Role of gga-miR-29b-3p in suppressing the proliferation, invasion and migration of MSB1 Marek's disease tumor cells by the targeting of the DNMT3B gene.

Background: Marek's disease (MD), a class II infectious, lymphoproliferative disease that mainly afflicts poultry, has been shown to cause wasting, limb paralysis, and often acute death. It is a neoplastic disease caused by a cell-binding herpesvirus that leads to the formation of tumors in various organs and tissues. Our previous reports have found that the microRNA, gga-miR-29b-3p, showed abnormal expression in MD lymphoma. However, it remains unknown whether gga-miR-29b-3p affects MD tumorigenesis.
Methods: The MD tumor cell line MSB1 was chosen to analyze the characteristics of gga-miR-29b-3p in tumors. Cell proliferation and migration were assessed by Cell Counting Kit-8 (CCK-8) and Transwell, respectively, and cell apoptosis and cycle were analyzed via fluorescent staining and flow cytometry, respectively. The regulation between gga-miR-29b-3p and its potential target genes was verified by dual luciferase results and loss-of-function assays. The effect of target genes was verified by examining the degree of RNA interference on MSB1 cells.
Results: Analysis revealed that gga-miR-29b-3p impaired the proliferation of the MSB1 MD tumor cell line, induced apoptosis without obvious effects on the cell cycle, and suppressed the expression of the invasion-associated MMP2 and MMP9 genes. It was concluded that DNMT3B is the direct target of gga-miR-29b-3p. As expected, the effects of DNMT3B knockdown with small interfering RNA (siRNA) on MSB1 cell proliferation, apoptosis, and cycle were associated with gga-miR-29b-3p overexpression. Moreover, BCL2 and BCL2L1 were downregulated and TNFSF10 was upregulated in both the gga-miR-29b-3p overexpression and DNMT3B knockdown groups. The expression levels of invasion-related genes were decreased post-DNMT3B knockdown. In both the gga-miR-29b-3p overexpression and DNMT3B knockdown conditions, a decrease in MEQ oncogene expression in MD virus was observed. Conclusions: Overall, gga-miR-29b-3p was demonstrated to have a suppressive effect in MD lymphoma progression via the targeting of the DNMT3B gene. Gga-miR-29b-3p overexpression and DNMT3B knockdown inhibited MSB1 cell proliferation through suppressing the pro-apoptotic gene expression and elevating the anti-apoptotic gene expression in the apoptosis pathway. Our study provides a theoretical basis for targeted treatment of MD. 2022 Annals of Translational Medicine. All rights reserved.

Introduction

Marek’s disease (MD) is infectious disease in poultry that is caused by a highly cell-bound herpesvirus [MD virus (MDV)], which was characterized by immunosuppression and polyneuritis (1,2). It has been found across a range of commercial poultry species, in chickens and quail initially and later in turkeys (3,4). Live attenuated vaccination is still used worldwide at this stage in the prevention of MD (5). However, the widespread use of the vaccine has also led to the increased virulence of MDV, from a strong strain (vMDV) in the 1970s that broke through the contemporaneous turkey herpesvirus (HVT) vaccine, to a super-virulent strain (vvMDV) in the 1980s, and then to an very virulent plus (vv+MDV) in the 1990s that also broke through the protection of vaccines such as bivalent vaccines HVT+MDV2 (SB1) (6). The emergence of extra-virulent viruses and the increased virulence of wild strains have emerged as the major main challenges to the sustained control of this disease, causing considerable harm to livestock and economic losses in the poultry farming industry (7,8).

MicroRNAs (miRNAs), are short-stranded RNAs that are found endogenously in eukaryotes, consist of highly conserved sequences, and participate in the regulation of numerous biological processes (9). MiRNAs contribute to gene transcriptional regulation by degrading messenger RNA (mRNAs), inhibiting protein synthesis and interacting with long non-coding RNA (10,11). Recent reports have revealed that aberrant miRNA expression in MD may be involved in the tumorigenesis process. For instance, Gallus gallus (gga)-miR-21 was found to be upregulated during gallid herpesvirus 2 (GaHV-2) infection driven by activator protein 1 (AP-1) and Ets-response elements and to promote tumor cell growth and apoptotic escape by targeting the programmed death cell 4 gene (12). Another study reported that gga-miR-26a was significantly downregulated in the spleen of MDV-infected chickens and that it inhibited MSB1 cell proliferation in a direct targeting relationship with NEK6, possibly by suppressing prematurely senescent cells to affect the proliferative potential of tumor cells (13). Other reports revealed that reduced expression of gga-miR-103-3p in MDV-infected tissues and this miRNA inhibited MSB1 migration but had no significant effect on proliferation, with the abnormal expression of 2 target genes, CCNE1 and TFDP2, being shown to disrupt the normal cell cycle and thus precipitating tumorigenesis (14). Downregulation of gga-miR-130b-3p in MD tumor may be directly attributed to the methylation of this miRNA (15). Indeed, gga-miR-155 is considered to be a potential marker for MD tumor, and studies suggest that the overexpression of this miRNA has a beneficial influence on MSB1 cell proliferation, migration, and invasion, but has a suppressive effect on cell apoptosis by targeting RORA (16,17). Our previous miRNAs profile found that gga-miR-29b-3p was significantly downregulated in MD tumorous tissues, implicating it may be involved in MD tumorigenesis (18). In chicken primordial germ cells, gga-miR-29b was found to downregulate the expression of DNMT3B, which provides more knowledge to explain the role of DNMT genes regulation in embryogenesis (19). In the jejunal mucosa of laying hens with different genetic backgrounds, the potential target genes of miR-29b-3p were enriched in energy pathways at different stages of production periods (20). In the present study, we examined a mechanism of gga-miR-29b-3p in MD tumor transformation and its effects on the characteristics of a MDV-transformed lymphoid cell line. It is hoped our findings can contribute to building a theoretical foundation and identifying molecular markers for the designed breeding of MD disease resistance. We present the following article in accordance with the MDAR reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-3519/rc).

Methods

Cell culture, miRNA transfection, and RNA interference

The MDV-transformed chicken lymphoblastoid cell line MDCC-MSB1 was kindly provided by Dr. C. Itakura in Tottori University and cultured at 37 ℃, under 5% CO2 in RPMI 1640 medium (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) (21,22). X-tremeGENE siRNA Transfection Reagent (Roche, Basel, Switzerland) was selected to transfect the corresponding miRNA or siRNA into MSB1. The gga-miR-29b-3p mimics, inhibitor, DNMT3B small interfering RNA (siRNA), and scramble nontarget negative control (NC) were synthesized by GenePharma Company (GenePharma Co. Ltd., Shanghai, China). The targeting sequence of DNMT3B was 5'-GCTGTGCCTTGAACATTGT-3'. As described previously, dosage of mimics, siRNA, and corresponding NC transfection was 100 nM, while that of the inhibitor and corresponding NC transfection was 150 nM (13,23).

Detection of tumorous cellular functions

The cells used for transfection were seeded into 96-well, 24-well, and 6-well plates with a total of 3×104, 1.5×105, and 5×105 cells per well, respectively. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8; Beyotime, Shanghai, China). The absorbance at 450 nm was examined at 24, 36, 48, 60, and 72 hours posttransfection. Cell apoptosis was detected using the TUNEL BrightRed Apoptosis Detection Kit (Vazyme, Nanjing, China). The cells were collected for apoptosis assay 48 hours posttransfection according to the manufacturer’s instructions (24). Briefly, the cells were fixed with 4% paraformaldehyde at 4 ℃ for 20 minutes and then permeabilized with 0.5% Triton X-100 at room temperature for 5 minutes. The cells were next labeled with tetramethylrhodamine (TMR) red deoxyuridine triphosphate (dUTP) which could bind with the broken DNA in the apoptotic cells, and the reaction was terminated with 20 mM of ethylenediaminetetraacetic acid (EDTA). Following this, the cells were stained with 1 µg/mL of DAPI. Finally, the cells were observed by fluorescence microscope (Olympus Corporation, Tokyo, Japan) and photographed. The procedures of the cell cycle assay were performed according the specifications of Li et al. [2017] and Zhao et al. [2017] (23,25). After fixation, the cells were treated with RNase A (20 µg/mL; Sigma-Aldrich, St. Louis, MO, USA) and then stained with propidium iodide (50 µg/mL; Sigma-Aldrich) for 30 minutes in the dark. The cell populations were quantified by FlowJo software (BD, Franklin Lakes, NJ, USA). The protocols of cell migration assay were also performed according to those of Li et al. [2014] (13).

RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using TRIzol Reagent (Invitrogen), and complement DNA (cDNA) was synthesized using a PrimeScript RT Reagent Kit (Takara Bio, San Jose, CA, USA). The RT-qPCR reactions were performed using the SYBR Premix Ex Taq II kit (Takara Bio) and detected on the ABI Prism 7500 HT sequence detection system (Applied Biosystems, Foster City, CA, USA). The primer information of matrix metallopeptidase (MMP) MMP2, MMP9, and β-actin can be found in the publication of Zhao et al. [2017] (Table S1) (23); that for MDV Eco Q fragment-encoded gene MEQ from Heidari et al. [2008] (26); that for BCL2 and BCL2L1 from Subramaniam et al. [2013] (27); that for TNFSF10 from Li et al. [2008] (28); and that for the DNMT3B gene from Tian et al. [2013] (Table S1) (29). The results were determined by the 2–ΔΔCt method (30).

Western blot

Western blot was conducted using standard methods (31) with specific primary antibody to DNMT3B (1:2,000; Abbkine, Wuhan, China) or β-actin antibody (1:1,000; Beyotime) overnight, along with anti-rabbit secondary antibody (1:1,000; Beyotime) and anti-mouse secondary antibody (1:1,000; Beyotime). The specific protein was visualized with SuperSignal West Pico PLUS (Life Technologies, Thermo Fisher Scientific). Grayscale analysis was performed using Image J software (National Institutes of Health, Bethesda, MD, USA).

Target gene prediction and dual-luciferase reporter assay

Potential miRNA targets of gga-miR-29b-3p were predicted the by online software platforms TargetScan (http://www.targetscan.org/) and miRDB (http://mirdb.org/miRDB/).

The 3 prime untranslated region (3'-UTR) fragments of DNMT3B covering the putative gga-miR-29b-3p binding sites were amplified to construct the wild-type and mutant-type luciferase reporter vectors. This region was amplified using forward primer 5'-GCCTCGAGCGTTTCAAAATGCTGCTG-3' and reverse primer 5'-CGGCGGCCGCTTTTTTTTTTCCTCTTATTTTAAAAGATTGTATA-3'. The mutant-type vector was also constructed to further verify the binding sites between gga-miR-29b-3p and the 3'-UTR of DNMT3B. The sequence of seed region in the 3'- UTR of DNMT3B was completely mutated with forward primer 5'-TCTAACTCTCCCACCACGATTTTTTTTAGTTAAAGGAT-3' and reverse primer 5'-TAACTAAAAAAAATCGTGGTGGGAGAGTTAGAAACCTT-3'. The amplified fragments were inserted into the psiCHECK2 plasmid.

The dual-luciferase reporter experiments were performed in the 293T cell line as described previously (14). The 293T cell line was cultured in DMEM medium (Invitrogen) with 10% FBS (Invitrogen), which was maintained at the condition of 37 ℃ and 5% CO2. The luciferase activity was measured using the dual-luciferase Reporter Assay System (Promega, Madison, WI, USA).

Statistical analysis

Numerical data are presented as mean ± standard error (SE). All statistical analyses were conducted with the SPSS 22.0 software (IBM Corp., Armonk, NY, USA). The Student’s t-test and ANOVA test were performed for data of two groups and over two groups., respectively. A P value <0.05 was considered to indicate a significant difference, while a P value <0.01 was considered to indicate an extremely significant difference.

Results

gga-miR-29b-3p inhibited MSB1 proliferation by promoting apoptosis-independent cell cycle retardation

To understand the role of gga-miR-29b-3p in the MSB1 cell line, we used gga-miR-29b-3p mimics or inhibitor to overexpress or suppress miRNA expression in MSB1 cells, respectively. First, high transfection efficiency was verified (). Transfection of gga-miR-29b-3p mimics significantly inhibited MSB1 cell proliferation compared with NC mimics at 24, 36, 48, and 72 hours. In contrast, the transfection of gga-miR-29b-3p inhibitor significantly increased MSB1 cell proliferation compared with NC inhibitor at 48 and 60 hours ().

Figure 1

Effects of gga-miR-29b-3p on MSB1 cell proliferation, apoptosis, and cycle. (A) Delineation for the transfection efficiency of miRNA mimics or inhibitor. FAM-labelled mimic NC was made for transfection in MSB1 cells. The scale bar is 100 µm. (B) Cell proliferation statistics after transfection of gga-miR-29b-3p mimics or inhibitor. (C) Immunofluorescent staining for TUNEL after transfection of gga-miR-29b-3p mimics or inhibitor. DAPI (blue color for nucleus) and BrightRed (red color for apoptotic cells). (D) Statistical analysis of apoptotic rates in the different groups. (E-G) RT-qPCR analysis of BCL2, BCL2L1, and TNFSF10 after transfection of gga-miR-29b-3p mimics or inhibitor. (H) Cell cycle analysis after transfection of gga-miR-29b-3p mimics or inhibitor. (I) Histogram of the proportion in the different phases of cell cycle after transfection. *, P<0.05; **, P<0.01. FAM, fluorescein phosphoramidite; NC, negative control; PE-A, phycoerythrin-area; RT-qPCR, real-time quantitative polymerase chain reaction.

To further analyze whether the gga-miR-29b-3p-impaired cell proliferation was caused by apoptosis in MSB1 cells, we performed fluorescent staining and analyzed the gene expression level of endogenous and exogenous apoptotic pathways. Using staining by cellular fluorescence, we found that the rates of apoptotic cells increased significantly after transfection with miRNA mimics and decreased significantly after transfection with miRNA inhibitor (). BCL2, BCL2L1, and TNFSF10 are key genes in the apoptotic pathway and necessary to induce cell apoptosis. We detected the expression levels of these genes. At 24 hours, the transcriptional expression of antiapoptotic BCL2 was significantly downregulated and that of antiapoptotic BCL2L1 was extremely significantly decreased; at 72 hours, after overexpression of gga-miR-29b-3p mimics, both the transcriptional expressions of BCL2 and BCL2L1 were significantly decreased (). Under the condition of transfection with gga-miR-29b-3p inhibitor, BCL2 was significantly increased at 24 hours and significantly increased at 72 hours, while BCL2L1 was significantly increased at 24 hours (). The expression of the proapoptotic gene TNFSF10 was significantly increased by transfection of gga-miR-29b-3p mimics at 24 hours and was extremely decreased by transfection of gga-miR-29b-3p inhibitor at 48 hours (). Moreover, there was no significant effect on cell cycle at 48 hours after gga-miR-29b-3p mimic or inhibitor transfection (). This suggested that gga-miR-29b-3p played a major role in promoting apoptosis through regulating the key gene expression level in the apoptotic pathway in MSB1 cells.

gga-miR-29b-3p inhibited the expression of invasion-related genes and MSB1 cell migration

Migration and invasion are among the malignant behaviors of tumor cells. Invasion and migration have different biological characteristics: the former involves the occupation of malignant tumors, both primary or secondary, to adjacent host tissues, while the latter involves the transfer of tumor cells to another site or organ by blood flow and the continued growth of tumors. To further clarify the roles of gga-miR-29b-3p, we selected the invasion markers MMP2 and MMP9 for comparing the ability of invasion between the transfection of gga-miR-29b-3p and controls. Interestingly, RT-qPCR showed the transcription level of MMP2 to be significantly decreased after transfection of gga-miR-29b-3p mimics at 48 hours, while the opposite phenomenon of significantly upregulated transcription levels in MMP2 was observed under transfection of gga-miR-29b-3p inhibitor at 24 and 48 hours (). Moreover, the transcription level of MMP9 was downregulated significantly at 72 hours, while the transcription level of MMP9 was significantly increased posttransfection with miRNA inhibitor at 48 and 72 hours (). The cell number of migrated MSB1 significantly decreased under transfection of gga-miR-29b-3p mimics and was rescued by gga-miR-29b-3p inhibitor (). This indicated that gga-miR-29b-3p restrained MSB1 cell invasion and migration.

Figure 2

Effect of gga-miR-29b-3p on MSB1 cell invasion and migration. (A,B) mRNA levels of MMP2 and MMP9 after transfection of gga-miR-29b-3p mimics and inhibitor. (C) Cell migration analysis after transfection of gga-miR-29b-3p mimics or inhibitor. (D) Bright field of cell migration after transfection of gga-miR-29b-3p mimics or inhibitor. After removal of the upper Transwell, the number of cells in the lower chamber was counted under the microscope. *, P<0.05; **, P<0.01. NC, negative control.

gga-miR-29b-3p suppressed DNMT3B gene expression upon binding to its 3'-UTR sequence

To elucidate effect of gga-miR-29b-3p in MD tumor formation, we next used online software and literature retrieval to select candidate gene DNMT3B. To reveal whether gga-miR-29b-3p could directly regulate its hypothetical candidate gene, DNMT3B, we constructed vectors including gga-miR-29b-3p wild binding site [wild type (WT)] and mutant binding site (MUT; ). Luciferase activity showed that gga-miR-29b-3p had a distinctly inhibitory role on the WT group but not the MUT group (). To further validate that DNMT3B was directly regulated by gga-miR-29b-3p, we collected MSB1 cells under transfection with gga-miR-29b-3p mimics, inhibitor, and respective NCs at different time points to compare the expression of DNMT3B at the transcription level and protein level. First, the transcription level of DNMT3B was remarkably suppressed in the mimic transfection group at all time points of received samples but was extremely elevated at 24 and 48 hours in the inhibitor transfection group (). Second, the expression at translational levels of DNMT3B was remarkably inhibited in the mimic transfection group and extremely elevated in the inhibitor transfection group at 96 hours (). Collectively, these results suggested that DNMT3B was directly prohibited by gga-miR-29b-3p and rescued through inhibitor.

Figure 3

DNMT3B was a target gene of gga-miR-29b-3p. (A) Schematic diagram illustrating the design of luciferase reporters with nucleotide sequences of the wild-type and mutant-type gga-miR-29b-3p binding sites located in the 3'-UTR of DNMT3B. (B) Luciferase activity analysis after transfection of mutant-type DNMT3B 3'-UTR. The Luciferase activity was normalized by transfection of mimics NC and empty vector psiCHECK2. (C) RT-qPCR analysis of DNMT3B after transfection of gga-miR-29b-3p mimics and inhibitor. (D) Western blot analysis of DNMT3B after transfection of gga-miR-29b-3p mimics and inhibitor. (E) Grayscale analysis of DNMT3B in the different groups. *, P<0.05; **, P<0.01. NC, negative control; RT-qPCR, real-time quantitative polymerase chain reaction; UTR, untranslated region; WT, wild type; MUT, mutant type.

DNMT3B knockdown inhibited cell proliferation by the promoting apoptosis of MSB1 cells

After identifying DNMT3B as a direct target gene of gga-miR-29b-3p, we analyzed its function in MD tumorigenesis by RNA interference. We designed 3 siRNA sequences to intervene with the DNMT3B gene, with the interference efficiency of the third sequence being almost 67% at the transcriptional level (). Using this RNA (hereafter namely siRNA-DNMT3B), the expression level of DNMT3B decreased by 87.5% at the translational level (). Transfection of siRNA-DNMT3B at 48 hours significantly inhibited cell proliferation (). TUNEL assay showed that the number of apoptotic cells increased significantly after transfection with siRNA-DNMT3B (). The DNMT3B gene intervention significantly downregulated the expression of the BCL2 and BCL2L1 genes but upregulated the mRNA expression of the TNFSF10 gene (). In contrast, knockdown of DNMT3B had no impact on cell cycle (). Taken together, our findings revealed that silencing DNMT3B could stimulate spontaneous apoptosis and lead to cell cycle-independent proliferation in MSB1 cells.

Figure 4

Effect of DNMT3B knockdown on MSB1 cell proliferation, apoptosis, and cell cycle. (A) Efficiency of knocking down DNMT3B with 3 different siRNAs. (B) The expression level of DNMT3B at the protein level with the effective siRNA. (C) Grayscale analysis of DNMT3B after knocking down DNMT3B with the most efficient siRNA. (D) Cell proliferation ability after knocking down DNMT3B. (E,F) Cell apoptosis ability and statistical analysis of apoptotic rates after knocking down DNMT3B. (G) RT-qPCR analysis of BCL2, BCL2L1, and TNFSF10 after knocking down DNMT3B. (H) Cell cycle analysis of knocking down DNMT3B. (I) Histogram of the proportion in different phases of cell cycle after DNMT3B knockdown. *, P<0.05; **, P<0.01. NC, negative control; RT-qPCR, real-time quantitative polymerase chain reaction; PE-A, phycoerythrin-area; siRNA, small interfering RNA.

Silencing of DNMT3B impaired the expression of invasion-related genes

To further verify whether the gga-miR-29b-3p target gene, DNMT3B, suppresses invasion and migration, we chose 2 indicators, cell migration and invasion-related genes (MMP2 and MMP9), to examine in follow-up experiments of DNMT3B knockdown of MSB1 cells. First, the transcription level of MMP2 and MMP9 was visibly downregulated in DNMT3B knockdown at 48 hours (). Second, the cell numbers of migration were not obviously different after transfection with siRNA-DNMT3B at 64 hours (). Therefore, we concluded that DNMT3B mediated invasion and exerted an enhanced effect on the viability MSB1 cells.

Figure 5

Effect of DNMT3B knockdown on MSB1 cell invasion and migration. (A) RT-qPCR analysis of MMP2 and MMP9 after DNMT3B knockdown. (B) Cell migration variations after DNMT3B knockdown. (C) Representative images depicting cell migration profiles of MSB1 cells after DNMT3B knockdown. After removal of the upper Transwell, the number of cells in the lower chamber was counted under the microscope. *, P<0.05; **, P<0.01. NC, negative control; RT-qPCR, real-time quantitative polymerase chain reaction.

gga-miR-29b-3p and DNMT3B gene affected the expression of the proto-oncogene MEQ

Among the genes encoded by MDV, MEQ is considered to be an important oncogene. We observed that gga-miR-29b-3p and its target gene DNMT3B could affect MEQ expression. MEQ expression was significantly downregulated after transfection with gga-miR-29b-3p mimics at 48 hours. Conversely, MEQ expression was visibly increased after transfection with gga-miR-29b-3p inhibitor at 48 and 72 hours (). Moreover, MEQ expression significantly decreased after DNMT3B interference at 48 hours (). These observations indicated that gga-miR-29b-3p and its target gene, DNMT3B, inhibited the expression of genes involved in proto-oncogenesis.

Figure 6

Effect of gga-miR-29b-3p and DNMT3B knockdown on the expression of the proto-oncogene MEQ. (A) RT-qPCR analysis of MEQ after transfection of gga-miR-29b-3p mimics and inhibitor. (B) mRNA expression level of MEQ after DNMT3B knockdown. *, P<0.05. NC, negative control; RT-qPCR, real-time quantitative polymerase chain reaction.

Discussion

MD is a type of lymphoproliferative disease resulting from a cell-associated pathogen MDV and is capable of causing massive damage to the poultry industry (7). Once birds are infected with MDV, culling and mortality occur in up to 80% of the population, and there remains no viable cure for this disease anywhere in the world (32). Therefore, there is an urgent need to conduct further research to discover any potential molecular mechanisms of MD and to develop more promising therapeutic approaches. In our previous study using Solexa deep sequencing, we observed the aberrant expression of gga-miR-29b-3p in the MD tumorous spleen and liver lymphoma, and its expression pattern was negatively correlated with MD (18). We conducted further study on gga-miR-29b-3p-mediated effects to better understand the link between this miRNA and MD tumorigenesis.

MiRNAs have important functions in the proliferation, migration, invasion, and chemoresistance of various solid tumors (33). Recent studies have indicated that miR-29b shows aberrant expression in a variety of tumorous tissues and tumorous cells, which indicates that miR-29b may play a vital role during carcinogenesis (34,35). As per previous reports, miR-29b participates in the differentiation and tumorigenesis of tumor in prostate cancer, breast cancer, glioblastoma multiforme (GBM), and non-small cell lung cancer (NSCLC), implicating miR-29b as a central component in tumor progression (36-39). A study in miR-29b in nude mice revealed that the overexpression of miR-29b could limit the metastasis of prostate tumor cells and inhibit the growth of prostate xenografts, which was consistent with our findings related to gga-miR-29b-3p (36). In another study, miR-29b was shown to inhibit antiangiogenesis and antitumorigenesis by targeting AKT3 and inducing the expression of VEGF and C-myc in breast cancer cells (38). MiR-29b was demonstrated to affect the self-renewal and proliferation in neural stem cells (NSCs) by regulating the β-catenin and T-cell factor-mediated Wnt/β-catenin signaling pathway (40). The genetic developmental mechanism of NSC-transformed GBM was similar with NSCs; it was reported that miR-29b had the ability to inhibit cell growth, induce apoptosis and produce anticancer effects in GBM cell lines (39). Moreover, gga-miR-29b-3p was found to inhibit the proliferation of an MDV-derived chicken lymphocyte line, MDCC-MSB1, and the decrease in cell proliferation rate resulted from an elevated number of apoptotic cells; the apoptotic pathways included exogenous pathway triggered by death receptors and endogenous pathway triggered by mitochondria (41). The exogenous apoptotic pathway primarily consists of a combination of death receptors and ligands leading to the receptor multimerization, in which TNFSF10 acts as a ligand that binds to death receptors DR4 and DR5 and activates caspase-8 to trigger the apoptotic cascade reaction (42). The B-cell lymphoma 2 (BCL2) family regulates the permeability of the mitochondrial outer membrane by polymerizing and depolymerizing with the components in this family (43).

Based on our findings, we concluded that the overexpression of gga-miR-29b-3p suppressed BCL2 and BCL2L1 and promoted the expression of the proapoptotic gene, TNFSF10. Invasion and migration are 2 powerful functions of tumor cells, in which MMPs are involved (44). Among the MMP family, MMP2 and MMP9 perform the effect of degrading type IV collagen, a major component of the basement membrane, prompting tumorous cell invasion in cancer (45). In one study, overexpression of miR-29b in vitro seemed to strengthen NSCLC cell proliferation, migration, and invasion by elevating cyclin D1 or MMP9 and reducing the expression of p21, Bax, and E-cadherin; moreover, the overexpression of miR-29b in vivo downregulated STRN4, inhibited cell proliferation, and delayed tumor progression (46). In our study, gga-miR-29b-3p inhibited the invasion and migration of MSB1 cells. Overall, gga-miR-29b-3p exerts a suppressive effect on MD tumor transformation.

Furthermore, we confirmed that gga-miR-29b-3p negatively regulated DNMT3B through directly binding to its 3'-UTR. DNMTs are the predominantly epigenetic modifier genes in mammals and are involved in DNA damage recognition, DNA recombination, and mutation repair (47). In previous studies, miR-29b targeting DNMT3B was also found to exert oncogenic and other effects. Cao et al. [2021] reported that miR-29b promoted cell cycle inhibitor CDKN2B demethylation and transcription by inhibiting DNMT3B activity, induced G1 phase block, inhibited cholangiocarcinoma cell proliferation, and promoted apoptosis (48). Another study reported that miR-29b induced miR-195 promoter demethylation and its re-expression by targeting DNMT3B, thereby inhibiting cell cycle progression and promoting apoptosis in tongue squamous cell carcinoma (TSCC) cell lines (49). In osteoarthritic (OA) cartilage tissue, DNMT3B was shown to induce hypermethylation of specific CpG sites in the miR-29b promoter region which contributed to miR-29b downregulation in OA chondrocytes, both of which were associated with OA-induced chondrocyte apoptosis (50). In our study, transfection of gga-miR-29b-3p mimics resulted in a significant decrease in the expression of DNMT3B at both the transcriptional and translational levels in MSB1. This may be attributed to the complementary pairing of the seed sequence for gga-miR-29b-3p to the 3'-UTR region for DNMT3B. However, further research concerning whether the methylation status of genes is affected by the abnormal regulation of DNMT3B in MSB1 cell is needed to confirm this speculation.

Some researchers have confirmed that DNA methylation is correlated with MD resistance and susceptibility. Yu et al. [2008] reported 2 line-specific DNA transition mutations, CpG→TpG (Chr20:10203733 and 10203778) in MD-susceptible line 72 when compared to the MD-resistance line 63 (51). Tian et al. [2013] found that both global methylation levels and the expression level of DNMT3B were higher in line 72 than in line 63 prior to MDV infection and that the transcriptional expression of DNMT3B was further induced in line 72 after MDV infection (29). These results indicate that the lower methylation in line 63 mostly results from silencing of DNMT3B and DNMT1. We found that interference with DNMT3B inhibited MSB1 cell proliferation and invasion and promoted apoptosis, indicating that gga-miR-29b-3p exert its effects during MD progression by targeting DNMT3B. DNMT3A and DNMT3B are often elevated in various tumors and tumorous cells (52). The role of DNMT3B in many other cancers and its transcriptional regulation by other molecules have been extensively studied (53,54).

MEQ, a 339-amino acid protein, confers an oncogenic property to MDV (55). The colocalization of MEQ with chicken histone deacetylase 1 (HDAC1) and HDAC2 was shown to result in proteasome-dependent degradation (56). The natural killer (NK) cells could be infected with MDV and produced antiviral response, in which MEQ induced NK cell activation (57). Therefore, we investigated whether gga-miR-29b-3p has a suppressive effect on MEQ expression. Both the overexpression of gga-miR-29b-3p and the blockade of DNMT3B decreased the expression of MEQ, suggesting that gga-miR-29b-3p and DNMT3B can regulate the expression of the proto-oncogene MEQ.

Conclusions

Gga-miR-29b-3p presumably acts as a tumor suppressor in MD tumorigenesis by negatively regulating the expression of DNMT3B. Gga-miR-29b-3p exhibited a suppressive effect on cell proliferation, migration, and invasion and facilitated cell apoptosis via the DNMT3B gene. Our study has identified a potential regulatory network of miR-29b-3p and its target gene that regulates the characteristics of MD lymphoma transformation. Our findings further provide information for elucidating the mechanics of MD resistance and susceptibility and for exploring new avenues in the intervention and treatment of MD.

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