Ssc-miR-92b-3p Regulates Porcine Trophoblast Cell Proliferation and Migration via the PFKM Gene

Embryo implantation, the pivotal stage of gestation, is fundamentally dependent on synchronous embryonic development and uterine receptivity. In the early gestation period, the uterus and conceptus secrete growth factors, cytokines, and hormones to promote implantation. Circulating exosomal miRNAs are potential indicators of normal or complicated gestation. Our previous study revealed that pregnant sows’ serum exosomes had upregulated miR-92b-3p expression compared to non-pregnant sows, and that the expression level progressively increased during early gestation. The present study’s findings indicate that, compared to the ninth day of the estrous cycle (C9), pregnant sows had upregulated miR-92b-3p expression in the endometrium and embryos during the implantation stage ranging from day 9 to day 15 of gestation. Additionally, our results demonstrate that miR-92b-3p promotes the proliferation and migration of Porcine Trophoblast Cells (PTr2). Dual-Luciferase Reporter (DLR) gene assay, real-time fluorescent quantitative PCR (RT-qPCR), and Western blotting (WB) confirmed the bioinformatics prediction that phosphofructokinase-M (PFKM) serves as a target gene of miR-92b-3p. Notably, interference of PFKM gene expression markedly promoted PTr2 proliferation and migration. Furthermore, mice with downregulated uterine miR-92b-3p expression had smaller rates of successful embryo implantation. In summary, miR-92b-3p putatively modulates embryo implantation by promoting PTr2 proliferation and migration via its target gene PFKM.


Introduction
Spontaneous embryonic loss is a long-standing major challenge impacting commercial swine production worldwide, with 20-45% of total embryonic losses occurring during gestation [1] and approximately 20-30% during the early stage of implantation (between days 12 and 30 of gestation) [2]. Around the 12th day of gestation, porcine embryos migrate from one uterine horn to the other to accommodate each other and successfully attach to the receptive uterine epithelium [3]. Steroid hormones [4] prepare the endometrium for embryo implantation, while growth factors, prostaglandins, and exosomes [5] facilitate specific dialogs.
During gestation, the placental tissue permits the maternal-fetal exchange of nutrients, oxygen, and waste products, provides a natural protective barrier for fetal growth and development, and produces essential hormones and growth factors to support gestational

The Role of PFKM in PTr2
To investigate the functional effects of PFKM on PTr2, we designed three interfering fragments at different positions for the PFKM gene and transfected these three si-PFKM into PTr2. All three interfering fragments downregulated PFKM gene expression, but si-PFKM-1586 interfered with PFKM gene expression most significantly ( Figure 4A); significant interference was equally observed for protein levels ( Figure 4B,C). Therefore, si-PFKM-1586 was selected for subsequent functional validation investigations. CCK-8 ( Figure 4F) and Edu ( Figure 4D,E) experiments demonstrated that si-PFKM significantly promoted PTr2 proliferation, and cell-scratch assays indicated that interference of PFKM expression significantly promoted PTr2 migration ( Figure 4G,H).

The Role of PFKM in PTr2
To investigate the functional effects of PFKM on PTr2, we designed three interfering fragments at different positions for the PFKM gene and transfected these three si-PFKM into PTr2. All three interfering fragments downregulated PFKM gene expression, but si-PFKM-1586 interfered with PFKM gene expression most significantly ( Figure 4A); significant interference was equally observed for protein levels ( Figure 4B,C). Therefore, si-PFKM-1586 was selected for subsequent functional validation investigations. CCK-8 (Figure 4F) and Edu ( Figure 4D,E) experiments demonstrated that si-PFKM significantly promoted PTr2 proliferation, and cell-scratch assays indicated that interference of PFKM expression significantly promoted PTr2 migration ( Figure 4G,H).

Si-PFKM Rescued the Influence of miR-92b-3p Antagomir in PTr2
Previous experiments revealed that miR-92b-3p targets and regulates the PFKM gene. To verify whether ssc-miR-92b-3p affects PTr2 proliferation and migration by targeting PFKM, si-PFKM was transfected into PTr2 to rescue the effect of miR-92b-3p antagomir on PTr2. The co-transfected miR-inhibitor NC and si-NC were used as the control group, and the results indicated that the groups co-transfected with miR-92b-3p antagomir and si-NC had inhibited cell proliferation ( Figure 5A-C) and migration ( Figure 5D,E) compared with the control group. Co-transfection with miR-inhibitor NC and si-PFKM-1586 promoted cell proliferation and migration, while for co-transfection with miR-92b-3p antagomir and si-PFKM-1586, the difference was not significant compared with the control group.
most potent interference and was therefore used in all subsequent experiments. (B,C) Protein blot analysis revealed that si-PFKM-1586 significantly interferes with PFKM protein expression. (D-F) CCK-8 and Edu results showed that interfering with the PFKM gene promotes PTr2 proliferation. (G,H) Interfering with PFKM gene expression promotes PTr2 migration. Data are expressed as the means ± SEM of three experiments. (* p < 0.05; ** p < 0.01).

Si-PFKM Rescued the Influence of miR-92b-3p Antagomir in PTr2
Previous experiments revealed that miR-92b-3p targets and regulates the PFKM gene. To verify whether ssc-miR-92b-3p affects PTr2 proliferation and migration by targeting PFKM, si-PFKM was transfected into PTr2 to rescue the effect of miR-92b-3p antagomir on PTr2. The co-transfected miR-inhibitor NC and si-NC were used as the control group, and the results indicated that the groups co-transfected with miR-92b-3p antagomir and si-NC had inhibited cell proliferation ( Figure 5A-C) and migration ( Figure 5D,E) compared with the control group. Co-transfection with miR-inhibitor NC and si-PFKM-1586 promoted cell proliferation and migration, while for co-transfection with miR-92b-3p antagomir and si-PFKM-1586, the difference was not significant compared with the control group.

Inhibition of miR-92b-3p Hindered Mouse Embryo Implantation in a Mouse Model
We further validated the role of miR-92b-3p in embryo attachment by utilizing an in vivo mouse model. On day 3 of gestation, miR-92b-3p antagomir was injected into one horn of the mouse uterus, and DEPC water was injected into the other horn of the uterus as a control. On day 7 of gestation, the mice were dissected and observed for embryo attachment; the number of embryos attached on the miR-92b-3p antagomir injection side of the uterus was significantly smaller than that in the control group on the other side ( Figure 6A,B).

Inhibition of miR-92b-3p Hindered Mouse Embryo Implantation in a Mouse Model
We further validated the role of miR-92b-3p in embryo attachment by utilizing an in vivo mouse model. On day 3 of gestation, miR-92b-3p antagomir was injected into one horn of the mouse uterus, and DEPC water was injected into the other horn of the uterus as a control. On day 7 of gestation, the mice were dissected and observed for embryo attachment; the number of embryos attached on the miR-92b-3p antagomir injection side of the uterus was significantly smaller than that in the control group on the other side ( Figure  6A,B).

Discussion
Embryo implantation is essential to the gestational process and strongly correlates with litter weight and size [28,29]. Successful embryo implantation partially depends on the information exchanged between embryos and the pregnant sow [30]. Compelling evidence indicates that miRNAs are involved in the regulation of embryonic implantation and development [18,[31][32][33]. In our previous study, miR-92b-3p expression was upregulated in the serum of pregnant sows, and the expression level was continuously upregulated during the process of embryo implantation [17]; however, the mechanism underlying miR-92b-3p modulation during early gestation remained elusive. Therefore, this study attempts to elucidate the mechanism of miR-92b-3p involvement in embryo implantation.
MiRNAs are confirmed to be vital for embryo implantation. Zhou et al. found that exosome concentration increased in the plasma of pregnant sows and that, in the fatter ones, plasma exosomes contained a high level of miR-221 that inhibited endothelial cell migration and angiogenesis by acting on the ANGPTL2 gene [31]. Upregulated miR-92b-3p expression reportedly supports cancer cell proliferation and migration in various human cancers [34,35]. On the other hand, miR-92b-3p is significantly enriched in human embryonic stem cells and is strongly associated with the regulation of embryonic stem cell proliferation [36]. In the present study, miR-92b-3p expression was significantly

Discussion
Embryo implantation is essential to the gestational process and strongly correlates with litter weight and size [28,29]. Successful embryo implantation partially depends on the information exchanged between embryos and the pregnant sow [30]. Compelling evidence indicates that miRNAs are involved in the regulation of embryonic implantation and development [18,[31][32][33]. In our previous study, miR-92b-3p expression was upregulated in the serum of pregnant sows, and the expression level was continuously upregulated during the process of embryo implantation [17]; however, the mechanism underlying miR-92b-3p modulation during early gestation remained elusive. Therefore, this study attempts to elucidate the mechanism of miR-92b-3p involvement in embryo implantation.
MiRNAs are confirmed to be vital for embryo implantation. Zhou et al. found that exosome concentration increased in the plasma of pregnant sows and that, in the fatter ones, plasma exosomes contained a high level of miR-221 that inhibited endothelial cell migration and angiogenesis by acting on the ANGPTL2 gene [31]. Upregulated miR-92b-3p expression reportedly supports cancer cell proliferation and migration in various human cancers [34,35]. On the other hand, miR-92b-3p is significantly enriched in human embryonic stem cells and is strongly associated with the regulation of embryonic stem cell proliferation [36]. In the present study, miR-92b-3p expression was significantly upregulated in the porcine endometrium and embryonic tissue and was significantly elevated in the endometrial tissue of pregnant sows compared with that of non-pregnant sows, with a progressive increase throughout the gestation period. Hence, miR-92b-3p is strongly correlated with embryo implantation. The results demonstrated that miR-92b-3p promotes PTr2 proliferation and migration. On the 12th day of gestation, porcine embryos undergo a swift transition from spherical to filamentous forms, and the specific sites of implantation are determined. During this process of morphological and functional alterations, embryo death reaches up to 30-40% due to epiboly failure and insufficient contact between the embryonic trophectoderm and the epithelial cells on the endothelium [37]. This implies that miR-92b-3p expression is upregulated during gestation to promote PTr2 proliferation and migration, thereby facilitating embryo implantation. After injection of a miR-92b-3p inhibitor into one side of the uterine horns of mice, the antago-miR-92b-3p group had significantly fewer implanted embryos than the control group.
In the preimplantation period, miRNAs modulate the implantation and growth of embryos by regulating target genes [31,32,38]. PFKM is a kinase that catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate, as well as the rate-limiting step of glycolysis [39]. During embryo implantation, the uterine cavity contains higher concentrations of glucose and fructose, two major energy substrates that fuel fetal growth and development [40]. In this study, we utilized three online tools, miRanda, RNAhybrid, and Targetscan, to analyze the suitability of PFKM as a potential target gene for ssc-miR-92b-3p, while the DLR gene assay, RT-qPCR, and WB analysis demonstrated the inhibitory effects of miR-92b-3p on PFKM mRNA and protein expression. Studies have reported that, under hypoxic conditions, PFKM Ser529 achieves glycosylation through post-translation modification of acetylglucosamine to inhibit PFKM activity and reroutes glucose flux away from the pentose phosphate pathway; this results in an increased Dribose 5-phosphate level supportive of rapid cancer cell growth through DNA and protein synthesis [41]. However, the role of PFKM in PTr2 is still unclear. In this study, subsequent to PTr2 treatment with small molecule inhibitors of PFKM (si-PFKM), it was observed that suppressing PFKM significantly promoted the proliferation and migration of PTr2. Successful embryo implantation and placental development require the ability of embryonic trophoblast cells to proliferate and migrate [42]. Furthermore, we demonstrated that interfering with PFKM expression reversed the effect of miR-92b-3p antagomir on PTr2 by conducting co-transfection experiments. These findings suggest that ssc-miR-92b-3p regulates the proliferation and migration of PTr2 by targeting PFKM.
In conclusion, miR-92b-3p expression is progressively upregulated in the endometrium and embryos from day 9 to day 15 of gestation, and the endometrial expression level is consistently higher in pregnant sows than on the ninth day of the estrous cycle (C9). Moreover, miR-92b-3p regulates PTr2 proliferation and migration via the PFKM gene ( Figure 7); thus, inhibiting miR-92b-3p expression adversely impacts mouse embryo implantation.

Sample Tissue Collection
In this study, healthy Large Yorkshire sows of similar age and genetic background were selected and treated to attain simultaneous estrus. They underwent artificial insemination after estrus was observed (recorded as day 0); one group was subjected to sham

Sample Tissue Collection
In this study, healthy Large Yorkshire sows of similar age and genetic background were selected and treated to attain simultaneous estrus. They underwent artificial insemination after estrus was observed (recorded as day 0); one group was subjected to sham insemination (non-pregnant group), while the other group was subjected to frozen fresh semen insemination. Day 1 post-insemination was identified as the first day of pregnancy. Uteri were harvested on day 9 of estrus and days 9, 12, and 15 of gestation, respectively. The uterus was incised longitudinally on its inner side, and endometrial tissue was collected. It was immediately snap-frozen in liquid nitrogen and transferred to a −80 • C refrigerator for storage. All procedures involving animals were conducted under a protocol approved by the Ethics Committees of the Laboratory Animal Center of South China Agricultural University (approval No. SYXK-2019-0136).

FISH Assay
The FISH method was employed to detect the localization and relative quantification of miR-92b-3p in endometrial and embryonic tissues. The probe sequence of miR-92b-3p was 5 -UAUUGCACUCGUCCCGGCCUCC-3 Cy3 with red labeling. The basic procedure was as follows: the tissues were washed and immediately placed into 4% paraformaldehyde (Servicebio, Wuhan, China) for more than 12 h. After the fixation was completed, the sections were dehydrated by gradient alcohol embedded in wax, and then incubated for 2 h at 62 • C. The sections were dewaxed and dehydrated in turn. After fixation, the sections were dehydrated using gradient alcohol, embedded in wax, and subsequently incubated for 2 h at 62 • C. The sections were dewaxed and dehydrated in turn. After natural drying, the sections were boiled in the repair solution for 10-15 min, cooled naturally, digested with proteinase K (20 µg/mL) dropwise at 37 • C, rinsed with pure water, and washed thrice with PBS (Servicebio, Wuhan, China) for 5 min each time. Pre-hybridization solution was added dropwise to the sections, followed by incubation at 37 • C for 1 h. The prehybridization solution was removed, and the hybridization solution containing the probe was added dropwise for overnight hybridization in a 37 • C thermostat. After incubation, the hybridization solution was washed off, and the sections were washed with 2 × SSC (Servicebio, Wuhan, China) at 37 • C for 10 min, then with 1 × SSC at 37 • C for 2 × 5 min, and lastly with 0.5 × SSC at room temperature for 10 min. The sections were incubated for 8 min in a light-proof environment following the dropwise addition of DAPI (Servicebio, Wuhan, China) staining solution, rinsed, then sealed with the dropwise addition of antifluorescence quenching sealer (Servicebio, Wuhan, China). The sections were placed under a fluorescent microscope (Nikon, Tokyo, Japan) for visualization and image acquisition.

Cell Culture and Transfection
PTr2 cells were provided by Dr. Yulong Yin from the Institute of Subtropical Agriculture, Chinese Academy of Sciences. PTr2 cells were isolated form porcine filamentous embryos at day 12 of pregnancy and have been extensively characterized in previous published paper [43]. PTr2 were cultured in DMEM/F12 basal medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 0.5% insulin (YEASEN, Shanghai, China), and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37 • C with 5% CO2 in a constant-temperature cell incubator. When the cell density reached 80%, 0.25% Trypsin (Gibco, Grand Island, NY, USA) was utilized to digest cells and plates were seeded with PTr2 in preparation for subsequent experiments. PK-15 cells were cultured in DMEM/F12 basal medium (Gibco, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA), and placed in a constanttemperature cell incubator maintained at 37 • C with 5% CO2. PTr2 cells in the logarithmic growth phase were inoculated into 6-well plates and cultured overnight until the cell density reached 50%-70%. miRNA mimics/antagomir/si-RNA/NC (GenePharma, Suzhou, China) were transfected with Lipofectamine 3000 Transfection Reagent (ThermoFisher, Waltham, MA, USA), following which they were cultured for 6-8 h in a cell incubator. The culture solution was changed after 6-8 h for subsequent experiments.

RNA Extraction and RT-qPCR
TRIzol (Ambion, Austin, TX, USA) was utilized to extract cell RNA, and RNA concentration was determined using a spectrophotometer. RNA was then reverse-transcribed into cDNA using a reverse transcription kit (TaKaRa, Kyoto, Japan). To establish the relative gene expression levels, a qPCR reaction system was implemented via a fluorescent quantitative PCR instrument. ssc-miR-92b-3p and gene expression levels were calculated according to the 2 −∆∆Ct formula using U6 and β-actin as internal controls.

Cell Counting Kit-8 (CCK-8) Assay
CCK-8 (YENSEN, Shanghai, China) was employed to detect the proliferation of cells in various periods. A 10 µL measure of CCK-8 solution was added to each well of a 96-well plate, and the plate was placed in a 37 • C cell incubator for 2 h. Each well's optical density (OD) at 450 nm was measured using a multifunctional enzyme marker.

EdU Assay
The BeyoClickTMEdu-555 Cell Proliferation Assay Kit (Beyotime, Shanghai, China) was utilized to evaluate cell proliferation status at various time points post-transfection. The prepared 2× Edu working solution was added to the cell culture plate (96-well plate) at 48 h after transfection, followed by incubation at 37 • C for 2 h in a 5% CO 2 cell culture incubator. Once Edu labeling was complete, the culture solution was removed, cells were washed twice with DPBS (Servicebio, Wuhan, China) for 3 min each time, 4% paraformaldehyde (Servicebio, Wuhan, China) was used for fixation at room temperature for 15 min, followed by washing with DPBS thrice, and cells were permeabilized for 10 min using a cell permeabilization solution. This was followed by washing twice with DPBS, the addition of 70 µL of prepared Click reaction solution to each well (96-well plate), and incubation for 35 min at room temperature and without light. The Click reaction solution was aspirated and discarded, and cells were washed twice with DPBS and incubated with DAPI (Servicebio, Wuhan, China) for 8 min at room temperature to stain cell nuclei. Subsequently, the DAPI solution was aspirated and discarded, and cells were washed twice with DPBS. Fluorescence detection was then performed.

Cell Wound-Healing Assay
After cells were transfected for 48 h, linear scratches were made vertically on 6-well plates with a large pipette tip, and the cell culture medium was changed to serum-free medium. Cell migration and scratch healing were visualized and photographed at 0 and 12 h. Three randomly selected visual fields were used to calculate the healing area using Image J software to obtain the cell migration rate.

Western Blotting
RIPA Lysis Buffer (CWBIO, Beijing, China) containing protease inhibitors was used to extract proteins. Samples were denatured by heating, proteins were separated via SDS-PAGE Gel (EpiZyme, Shanghai, China) electrophoresis and transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany), sealed with 5% skimmed milk (BD, Franklin Lakes, NJ, USA) for 2 h in a shaker at room temperature, washed with TBST, and incubated overnight at 4 • C with PFKM (Abcam, ab154804, 1:1000, Cambridge, UK) and Tubulin (Servicebio, GB11017, 1:1000, Wuhan, China) antibodies. Next, the samples were washed thrice with TBST and incubated with the secondary antibody (HRP-conjugated Goat Anti-Rabbit IgG, BBI Life Sciences D110058, 1:10,000, Shanghai, China) at 37 • C for 1 h. The target protein bands were visualized using chemiluminescent solution (CWBIO, Beijing, China), and the relative protein levels were quantified via Image J software.

Dual-Luciferase Reporter Gene Assay
Using PITA, miRanda, and RNAhybrid, the target gene of miR-92b-3p was predicted, the porcine PFKM gene's 3 -UTR binding site that binds to miR-92b-3p was cloned into the pmirGLO vector (Promega, USA), and the mutant plasmid was constructed by targeted mutagenesis. PK-15 cells were inoculated into cell culture plates at 1 × 104 per well, and miR-92b-3p-mimics and dual-luciferase reporter plasmids (pmirGLO-PFKM-WT, pmirGLO-PFKM-MUT) were transfected into PK-15 cells using Lipofectamine 3000 transfection reagent when cell confluence reached 60-80%. The solution was changed after 6 h of transfection. After 24 h of transfection, the treated cells were harvested and assessed for luciferase activity using the Dual-Luciferase Assay Kit (YENSEN, Shanghai, China).

Intrauterine Injection in Mice
We selected 6-8-week-old ICR mice as an experimental model, and after mating, vaginal plugs were observed on the first day of gestation. At day 3 of gestation, pregnant mice were anesthetized, and 10 µL of 20 µM miR-92b-3p antagomir solution and 10 µL of DEPC water (GeneStar, Nanjing, China) were injected into both uterine horns; the mice were sutured and placed in a 37 • C environment for awakening. Mice were dissected on day 7 of gestation to observe the embryo attachment rate.

Statistical Analysis
All data were expressed as the mean ± standard deviation of at least three independent experiments, and the data were analyzed and plotted using the GraphPad Prism 8.0 software. T-test for independent samples was used for comparisons between two groups, with p < 0.05 indicating statistically significant differences and p < 0.01 indicating highly statistically significant differences.