The Mechanisms of BDNF Promoting the Proliferation of Porcine Follicular Granulosa Cells: Role of miR-127 and Involvement of the MAPK-ERK1/2 Pathway

Simple Summary Recently, increasing the efficiency of porcine embryo cultures by promoting oocyte maturation in vitro has attracted much attention. Brain-derived neurotrophic factor (BDNF) was beneficial to oocyte maturation and increased the developmental potential of porcine embryos. Although the effects of BDNF on porcine follicular development and the maturation of oocyte have been previously demonstrated, no literature was available, at the time of this work, relating to miRNA-regulated gene expression and signal pathways in mechanisms of BDNF, promoting porcine GCs proliferation. Therefore, this study explored the miRNAs involved in BDNF-induced proliferation of porcine GCs, as well as the involvement of the MAPK-ERK signaling pathway. Abstract As a member of the neurotrophic family, brain-derived neurotrophic factor (BDNF) provides a key link in the physiological process of mammalian ovarian follicle development, in addition to its functions in the nervous system. The emphasis of this study lay in the impact of BDNF on the proliferation of porcine follicular granulosa cells (GCs) in vitro. BDNF and tyrosine kinase B (TrkB, receptor of BDNF) were detected in porcine follicular GCs. Additionally, cell viability significantly increased during the culture of porcine GCs with BDNF (100 ng/mL) in vitro. However, BDNF knockdown in GCs decreased cell viability and S-phase cells proportion—and BDNF simultaneously regulated the expression of genes linked with cell proliferation (CCND1, p21 and Bcl2) and apoptosis (Bax). Then, the results of the receptor blocking experiment showed that BDNF promoted GC proliferation via TrkB. The high-throughput sequencing showed that BDNF also regulated the expression profiles of miRNAs in GCs. The differential expression profiles were obtained by miRNA sequencing after BDNF (100 ng/mL) treatment with GCs. The sequencing results showed that, after BDNF treatment, 72 significant differentially-expressed miRNAs were detected—5 of which were related to cell process and proliferation signaling pathways confirmed by RT-PCR. Furthermore, studies showed that BDNF promoted GCs’ proliferation by increasing the expression of CCND1, downregulating miR-127 and activating the ERK1/2 signal pathway. Moreover, BDNF indirectly activated the ERK1/2 signal pathway by downregulating miR-127. In conclusion, BDNF promoted porcine GC proliferation by increasing CCND1 expression, downregulating miR-127 and stimulating the MAPK-ERK1/2 signaling cascade.


Introduction
Ovarian follicle development is a fundamental process of reproductive physiology in female mammals. Germ stem cells differentiate into oogonia in the genital ridge and the normal saline after being washed three times with 75% ethanol for disinfection. Follicular fluid was aspirated between 3 mm and 5 mm follicles using a 10 mL sterile syringe, and about 8-10 mL was acquired from twenty porcine ovaries. The collected follicular fluid was transported into 20 mL PBS and centrifuged at 300× g for 1 min to eliminate the oocytes. The isolated supernatant was transferred into a 50 mL sterile centrifuge tube, followed by centrifugation at 500× g for 6 min to separate GCs. The trypan blue exclusion test was performed to examine the viability of GCS. Approximately 1 × 10 6 GCs per well were seeded in 6-well plates within 2 mL culture medium (Dulbecco's Modified Eagle Media/Nutrient Mixture F12 (DMEM/F12)), comprising of 10% fetal bovine serum (FBS) for 24 h at 37 • C with 5% CO 2 .

Immunofluorescence Staining
Fresh porcine follicle granulosa cells were cultured in 24-well (2 × 10 5 cells/well) optical bottom plates for 24 hand then triple-washed with PBS (5 min each). Cells were preserved for 20 min in precooled absolute methanol and then permeabilized with 0.1% Triton X-100 for 30 min. Cells were blocked with 1% goat serum at ambient temperatures for 30 min, followed by incubation at 4 • C overnight in the following primary antibodies: anti-FSHR (BS2618, Bioworld, Nanjing, China, 1:200 for rabbit anti-mouse), anti-BDNF (BS6533, Bioworld, 1:200 for rabbit anti-mouse), or anti-TrkB (BS94070, Bioworld, 1:100 for rabbit anti-mouse). They were then rinsed three times with PBS. After that, the cells were incubated in goat anti-rabbit immunoglobulin G (IgG) combined with fluorescein (FITC) antibody (BS10950, Bioworld, 1:1000) at ambient temperatures for 1 h and triple-washed with PBS. After that, cells were stained for 10 min using PI solution (1:1000; Beyotime, Shanghai, China) and observed with a fluorescence microscope (IX71; Olympus, Tokyo, Japan). The porcine GCs were presented in two colors: green for FSHR and red for nucleus.

Cell Transfection
The siRNA for BDNF (si-BDNF sequence: 5 -GCGGTTCATAAGGATAGAC-3 ) was synthesized from RiboBio (Guangzhou, China). Transfection of porcine GCs with 50 nM siRNA was accomplished using FuGENE ® HD (Roche, Basel, Switzerland) reagent for transfection and Opti-MEM medium (Gibco, Paisley, Scotland, UK), as directed by the manufacturing company. GCs were transfected with siRNA for 24 h to extract RNA and protein.
The mimics and inhibitors of miRNAs (miR-185, miR-1273, miR-7047, miR-127 and miR-532) were acquired from Genepharma (Shanghai, China). Based on the manufacturer's guidelines, mimics of the five miRNAs or miR-127 inhibitors were transfected into the GCs using FuGENE ® HD transfection reagent for 24 h. The transfection concentration was 50 nM.

RNA Extraction and Reverse Transcripton-Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA was extracted from GCs using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription was done using a PrimeScript RT Reagent Kit (Takara, Tokyo, Japan) based on manufacturer instructions. qRT-PCR was performed with a SYBR Green Master Mix (Takara, Tokyo, Japan) and real-time quantitative fluorescence PCR instrument (Mx3005P; Agilent, Santa Clara, CA, USA). The reaction criteria were as follows for temperature profile: denaturation at 95 • C for 15 min, then amplification for 40 cycles of 35 s at 95 • C and 40 s at 60 • C. Using GAPDH or U6 as reference genes, the relative mRNA expressions of indicated genes were calculated using 2 −∆∆CT methods. All sequences of primer synthesis were completed by Comate Bioscience Co., Ltd. (Changchun, China) as shown in Supplementary Table S1.

Flow Cytometric Analysis (FACS)
FACS was used to measure the cell cycles of the porcine GCs. GCs were digested by trypsin and the collected cells were twice-rinsed with precooled PBS, then fixed and refrigerated in 70% ethanol for more than 24 h. The fixed cells were resuspended in 500 µL staining buffers containing PI (50 µg/mL) and RNaseA (1 mg/mL) after rinsing with PBS. Subsequently, after 30 min incubation in the dark, BD-LSR flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) was performed to determine cell cycle kinetics.

Sequencing of miRNAs
GCs were collected after treatment with BDNF (100 ng/mL) for 24 h. The library operation and sequencing experiments were performed following standard Illumina procedures. The Small RNAs Sample Pre Kit (Illumina, San Diego, CA, USA) was used to structure libraries of small RNA sequencing and Illumina HiseQ2000/2500 was performed for sequencing. A sequence length of 1 × 50 bp was collected for bioinformatics analysis.

Bioinformatics Analysis
The Illumina HiSeq 4000 sequencing platform (Illumina, Inc., San Diego, CA, USA) was used to sequence RNA from pig GC specimens and construct cDNA libraries. Denatured libraries were converted into single-stranded DNA molecules and sequenced over 51 cycles on Illumina HiSeq, based on the manufacturer's directions. The analyses were performed with the help of Beijing Yuanyi Gene Technology Co., Ltd. (Beijing, China). After sequencing, Solexa CHASTITY [41] quality filtered reads were harvested as clean reads. Then, clean reads were screened for siRNA through BLAST and Rfam databases and classified siRNA were annotated or predicted as mature miRNA. The miRBase database was used for sequence alignment of mature miRNA. We calculated miRNA expression using the most readily available isoforms; miRBase was used to identify the mature miRNAs and all miRNA isoforms (5p or 3p). When miRNA profiles were differentially-expressed, comparisons between the two groups, fold changes, p-values and FDRs were calculated and used to identify significantly differentially-expressed miRNAs. log2 (fold change) > 1 or log2 (fold change) < −1 were selected for differentially-expressed miRNAs. The differences were statistically significant (p < 0.05) after R package comparison.

Data Analysis
Statistical analyses were performed with SPSS (Windows version 19). Data analysis between the two groups was done using the unpaired t-test. Three or more groups of data were evaluated for significant differences using ANOVA. Statistical significance was shown at * p < 0.05 and ** p < 0.01. Data were shown as mean ± standard deviation (SD).

Effects of BDNF on the Proliferation of Porcine GCs
The effects of BDNF on GC proliferation were determined. Figure 1A revealed that BDNF increased porcine GCs' viability in a dose-dependent manner (p < 0.05) and a concentration of 100 ng/mL was the most effective after 24 h. Unless otherwise noted, all BDNF described in our results were treated at 100 ng/mL for 24 h.  In addition, BDNF knockdown ( Figure 1B) in GCs significantly decreased GC viability ( Figure 1C). The proportion of S-phase cells in the si-BDNF group (7.55 ± 0.1%) was considerably reduced, compared with the si-NC group (9.47 ± 0.42%) ( Figure 1D). The levels of proliferation-related genes' (including CCND1, p21 and Bcl2) expression and apoptosis-related genes (Bax) in porcine GCs cells were evaluated. As shown in Figure 2E, after BDNF knockdown, the mRNA and protein expression levels of CCND1, p21 and Bcl2 were significantly reduced. On the contrary, the expression levels of Bax were considerably elevated ( Figure 1E).

The BDNF/TrkB Pathway Affects the Proliferation of Porcine GCs
The effects of BDNF on GC proliferation after K252α treatment were assessed. The results showed that the viability of porcine GCs (Figure 2A) and the distribution of cells in S-phase ( Figure 2B) were significantly decreased with K252α treatment alone. Moreover, K252α reduced BDNF-induced porcine GC viability, and the proportion of cells in Sphase, when K252a and BDNF treatment were undertaken together in GCs (Figure 2A, B).
The expression levels of genes connected with cell proliferation and apoptosis were measured. BDNF significantly increased mRNA (Figure 2(C-a)) and protein (Figure 2(Cb,C-c)) expression levels for CCND1, p21 and Bcl2. Additionally, the Bax mRNA and protein expression levels were reduced dramatically. Figure 2C shows that BDNF, in combination with K252a, weakened the effects of BDNF on the expression of the above genes in GCs.
Subsequently, to ascertain the consequences of these miRNAs on GC proliferation, GCs were transfected with the above 5 miRNA mimics for 48 h, respectively. Overexpression of miR-185 and miR-532 inhibited GC viability, while overexpression of miR-127 significantly reduced the viability of GCs and the proportion of cells in S-phase simultaneously ( Figure 3C). Therefore, we selected miR-127 as the key miRNA in BDNF-induced GC proliferation to continue our research.

The BDNF/TrkB Pathway Affects the Proliferation of Porcine GCs
The effects of BDNF on GC proliferation after K252α treatment were assessed. The results showed that the viability of porcine GCs (Figure 2A) and the distribution of cells in S-phase ( Figure 2B) were significantly decreased with K252α treatment alone. Moreover, K252α reduced BDNF-induced porcine GC viability, and the proportion of cells in S-phase, when K252a and BDNF treatment were undertaken together in GCs (Figure 2A,B).
The expression levels of genes connected with cell proliferation and apoptosis were measured. BDNF significantly increased mRNA ( Figure 2C-a) and protein ( Figure 2C-b,C-c) expression levels for CCND1, p21 and Bcl2. Additionally, the Bax mRNA and protein expression levels were reduced dramatically. Figure 2C shows that BDNF, in combination with K252a, weakened the effects of BDNF on the expression of the above genes in GCs.

BDNF Promotes GCs Proliferation through Increase of CCND1 by Downregulating miR-127
miRNA sequencing was performed. According to the sequencing results, 72 differentiallyexpressed miRNAs were detected, including 34 upregulated and 38 downregulated, as shown in Figure 3A-a,A-b (fold change > 2, p < 0.05). Additionally, 5 miRNAs (miR-185, miR-1273, miR-7047, miR-127 and miR-532) associated with cellular processes (Figure 3A-c) and cell proliferation signaling pathways ( Figure 3A-d) were significantly downregulated in GCs after BDNF treatment ( Figure 3B). miR-127 inhibited the expression of CCND1. The protein expression levels of CCND1 were compatible with RT-qPCR after miR-127 was overexpressed or inhibited in porcine GCs ( Figure 3D,E).  Subsequently, to ascertain the consequences of these miRNAs on GC proliferation, GCs were transfected with the above 5 miRNA mimics for 48 h, respectively. Overexpression of miR-185 and miR-532 inhibited GC viability, while overexpression of miR-127 significantly reduced the viability of GCs and the proportion of cells in S-phase simultaneously ( Figure 3C). Therefore, we selected miR-127 as the key miRNA in BDNF-induced GC proliferation to continue our research.

BDNF Promotes GC Proliferation via the ERK1/2 Signaling Pathway Mediated by miR-127
BDNF significantly decreased miR-127 expression ( Figure 3B) and increased CCND1 expression ( Figure 2C). To confirm the regulatory relationship between miR-127 and Animals 2023, 13, 1115 8 of 13 CCND1, the mRNA expression relationship was determined. As shown in Figure 3C, miR-127 inhibited the expression of CCND1. The protein expression levels of CCND1 were compatible with RT-qPCR after miR-127 was overexpressed or inhibited in porcine GCs ( Figure 3D,E).

BDNF Promotes GC Proliferation via the ERK1/2 Signaling Pathway Mediated by miR-127
To determine whether the MAPK-ERK1/2 pathway was entangled in proliferation by porcine GCs after BDNF treatment, the phosphorylation and total protein levels of ERK1/2 in porcine GCs were assessed. The results indicated the phosphorylation levels of ERK1/2 increased after BDNF supplementation for 15 (p < 0.05), 30 (p < 0.01) and 60 min (p < 0.05; Figure 4A). Moreover, GCs were treated with BDNF in combination with PD98059; the effects of BDNF on the viability of GCs (Figure 4B-a) and the distribution of cells in S-phase ( Figure 4B-b) were diminished by a blockade of the MAPK-ERK1/2 pathway. Secondly, the expression levels of CCND1 were assessed. Inhibition of ERK1/2 decreased the expression levels of CCND1 mRNAs and proteins ( Figure 4B-c-B-e). Thirdly, MAPK-ERK1/2 suppression had no impact on the expression levels of miR-127 ( Figure 4B-f), but the phosphorylation of ERK1/2 was stimulated after transfection of miR-127 inhibitor with GCs. Then, after treatment with BDNF, the expression level of p-ERK1/2 was further increased ( Figure 4C).

Discussion
In ovaries, BDNF is mainly presented in follicular granulosa cells and oocytes [16], while BDNF and its receptor TrkB are mainly expressed in granulosa cells and membrane cells of porcine follicles [42]. In this study, the expression levels of BDNF and trkB in GCs were verified by immunofluorescence (Supplementary Figure S1). BDNF regulates early follicle development in various mammals and directly affects ovulation, as shown in previous studies [10,17]. BDNF has also been associated with different causes of infertility during in vitro fertilization [12,[43][44][45]. Additionally, BDNF regulates the maturation of cytoplasmic and nuclear in porcine oocytes by paracrine and/or autocrine signaling systems and promotes potential embryo growth following in vitro fertilization and somatic cell nuclear transfer [10]. Thus, elucidating the transcriptional mechanisms regulated by BDNF in GCs will be beneficial for improving the IVM of porcine oocytes.
Transfection of specific siRNA interfering with BDNF expression in cells will be beneficial to studies of the role of BDNF in the proliferation of porcine GCs. With the extension of transfection time, the interference efficiency of siRNA was enhanced, but further inhibition of GCs proliferation had no significant effect. These findings suggested that the continuous decreases in the secretion of BDNF in porcine GCs did not inhibit cell proliferation at all times. miRNAs are small noncoding RNAs. They play a vital role in hormone-induced ovarian development [46,47]. In previous studies, the primary role of miR-127 was described as a tumor inhibitor, involved in a series of cellular processes-for instance proliferation, senescence, migration and invasion [48][49][50]. Moreover, miR-127 mediated the differentiation of mouse embryonic endoderms and promoted placental development [38,51]. However, the potential functions of miR-127 in porcine reproduction remain ambiguous. To our knowledge, this study revealed the expression of miR-127 in porcine GCs for the first time, and demonstrated that miR-127 was involved in porcine granulosa cell proliferation as a negative regulator.
Cell proliferation and differentiation are regulated by the progression of the cell cycle. This progression is controlled by cyclins and Cdks complexes [52]. Cyclin D1 (CCND1), a member of the cyclin family, promotes the transition of cells from G1 phase to S phase by interacting with CDK4 and CDK6 [53][54][55][56][57]. CCND1 is also a key regulator of cell proliferation [58]. Due to the inhibition of miR-127 on the proportion of cells in S-phase, the regulatory relationship between miR-127 and CCND1 was examined. The overexpression and knockdown of miR-127 showed that miR-127 significantly negatively regulated CCND1. It also revealed the regulatory pathway, in which BDNF downregulated miR-127 to promote CCND1 expression during porcine GC cell proliferation.
MAPK-mediated signal transduction is a key factor affecting cell fate processes [59]. As the core module of the MAPK signal cascade, the MAPK-ERK1/2 signaling pathway is highly conservative [60]. The extracellular signals are transmitted into the nucleus by ERK1/2 and trigger modifications in the expression of certain proteins in cells [61]. MAPK-ERK12 is also connected with several cellular activities, comprising differentiation, proliferation, apoptosis, transcription and adhesion [62][63][64][65][66][67]. Hence, it was necessary to examine this intracellular signaling pathway to further substantiate the molecular mechanisms underlying BDNF-induced cell proliferation and increased CCND1 expression in GCs. According to our data, phosphorylation of ERK1/2 was increased by BDNF, and porcine GC proliferation was inhibited by blockades of TrkB or ERK1/2. Thus, we concluded that BDNF-induced cell proliferation also depended on MAPK-ERK1/2 signaling activation and that BDNF promoted GC proliferation by regulating CCND1 through MAPK-ERK1/2 signaling cascade. A study on the promotion of bovine GC proliferation by the BDNF-MAPK-ERK1/2 signaling pathway was previously reported [15]. Notably, however, miRNAs (especially miR-127) were found to be involved in the transduction of the BDNF-MAPK-ERK1/2 signaling pathway in this study. Downregulating miR-127 facilitated the stimulation of the MAPK-ERK1/2 signaling pathway. This result suggested that CCND1 upregulation by BDNF indirectly actuated the MAPK-ERK1/2 pathway by downregulating miR-127. These findings provided novel insights for future studies on the function of BDNF and revealed the molecular mechanisms underlying mammalian follicle development. In addition, these findings could provide a new target for the treatment of follicular dysplasia, such as PCOS.

Conclusions
In conclusion, we determined that BDNF stimulated GC proliferation by increasing CCND1 expression through miR-127 downregulation and MAPK-ERK1/2 pathway activation ( Figure 5). Notably, BDNF stimulated the MAPK-ERK1/2 pathway directly and indirectly by downregulating miR-127. Our current findings provided insights that could aid in efforts to effectively promote porcine oocyte maturation and improve embryo production efficiency in vitro. 023, 13, x FOR PEER REVIEW 12 of MAPK-ERK1/2 signaling pathway in this study. Downregulating miR-127 facilitated t stimulation of the MAPK-ERK1/2 signaling pathway. This result suggested that CCND upregulation by BDNF indirectly actuated the MAPK-ERK1/2 pathway by downregul ing miR-127. These findings provided novel insights for future studies on the function BDNF and revealed the molecular mechanisms underlying mammalian follicle develo ment. In addition, these findings could provide a new target for the treatment of follicul dysplasia, such as PCOS.

Conclusions
In conclusion, we determined that BDNF stimulated GC proliferation by increasi CCND1 expression through miR-127 downregulation and MAPK-ERK1/2 pathway ac vation ( Figure 5). Notably, BDNF stimulated the MAPK-ERK1/2 pathway directly and i directly by downregulating miR-127. Our current findings provided insights that cou aid in efforts to effectively promote porcine oocyte maturation and improve embryo pr duction efficiency in vitro.  Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani13061115/s1, Figure S1: Porcine follicular granulosa cells identification and the expression of BDNF and TrkB in GCs; Table S1: Primers used for qRT-PCR.