Cell culture-related chemicals and media were purchased from Life Technologies (Gaithersburg, MD, USA) unless otherwise stated. All animal treatment complied with a protocol that was approved by the Institutional Animal Care and Use Committee of Jilin University (Number of permit: 201812061).

PFFs were derived from whole conceptuses of Large White pigs at day 30 of pregnancy [15]. Briefly, the heads and internal organs were removed from the conceptuses and washed three times with Ca²⁺ and Mg²⁺ free phosphate-buffered saline supplemented with 1% penicillin and streptomycin. Then, the remnants were minced and digested with 0.25% trypsin-ethylenediaminetetraacetic acid. Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) was added to the medium to inactivate the enzyme. The suspended cells were centrifugated (1,000×g) for 5 min and seeded into plastic culture dishes (Corning, New York, USA). The cells were cultured in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 1% nonessential amino acids, and 10 mg/mL penicillin–streptomycin in a humidified atmosphere of 5% CO₂ at 39°C. Unattached cells were removed and attached cells were further cultured until confluency. PFFs were stored in liquid nitrogen with a freezing medium, which containing 80% DMEM, 10% dimethyl sulfoxide (Sigma, St. Louis, MO, USA), and 10% FBS.

The genomic DNA was extracted from PFFs according to the manufacturer’s introductions of Multisource Genomic DNA Miniprep Kit (Axygen, Shanghai, China). Total RNA was extracted from PFFs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA concentration for each sample was quantified by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). The cDNAs from 1,000 ng of total RNA were obtained by PrimeScript RT reagent kit (TaKaRa, Dalian, China) according to the manufacturer’s introductions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using TransStart Green qPCR SuperMix (Transgen Biotech, Beijing, China) according to the manufacturer’s introductions. The primers were synthesized by Genewiz (Genewiz, Jiangsu, China) (Table 1). Gene expression levels were normalized to the expression of β-actin using the comparative Ct (2^−ΔΔCt) method.

psiCHECK-2 reporter vectors were constructed as follows. The fragments (90 bp) of wild-type IGF1R 3′ UTR, mutant type IGF1R 3′ UTR, wild-type SRY-box 4 (SOX4) 3′ UTR and mutant type SOX4 3′ UTR were synthesized by Genewiz (China) and contained NotI and XhoI restriction sites. Wild-type IGF1R 3′ UTR and SOX4 3′ UTR contain putative miR-140 binding sites. Mutant type IGF1R 3′ UTR and SOX4 3′ UTR mutant miR-140 binding sites contained the mutation of AACCACT to ATCGAGT. The fragments were respectively ligated into the psiCHECK-2 vector (Promega, Guangzhou, China) at corresponding restriction sites to generate four kinds of psiCHECK-2 reporter vectors (WT-IGF1R, Mut-IGF1R, WT-SOX4, and Mut-SOX4).

pGL4.2 reporter vectors were constructed as follows. Wild-type IGF1R promoter fragments containing the binding site of SOX4 were amplified by PCR with primer WT, which contains a XhoI site (CTCGAG) and an EcoRV site (GATATC) as underlined in Table 1. Mutant-type IGF1R promoter fragments, whose binding site of SOX4 were mutated from CC AAAACAAGGGCGAA to CGATATCTACGCCCAT, were synthesized by Genewiz (Genewiz, China) and contained a XhoI site (CTCGAG) and an EcoRV site (GATATC) at each end, respectively. The products were obtained and cloned into corresponding sites of pGL4.20-basic vector (Promega, Madison, WI, USA) to generate pGL4.2 reporter vectors (WT-Promoter and Mut-Promoter).

SOX4 expression vectors (pcDNA3.1-SOX4) were constructed as follows. The coding sequence of SOX4 was amplified by PCR with cDNA synthesized from PFF with primer_SOX4-cds containing a XhoI site (CTCGAG) and an EcoRI site (GAATTC) as underlined in Table 1. The PCR product was cleaved and cloned into corresponding sites of the pcDNA3.1 vector.

All clones were subjected to sequencing analysis (Genewiz, China) after construction.

PFFs (5×10³) were seeded in each well of 96-well plates with growth medium without antibiotics at 1 d before transfection to reach a confluency of 80% at transfection. 100 nM of miR-140 mimic, miR-control, miR-140 inhibitor, inhibitor control, siRNA for SOX4 (si-SOX4), siRNA for IGF1R (si-IGF1R) or siRNA control (si-control) (GenePharma, Shanghai, China) (Table 2) were transfected into PFFs (100 μL medium), respectively, using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s protocol.

To investigate the target genes of miR-140, the PFFs (100 μL medium) were cotransfected with the dual-luciferase reporter vectors (180 ng of WT-IGF1R, Mut-IGF1R, WT-SOX4, or Mut-SOX4, respectively, and 20 ng of Renilla luciferase vector) and 100 nM of miR-140 mimic or miR-140 inhibitor. After 48 h transfection, cells were collected for luciferase activity assays with the Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer’s protocol.

To detect the effects of SOX4 on the transcriptional activity of IGF1R promoter, 200 ng of pcDNA3.1-SOX4 or pcDNA3.1-control were cotransfected with WT-Promoter or Mut-Promoter into PFFs (100 μL medium). 48 h after transfection, the luciferase activity was measured.

The 96-well plates were incubated at 39°C and proliferation of PFFs was assayed at 24, 48, and 72 h after transfection using a CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, CCK-8 solution was added in each well and incubated for 1 h at 37°C. The optical density was measured at 450 nm using Victor3TM multilabel plate counter (PerkinElmer, Waltham, MA, USA).

PFFs were thoroughly minced in lysis buffer contained 0.1 mM phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Shanghai, China). Lysates (25 μg) were separated on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and then electrotransferred to nitrocellulose membranes (Whatman, Maidstone, UK). Membranes were blocked for 2 h at room temperature with 5% nonfat dried milk solution and then immunoblotted overnight at 4°C with primary antibodies against SOX4, IGF1R, and β-actin (1:2,000, Bioworld Technology CO., Ltd., Nanjing, China). After washing, the membranes were probed with HRP-conjugated secondary antibodies (1:2,000, Bioworld Technology CO., Ltd., USA). The enhanced chemiluminescence plus Western blotting detection system (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) was used for protein detection.

Chromatin immunoprecipitation (ChIP) assay was performed according to the protocol of ChIP Assay Kit (Beyotime, Nanjing, China). In brief, cells were cross-linked with 37% formaldehyde for 10 min at room temperature. The reaction was stopped by 125 mM glycine for 5 min at room temperature. The cells were scraped and lysed in the SDS lysis buffer containing 1 mM PMSF. The lysate was sonicated and centrifugated. Then, the supernatant was immunoprecipitated overnight at 4°C with antibody anti-SOX4 (1:100, Cell Signaling Technology, Danvers, MA, USA). An equal mass of Normal Rabbit immunoglobulin G (1:1,000, Cell Signaling Technology, USA) was used as negative control. Following immunoprecipitation, the supernatant was transferred to protein A/G-agarose beads and rotated at 4°C for 45 min. The DNA was collected from beads and used as the template for real-time PCR using Primer_ChIP (Table 1). The relative quantity of DNA was determined by comparing the sample fluorescence to the fluorescence values measured from the total chromatin input dilution series.

The miR-140 targets were predicted by TargetScan (http://www.targetscan.org/vert_72/). Transcription factors binding sites were predicted with Jaspar database (http://jaspar.genereg.net/). Data were expressed as the mean±standard deviation from at least three independent experiments. Differences among groups were tested by one-way analysis of variance and p<0.05 was considered statistically significant. Comparisons between two groups were evaluated using least significant difference t-test.

To detect the effects of IGF1R on PFFs proliferation, si-IGF1R was transfected into PFFs to inhibit the expression of IGF1R. Western blotting showed that si-IGF1R effectively inhibited the protein expression level of IGF1R (Figure 1A). The CCK8 assay showed PFFs proliferation was effectively inhibited via knockdown of IGF1R (Figure 1B). In addition, miR-140 was predicted to bind with IGF1R 3′UTR (Figure 1C). miR-140 mimic or miR-140 inhibitor were used to overexpress or suppress the expression of miR-140 in PFFs, respectively (Figure 1D, E). To validate this prediction, WT-IGF1R or Mut-IGF1R was cotransfected with miR-140 mimic or miR-140 inhibitor into PFFs. The luciferase activity of WT-IGF1R was significantly suppressed in PFFs transfected with miR-140 mimic, while increased in PFFs transfected with miR-140 inhibitor (Figure 1F). However, the luciferase activity of Mut-IGF1R was not significantly changed in PFFs transfected with miR-140 mimic or miR-140 inhibitor (Figure 1G). IGF1R mRNA level and protein level were suppressed (or increased) in PFFs transfected with miR-140 mimic (or miR-140 inhibitor) (Figure 1H, I). These results suggested that IGF1R was a direct target gene of miR-140. Furthermore, IGF1R is a strong activator of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-Akt pathway. The evolutionarily ancient IGF1R-PI3K-AKT pathway plays a key role in regulating cell proliferation [16–18]. In our study, overexpression of miR-140 increased AKT phosphorylation (P-AKT) level and inhibition of miR-140 decreased P-AKT level (Figure 1J). Overexpression of miR-140 suppressed proliferation of PFFs (Figure 1K). In contrast, knockdown of miR-140 promoted proliferation of PFFs (Figure 1L). This suggested that PI3K-AKT pathway was associated with miR-140 inhibiting PFFs proliferation. Taken together, these results demonstrated that miR-140 was able to inhibit PFFs proliferation by targeting directly to IGF1R.

Meanwhile, SOX4 was predicted as a target gene of miR-140 (Figure 2A). This prediction was validated by the same way with validating IGF1R as a target gene of miR-140. To validate this prediction, WT-SOX4 or Mut-SOX4 was cotransfected with miR-140 mimic or miR-140 inhibitor into PFFs. The luciferase activity of WT-SOX4 was significantly suppressed in PFFs transfected with miR-140 mimic, while increased in PFFs transfected with miR-140 inhibitor (Figure 2B). However, the luciferase activity of Mut-SOX4 was not significantly changed (Figure 2C). Furthermore, SOX4 mRNA level and protein level were suppressed (or increased) in PFFs transfected with miR-140 mimic (or miR-140 inhibitor), respectively (Figure 2D, E).

Using bioinformatic prediction, IGF1R was predicted to be a potential target of transcription factor SOX4. To detect the effects of SOX4 on transcriptional activity of IGF1R promoter, pcDNA3.1-SOX4 was cotransfected with pGL4.2-WT or pGL4.2-Mut. The luciferase activity showed overexpression of SOX4 increased transcriptional activity of IGF1R promoter. However, the effect of SOX4 on IGF1R promoter was abrogated when binding site of SOX4 in IGF1R promoter was mutated (Figure 2F). Meanwhile, ChIP assay confirmed the SOX4 bind with IGF1R promoter (Figure 2G). These data suggested that transcription factor SOX4 bind to IGF1R promoter to improve transcriptional activity of IGF1R promoter. To investigate the effect of transcription factor SOX4 on miR-140 inhibiting IGF1R expression, miR-140 mimic was cotransfected into PFFs with pcDNA3.1-SOX4 or pcDNA3.1-control, respectively. Our results showed that SOX4 abrogated the effect of miR-140 on IGF1R expression and played a key role in miR-140 inhibiting PFFs proliferation.

Next we examined whether SOX4 and IGF1R were involved in the effect of miR-140 on PFFs proliferation. The expression level of SOX4 was suppressed by si-SOX4 in PFFs. Western blotting showed that si-SOX4 effectively inhibited the protein expression level of SOX4 (Figure 3A). miR-140 inhibitor was cotransfected with si-SOX4, si-IGF1R, or si-control into PFFs. CCK8 assays results showed that miR-140 inhibitor induced PFFs proliferation, which was abrogated by si-SOX4 and si-IGF1R (Figure 3B). So SOX4 and IGF1R reversed the effects of miR-140 on PFFs proliferation, which suggested that miR-140, promoted PFFs proliferation by targeting SOX4 and IGF1R.