CO 2 ). When the cells grew to the right density, 7.5 pmol of miR-146c, miR-146c-NC, miR-146c-Inh, or miR-146c-Inh-NC and Lipofectamine 3000 were added to the six-well plates. In addition, the blank control was a mock transfection (marked as blank). The cells were collected after transfection of 48 h. Total RNA was extracted and purified, TransStart Top Green qPCR SuperMix (TRANSGEN) was used to mix with total RNA, then each RNA sample was tested by RT-qPCR on the CFX96 or CFX384 TouchTM (Bio-Rad). There were three independent transfections. We used the 2 −∆∆Ct method to calculate the relative mRNA expression of TLR6, NF-κB p65, MyD88, and TNF-α [35].

ELISA
There were 6 × 10 3 DF-1 cells and 100 µL DMEM in each well on six-well plates. Then, plates were placed into an incubator overnight (39 • C, 5% CO 2 ). Before transfection, phosphate-buffered saline (PBS) was used to wash DF-1 cells twice, and Opti-MEMI ReduMced Serum was added to the plates. When the cells grew to the right density, 7.5 pmol of miR-146c, miR-146c-NC, miR-146c-Inh, or miR-146c-Inh-NC and Lipofectamine 3000 were added to the six-well plates. In addition, the blank control was a mock transfection (marked as the blank group). The cells were gathered after 48 h transfection. Then, cell culture supernates were collected in the six-well plates and centrifugation at 3000× g, 4 • C, for 10 min. The MyD88, TLR6, and TNF-α were tested by ELISA kit instruction (YUANYE, Shanghai, China).

Western-Blot Analysis
When cells plated in 24-well plates grew to the right density, indicated RNA oligonucleotides were transfected into cells. After transfection of 48 h, RIPA-buffer (Beyotime, Beijing, China) with 100 mM phenylmethanesulfonyl fluoride (PMSF) was used to isolate the total proteins from the DF-1 cells. Bicinchoninic acid (BCA) protein assay reagent kit (Beyotime) was used to test the Protein concentrations. Then, total protein was separated with 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein was transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime) for 2 h at 80 mA. Five percent (w/v) fat-free milk was used to block the membranes at normal atmospheric temperature for 1 h. Next, anti-β-actin served as a loading control, and the membrane was incubated with goat polyclonal anti-MMP16 (Sigma-Aldrich, St. Louis, MO, USA) and rabbit anti-β-actin overnight at 4 • C. The membrane was incubated with rabbit anti-goat secondary antibody for 1 h after TBST washing. The enhanced chemiluminescence (ECL) detection system (Bio-Rad) was used to detect antigen-antibody complexes on the membranes. All the tests were triplicated.

Cell Proliferation, Cycle, and Apoptosis
DF-1 cells were seeded in six-well plates at 6 × 10 3 cell/well 100 µL of DMEM containing 10% (v/v) FBS, then they were placed into an incubator overnight at 39 • C with 5% CO2. Then, the miR-146c, miR-146c-NC, miR-146c-Inh, or miR-146c-Inh-NC was transfected into the DF-1 cells as described above. When the cells grew to the right density, cells were infected by MG-HS (7 µL, 10 10 CCU/mL). Ten microliter CCK-8 solution was each added 24 h, 48 h, and 72 h after transfection, and incubated at 39 • C for 4 h. Uninfected MG cells (blank MG-) and the infected MG cells (miR-free MG+) were regarded as controls. The Cell Counting Kit-8 (CCK-8, DOJINDO, Shanghai, China) was adopted to test cell proliferation. The optical density of each well plate at 450 nm was measured by microplate reader (Bio-Rad).
Transfection treatments were described above. Cells were infected by MG-HS (7 µL, 10 10 CCU/mL) after transfection for 4 h. After transfection for 48 h, 70% ethanol was used to immobilize cells. MiR-free-MG+ and blank MGserved as controls. Cell cycle detection kit (KeyGEN, Nanjing, China) was used to analyze cell cycle and flow cytometer calculated percentages of cells in G1, S, and G2 phases. Annexin V, FITC apoptosis detection kit (DOJINDO) was used to test the cell apoptosis. Percentages of cells in four quadrants were calculated. All the tests were triplicated.

Statistical Analysis
All experiments were carried out three times. Data were mean ± SD and performed using Student's t-test. * p < 0.05, ** p < 0.01 mean significant differences.

gga-miR-146c Expression Was Upregulated after MG Infection
The qRT-PCR results showed gga-miR-146c expression was significantly upregulated in MG-infected DF-1 cells comparing with noninfected ones ( Figure 1A). Subsequently, we further identified gga-miR-146c expression in chicken embryos upon MG-HS infection on the ninth day of hatching (total 21 days of egg hatching). Eight to eleven days after infection (amount to 17 to 20 days of egg hatching), gga-miR-146c expression was remarkably upregulated in MG-infected embryonic chicken lungs comparing with its expression in the noninfected lungs of chicken embryos ( Figure 1B).
tistical Analysis experiments were carried out three times. Data were mean ± SD and performe 's t-test. *P ＜ 0.05, **P ＜ 0.01 mean significant differences. ts miR-146c Expression Was Upregulated after MG Infection e qRT-PCR results showed gga-miR-146c expression was significantly upregul cted DF-1 cells comparing with noninfected ones ( Figure 1A). Subsequently, we d gga-miR-146c expression in chicken embryos upon MG-HS infection on the ninth (total 21 days of egg hatching). Eight to eleven days after infection (amount to 17 to 20 ching), gga-miR-146c expression was remarkably upregulated in MG-infected em lungs comparing with its expression in the noninfected lungs of chicken embryos (Fig . RT-qPCR was performed to ass -miR-146c expression that normalized to 5S-rRNA. Uninfected groups served as nega trols. Values are mean ± SD, *P < 0.05, **P < 0.01.

miR-146c Contains Conserved Target Site of MMP16
th a combined use of online software (miRDB, miRbase, and TargetScan) to -146c, MMP16 was selected as a possible target of gga-miR-146c due to a high pr core 94). Target site sequence in the MMP-16 3′-UTR was conserved across human, etc. (Figure 2A). The predicted target site is located at 1371-1393 bps, and the see 1386-1392 bps ( Figure 2B). The minimum free energy (mFE) between MMP16 3′-U . RT-qPCR was performed to assess gga-miR-146c expression that normalized to 5S-rRNA. Uninfected groups served as negative controls.

gga-miR-146c Contains Conserved Target Site of MMP16
With a combined use of online software (miRDB, miRbase, and TargetScan) to analysis gga-miR-146c, MMP16 was selected as a possible target of gga-miR-146c due to a high prediction score (score 94). Target site sequence in the MMP-16 3 -UTR was conserved across human, mouse, rat, pig, etc. (Figure 2A). The predicted target site is located at 1371-1393 bps, and the seed site is between 1386-1392 bps ( Figure 2B). The minimum free energy (mFE) between MMP16 3 -UTR and gga-miR-146c is about −24.1 kCal/mol, which indicates high stability ( Figure 2C). contained three mutations in the seed region into DF-1 cells. As expected, no remarkable effect was observed when applied with miR-146c mimics ( Figure 2D). gga-miR-146c The highlighted sequence is conserved region; (B) Alignments of gga-miR-146c and the target site in MMP16 3′-UTR. The highlighted part is gga-miR146c seed sequence; (C) Structure of gga-miR-146c and MMP16 3′-UTR target site. Red strand represents target sequence and green represents gga-miR146c; (D) Luc-MMP16 (3′-UTR) and either gga-miR-146c or gga-miR-146c-NC were co-transfected into cells. The dual-luciferase glow assay was performed 24 h transfection later. Data from three experiment results were mean ± SD. Two-tailed Student's t-test, **P < 0.01.
The highlighted sequence is conserved region; (B) Alignments of gga-miR-146c and the target site in MMP16 3 -UTR. The highlighted part is gga-miR146c seed sequence; (C) Structure of gga-miR-146c and MMP16 3 -UTR target site. Red strand represents target sequence and green represents gga-miR146c; (D) Luc-MMP16 (3 -UTR) and either gga-miR-146c or gga-miR-146c-NC were co-transfected into cells. The dual-luciferase glow assay was performed 24 h transfection later. Data from three experiment results were mean ± SD. Two-tailed Student's t-test, ** p < 0.01.

MMP16 Is a Direct Target Gene of gga-miR-146c
To validate the direct binding of gga-miR-146c to 3 -UTR of MMP16 gene, a luciferase reporter gene analysis (psi-CHECK™-2) was performed. The luciferase reporter vector Luc-MMP16 containing a potential seed sequence of MMP16 3 -UTR was constructed and co-transfected with gga-miR-146c mimics (miR-146c) for 48 h. MMP16 3 -UTR luciferase activity was effectively decreased with the application of gga-miR-146c mimics, while no effect on luciferase activity was observed in negative control (nontargeting) (miR-146c-NC) ( Figure 2D). To determine that interaction between miR-146c with the 3 -UTR of MMP16 caused the reduction of luciferase activity, at the same time, we co-transfected miR-146c mimics and a mutant dual luciferase reporter that contained three mutations in the seed region into DF-1 cells. As expected, no remarkable effect was observed when applied with miR-146c mimics ( Figure 2D).

MG Infection Downregulates MMP16 Expression
To further identify the role of MMP16 in MG-HS infection, quantitative polymerase chain reaction (qRT-PCR) was adopted to measure MMP16 expression. It was significant that MMP16 expression level was lower in the infection group ( Figure 4A). Eight to eleven days after infection (amount 17 to 20 days of egg hatching), MMP16 expression was significantly decreased in MG-infected lungs of chicken embryos ( Figure 4B). These results further demonstrate that MMP16 expression is repressed within limits by gga-miR-146c in vivo and in vitro. (B and C) DF-1 cells were transfected with gga-miR-146c mimics. MMP16 messenger RNA (mRNA) and protein expression was respectively measured by reverse transcription polymerase chain reaction (RT-qPCR) or Western blot, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) orβ-actin; (D) gga-miR-146c expression level was reduced with inhibitor transfection; (E,F) MMP16 mRNA and protein expression with gga-miR-146c inhibitor transfection was detected by RT-qPCR or Western blot respectively, normalized to GAPDH or β-actin; Values are mean ± SD, * p < 0.05, ** p < 0.01.

MG Infection Downregulates MMP16 Expression
To further identify the role of MMP16 in MG-HS infection, quantitative polymerase chain reaction (qRT-PCR) was adopted to measure MMP16 expression. It was significant that MMP16 expression level was lower in the infection group ( Figure 4A). Eight to eleven days after infection (amount 17 to 20 days of egg hatching), MMP16 expression was significantly decreased in MG-infected lungs of chicken embryos ( Figure 4B). These results further demonstrate that MMP16 expression is repressed within limits by gga-miR-146c in vivo and in vitro.

gga-miR-146c Is of Importance in NF-κB Pathway Regulation
Our previous study suggests that IL2/IL6-mediated inflammatory responses are induced with MG infection via TLR6/MyD88/NF-κB pathway [36]. To identify whether gga-miR-146c activates the TLR6/MyD88/NF-κB pathway to defend against MG infection, gga-miR-146c, miR-146c-NC, and miR-146c-Inh or miR-146c-Inh-NC were transfected into DF-1 cells for 48 h. The qRT-PCR results revealed that it is the gga-miR-146c expression, but not miR-146c-NC, significantly increased TLR6, NF-κB p65, MyD88, and TNF-α expression levels ( Figure 5A), whereas miR-146c inhibitor significantly decreased the expression of these four genes ( Figure 5C). The results of immunofluorescence showed NF-κB p65 protein was in the nucleus of DF-1 cells which transfected with gga-miR-146c, whereas the other groups had no clear change ( Figure 5E). The proteins of TLR6, MyD88, and TNF-α in the cell supernatant treated with gga-miR-146c were higher than the cells treated with miR-146c-NC ( Figure 5B), whereas the miR-146c inhibitor significantly decreased the proteins expression of TLR6, MyD88, and TNF-α ( Figure 5D), and qRT-PCR results have the same trend with the ELISA results. These data indicate that gga-miR-146c inhibit MMP16 expression within limits, and then activate the NF-κB signaling pathway in inflammation response.

gga-miR-146c Is of Importance in NF-κB Pathway Regulation
Our previous study suggests that IL2/IL6-mediated inflammatory responses are induced with MG infection via TLR6/MyD88/NF-κB pathway [36]. To identify whether gga-miR-146c activates the TLR6/MyD88/NF-κB pathway to defend against MG infection, gga-miR-146c, miR-146c-NC, and miR-146c-Inh or miR-146c-Inh-NC were transfected into DF-1 cells for 48 h. The qRT-PCR results revealed that it is the gga-miR-146c expression, but not miR-146c-NC, significantly increased TLR6, NF-κB p65, MyD88, and TNF-α expression levels ( Figure 5A), whereas miR-146c inhibitor significantly decreased the expression of these four genes ( Figure 5C). The results of immunofluorescence showed NF-κB p65 protein was in the nucleus of DF-1 cells which transfected with gga-miR-146c, whereas the other groups had no clear change ( Figure 5E). The proteins of TLR6, MyD88, and TNF-α in the cell supernatant treated with gga-miR-146c were higher than the cells treated with miR-146c-NC ( Figure 5B), whereas the miR-146c inhibitor significantly decreased the proteins expression of TLR6, MyD88, and TNF-α ( Figure 5D), and qRT-PCR results have the same trend with the ELISA results. These data indicate that gga-miR-146c inhibit MMP16 expression within limits, and then activate the NF-κB signaling pathway in inflammation response.

gga-miR-146c Is of Importance in NF-κB Pathway Regulation
Our previous study suggests that IL2/IL6-mediated inflammatory responses are induced with MG infection via TLR6/MyD88/NF-κB pathway [36]. To identify whether gga-miR-146c activates the TLR6/MyD88/NF-κB pathway to defend against MG infection, gga-miR-146c, miR-146c-NC, and miR-146c-Inh or miR-146c-Inh-NC were transfected into DF-1 cells for 48 h. The qRT-PCR results revealed that it is the gga-miR-146c expression, but not miR-146c-NC, significantly increased TLR6, NF-κB p65, MyD88, and TNF-α expression levels ( Figure 5A), whereas miR-146c inhibitor significantly decreased the expression of these four genes ( Figure 5C). The results of immunofluorescence showed NF-κB p65 protein was in the nucleus of DF-1 cells which transfected with gga-miR-146c, whereas the other groups had no clear change ( Figure 5E). The proteins of TLR6, MyD88, and TNF-α in the cell supernatant treated with gga-miR-146c were higher than the cells treated with miR-146c-NC ( Figure 5B), whereas the miR-146c inhibitor significantly decreased the proteins expression of TLR6, MyD88, and TNF-α ( Figure 5D), and qRT-PCR results have the same trend with the ELISA results. These data indicate that gga-miR-146c inhibit MMP16 expression within limits, and then activate the NF-κB signaling pathway in inflammation response.

Upregulation of gga-miR-146c Promotes Proliferation and Cycle Progression by Repressing Apoptosis
To explore biological functions of gga-miR-146c in MG-HS infection, we focused on influences of gga-miR-146c on cell proliferation, apoptosis, and cycle. Four groups of transfection treatments were carried out. Cells transfected with gga-miR-146c mimics before MG infection were marked as miR-146c (MG+); cells transfected with miR-146c-NC were marked as miR-146c-NC (MG+); non-transfected cells were marked as miR-free (MG+). All above groups were infected with MG. Uninfected DF-1 cells were marked as blank (MG-). Cell Counting Kit-8 was adopted to calculate the results. It is showed that overexpressed miR-146c (MG+) group and the blank (MG-) group significantly increased the cell multiplication at 48 h and 72 h post-transfection compared to miR-146c-NC (MG+) group or miR-free (MG+) group ( Figure 6A). At the same time, miR-146c inhibitor was assayed for further validation on cell proliferation. As expected, it led to significant reduction in cell proliferation after 48 h and 72 h transfection compared to each other three groups. A significant reduction of cell proliferation was also observed in miR-146c-Inh-NC (MG+) or miR-free (MG+) group ( Figure 6B). miR-146c-Inh-NC.The mock transfection served as blank. Stained macrophages were magnified 200×. gga-miR-146c overexpression made NF-κB p65 (red) turning into the cell nucleus.

Upregulation of gga-miR-146c Promotes Proliferation and Cycle Progression by Repressing Apoptosis
To explore biological functions of gga-miR-146c in MG-HS infection, we focused on influences of gga-miR-146c on cell proliferation, apoptosis, and cycle. Four groups of transfection treatments were carried out. Cells transfected with gga-miR-146c mimics before MG infection were marked as miR-146c (MG+); cells transfected with miR-146c-NC were marked as miR-146c-NC (MG+); non-transfected cells were marked as miR-free (MG+). All above groups were infected with MG. Uninfected DF-1 cells were marked as blank (MG-). Cell Counting Kit-8 was adopted to calculate the results. It is showed that overexpressed miR-146c (MG+) group and the blank (MG-) group significantly increased the cell multiplication at 48 h and 72 h post-transfection compared to miR-146c-NC (MG+) group or miR-free (MG+) group ( Figure 6A). At the same time, miR-146c inhibitor was assayed for further validation on cell proliferation. As expected, it led to significant reduction in cell proliferation after 48 h and 72 h transfection compared to each other three groups. A significant reduction of cell proliferation was also observed in miR-146c-Inh-NC (MG+) or miR-free (MG+) group ( Figure 6B). To explore how gga-miR-146c promotes cell proliferation, the cell cycle and apoptosis of DF-1 cells were assayed by a flow cytometer. Similarly, DF-1 cells were transfected with the synthetic RNA oligonucleotides. MG infection disturbed mitosis by guiding cells arrested in the G1 cell cycle. The number of cells that stayed in S and G2 phases increased remarkably due to gga-miR-146c overexpression ( Figure 7A). On the contrary, miR-146c-Inh led to an opposite result ( Figure 7B). To explore how gga-miR-146c promotes cell proliferation, the cell cycle and apoptosis of DF-1 cells were assayed by a flow cytometer. Similarly, DF-1 cells were transfected with the synthetic RNA oligonucleotides. MG infection disturbed mitosis by guiding cells arrested in the G1 cell cycle. The number of cells that stayed in S and G2 phases increased remarkably due to gga-miR-146c overexpression ( Figure 7A). On the contrary, miR-146c-Inh led to an opposite result ( Figure 7B).
According to cell apoptosis analysis results, gga-miR-146c overexpression effectively decreased apoptosis rates compared to the miR-146c-NC (MG+) group or the miR-free group (MG+) ( Figure 8A). Opposite results were obtained when cells were treated with a miR-146c inhibitor ( Figure 8B).
The above results indicate that upregulation of gga-miR-146c by MG-HS significantly enhances DF-1 cells proliferation upon MG infection by inhibiting cell apoptosis and enhancing cell cycle progression to defend against host MG infection. According to cell apoptosis analysis results, gga-miR-146c overexpression effectively decreased apoptosis rates compared to the miR-146c-NC (MG+) group or the miR-free group (MG+) ( Figure  8A). Opposite results were obtained when cells were treated with a miR-146c inhibitor ( Figure 8B).  According to cell apoptosis analysis results, gga-miR-146c overexpression effectively decreased apoptosis rates compared to the miR-146c-NC (MG+) group or the miR-free group (MG+) ( Figure  8A). Opposite results were obtained when cells were treated with a miR-146c inhibitor ( Figure 8B).  The above results indicate that upregulation of gga-miR-146c by MG-HS significantly enhances DF-1 cells proliferation upon MG infection by inhibiting cell apoptosis and enhancing cell cycle progression to defend against host MG infection.
The miR-146 family has important regulatory functions in many diseases, including influenza, virus infection, and cancer [44]. gga-miR-146c was upregulated in chicken embryos lungs upon MG infection according to deep sequencing data [31]. In this study, we demonstrated that gga-miR-146c was remarkably upregulated in both cells ( Figure 1A) and lungs ( Figure 1B). It is further indicated that MMP16 is the target gene of gga-miR-146c ( Figures 2D and 3). MMP16 expression was significantly decreased in MG-infected DF-1 cells and chicken embryos (Figure 4). These results indicate that miR-146c might participate in MG infection by binding to 3′-UTR region of MMP16. Meanwhile, MG infection is a complex process, so there may be other factors regulating MMP16 expression. It remains to be further studied.
The significant upregulation of gga-miR-146c by MG prompted us to investigate the biological function of gga-miR-146c in DF-1 cell line. MMPs, a family of zinc-dependent endopeptidases, are of importance in pathological processes, including inflammation, pulmonary diseases, cardiovascular
The miR-146 family has important regulatory functions in many diseases, including influenza, virus infection, and cancer [44]. gga-miR-146c was upregulated in chicken embryos lungs upon MG infection according to deep sequencing data [31]. In this study, we demonstrated that gga-miR-146c was remarkably upregulated in both cells ( Figure 1A) and lungs ( Figure 1B). It is further indicated that MMP16 is the target gene of gga-miR-146c ( Figures 2D and 3). MMP16 expression was significantly decreased in MG-infected DF-1 cells and chicken embryos (Figure 4). These results indicate that miR-146c might participate in MG infection by binding to 3 -UTR region of MMP16. Meanwhile, MG infection is a complex process, so there may be other factors regulating MMP16 expression. It remains to be further studied.
The significant upregulation of gga-miR-146c by MG prompted us to investigate the biological function of gga-miR-146c in DF-1 cell line. MMPs, a family of zinc-dependent endopeptidases, are of importance in pathological processes, including inflammation, pulmonary diseases, cardiovascular diseases, and cancer. [45,46]. MMP9 promotes Hepatitis B virus replication [47], and is of significant importance in response to gonococcal infection [48]. In melanoma, MMP16 mediates a proteolytic switch that promotes cell adhesion and lymphatic invasion [49]. MiR-146b-5p targets MMP16 to dramatically inhibit glioma and pancreatic cancer migration and invasion [50]. MiR-146a downregulated the expression of MMP16 in caco-2 cells [51]. MiR-145 suppresses osteosarcoma metastasis via targeting MMP16 [52].
NF-κB, a major transcription factor related to inflammatory signaling, is primarily involved in regulation of response to inflammatory and pathogens [53]. Bacteria challenged with Mycoplasma pneumoniae can release classical proinflammatory cytokines [54], which was shown to active NF-κB [55]. It is well-known that many microRNAs participate in the NF-κB pathway [56]. For example, the NF-κB pathway is activated due to the loss of miR-514a-3p in human testicular germ cell tumors [57]. The upregulation of miR-155 and miR-21 suppress the NF-κB pathway in HCV-or HIV-infected cells [58]. MiR-17-92 promotes leukemogenesis and activation of the NF-κB signaling [45]. Upregulated miR-429 inhibits the migration of HCC cells via the NF-κB pathway [59]. MiR-191 represses angiogenesis by the NF-κB pathway activation [60]. The NF-κB pathway was drastically ameliorated when MMP3 was decreased [61]. Multiple pathogens ligands, including bacteria, viruses, fungi, and protozoa, can be recognized by Toll-like receptors (TLRs) [62,63]. TLRs activate NF-κB-mediated inflammatory responses when stimulated by carcinogenic microbes and endogenous molecules [64]. NF-κB was found to mainly interact with TLRs [65]. The NF-κB pathway was inactivated for TLR6 knockout [66]. In our previous study, TLR6 were upregulated upon MG infection, followed by the upregulation of downstream NF-κB-mediated inflammatory responses. After TLR6 knockdown, DF-1 cells cannot make inflammatory response to MG infection via TLR6/NF-κB pathway [36]. In this study, the overexpression of gga-miR-146c led to a remarkable increase the expression of TLR6, NF-κB p65, TNF-α, and MyD88 in DF-1 cells ( Figure 5A,B,E), whereas gga-miR-146c inhibitor extremely repressed their expression ( Figure 5C-E). gga-miR-146c may affect downstream genes TLR6 and MyD88 to active TLR6/MyD88/NF-κB pathway through its target genes. TLR6 signaling pathway is activated to produce TNF-α which is one of inducible factors of NF-κB. We demonstrate that MMP16 is a direct target gene of gga-miR-146c, while it is still possible that other target genes of gga-miR-146c contribute to the observation. Recently, we reported that expression of NF-κB, TNF-α, and MyD88 was increased in chicken embryonic lungs upon MG infection as well as in DF-1 cells [17]. Together, it is rational to believe that upregulation of gga-miR-146c is involved in pathogenesis of MG infection to activate TLR6/MyD88/NF-κB pathway.
The NF-κB signaling pathway is closely associated with DF-1 cells proliferation [67]. The miR-146a could promote cell proliferation by suppressing activation of the NF-κB signaling pathway [68][69][70]. The miR-146a could suppress cell cycle progression in non-small cell lung cancer [71]. Downregulation of miR-146b promotes the proliferation rate of fusion cells [72]. Inhibition of NF-κB significantly suppressed MMP9 gene expression [73]. The metastatic thyroid cancer was related with NF-κB/MMP9 high expression [74]. MiR-132 affects glima cell migration and invasion by MMP16 [75]. Silence of MMP16 expression significantly decreased the invasion and proliferation capacity of gastric cancer (GC) [76]. Our results indicate that MG infection inhibits DF-1 cells multiplication by blocking the transition from the G1 phase to S and G2 phases, and upregulated miR-146c significantly promotes cell proliferation by inhibiting cell apoptosis and enhancing cell cycle progression ( Figure 6A, Figure 7A, and Figure 8A), whereas the miR-146c inhibitor restrained cell proliferation by promoting cell apoptosis and inhibiting cell cycle progression ( Figure 6B, Figure 7B, and Figure 8B). As we can see, MG infection and gga-miR-146c have opposite effect on cell proliferation and apoptosis. In the miR-free (MG+) group, cells were infected with MG only. DF-1 cells were damaged upon MG infection. Then, gga-miR-146c was upregulated to react to MG infection. In the miR-146c (MG+) group, gga-miR-146c mimics was transfected to DF-1 cells firstly, and after 4 h, cells were infected with MG. Therefore, gga-miR-146c in miR-146c (MG+) group worked earlier and more effective compared with miR-free (MG+) group. In general, gga-miR-146c promotes proliferation, inhibits cell apoptosis, and finally, prevents MG infection. In conclusion, gga-miR-146c upregulation upon MG infection inhibits MMP16 expression within limits, in turn activating TLR6/MyD88/NF-κB pathway to promote the cell proliferation and the cell cycle progression by decreasing cell apoptosis to defend against MG infection. As both gga-miR-146c and MMP16 are highly conserved in multiple species ( Figure 2C), we propose that there may be a similar strategy for the other species to defend against Mycoplasma infection. However, further studies are needed to support this prediction.