miR-126 Controls the Apoptosis and Proliferation of Immature Porcine Sertoli Cells by Targeting the PIK3R2 Gene through the PI3K/AKT Signaling Pathway

Simple Summary MicroRNAs (miRNAs) have been reported with potential regulatory roles in spermatogenesis. In the present study, we demonstrated that miR-126 can stimulate cell proliferation and restrain the apoptosis of immature porcine Sertoli cells by targeting the PIK3R2 gene. Through this process, miR-126 further activates the PI3K/AKT signaling pathway. These results indicated that miR-126, PIK3R2, and the PI3K/AKT signaling pathway might play pivotal regulatory roles in porcine spermatogenesis by deciding the destiny of immature Sertoli cells. Abstract The quantity of Sertoli cells in the adult testis decides the daily gamete formation, and accumulating evidence indicates that epigenetic factors regulate the proliferation of Sertoli cells. Research on the function and regulatory mechanism of microRNAs (miRNAs) in Sertoli cells has not been comprehensive yet, especially on domestic animals. In this article, we report that miR-126 controls the proliferation and apoptosis of immature porcine Sertoli cells based on previous studies. Our results confirmed that miR-126 elevation promotes cell cycle progression, cell proliferation and represses cell apoptosis; on the contrary, the inhibitory effects of miR-126 result in the opposite. The phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) gene, a member of the PI3K family, was verified as a direct target of miR-126 using the dual-luciferase reporter analysis. miR-126 negatively regulated the mRNA and protein expression level of PIK3R2 in immature porcine Sertoli cells. siRNA-induced PIK3R2 inhibition caused similar effects as miR-126 overexpression and eliminated the influences of miR-126 knockdown in immature porcine Sertoli cells. In addition, both miR-126 overexpression and PIK3R2 inhibition elevated the phosphorylation of PI3K and AKT, whereas the miR-126 knockdown demonstrated the contrary result. In short, miR-126 controls the proliferation and apoptosis of immature porcine Sertoli cells by targeting the PIK3R2 gene through the PI3K/AKT signaling pathway. The research supplies a theoretical and practical foundation for exploring the functional parts of miR-126 in swine sperm by defining the destiny of immature Sertoli cells.


Introduction
Spermatogenesis is a continuous and productive procedure that generates male gametes. Sertoli cells, the only somatic cell in the seminiferous tubules, play the crucial role of maintaining and regulating the normal process of spermatogenesis by producing an impermeable immune barrier (blood-testis barrier), providing multiple nutritional factors, and maintaining immune tolerance [1]. However, the number of mature Sertoli cells of adult testes decides the gamete manufacture, as each Sertoli cell regulates a certain amount of germ cells [2]. Sertoli cells only undergo proliferation and apoptosis during the fetal, collected and measured for luciferase activity assay with the Dual-Glo ®Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. Firefly luciferase (Promega, Madison, WI, USA) activities were normalized to the Renilla luciferase (Promega, Madison, WI, USA) activities.

Real Time qPCR
Total RNA was extracted from Sertoli cells after transfection for 48 h, using TRIzol Reagent (Invitrogen, USA). The quality and concentration of RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Primers used in the RT-qPCR (Table 1) were designed based on Primer-BLAST (https://www.ncbi.nlm.nih.gov/ tools/primeblast/idex.cgi?LINK_LOC=BlastHome), and synthesized by Sango Biotech (Shanghai, China). The total RNA was reverse transcribed for each sample, using a Primescript First Strand cDNA Synthesis Kit (Takara Bio, Beijing, China). The qPCR amplifications were performed on a Thermo Scientific PIKO REAL 96 real-time PCR System using a SYBR Green kit (TaKaRa, China). Individual qPCR samples were performed in triplicates. The relative expression levels of miR-126 and PIK3R2 genes were normalized to U6 and pig-TBP genes and calculated using the 2 − Ct value.

Statistical Analysis
All of the data are expressed as the means ± SEM from three independent replicates for each experiment. Data from the experiments were analyzed by t-test or one-way analysis of variance (ANOVA) followed by Duncan's multiple range test comparisons using SPSS 17.0 software; p values < 0.05 or p values < 0.01 were indicated statistically significant.

Statistical Analysis
All of the data are expressed as the means ± SEM from three independent replicates for each experiment. Data from the experiments were analyzed by t-test or one-way analysis of variance (ANOVA) followed by Duncan's multiple range test comparisons using SPSS 17.0 software; p values < 0.05 or p values < 0.01 were indicated statistically significant.

miR-126 Promotes the Cell Cycle Progression and Proliferation of Immature Porcine Sertoli Cells
In our previous study, the effect of 60 miRNAs on immature porcine Sertoli cell proliferation was validated by the EdU-based high-content screening assay. miR-126 was identified as a candidate to promote immature porcine Sertoli cell proliferation. In order to inquire into the effect of miR-126 on cell cycle progression, immature porcine Sertoli cells were transfected with miR-126 mimic or inhibitor. The results demonstrated that miR-126 overexpression significantly decreased the percentage of cells in the G1 phase (p < 0.05) and increased the S phase cell population (p < 0.05) ( Figure 1A). Conversely, more miR-126 inhibitor-transfected cells were detected in the G1 phase (p < 0.05), whereas fewer cells were found in the G2 phase compared with the inhibitor NC-transfected cell group (p < 0.05) ( Figure 1C). In addition, miR-126 overexpression significantly elevated the relative mRNA expression level of cell cycle-related genes, c-MYC, CNNE1, and CDK4 (p < 0.05) ( Figure 1B), whereas miR-126 inhibition significantly decreased the relative mRNA expression levels of CCNE1, CCND1, and CDK4 (p < 0.05) ( Figure 1D). The CCK-8 detection revealed that miR-126 overexpression significantly increased cell proliferation from 48 to 72 h (p < 0.01) (Figure 2A), whereas knockdown of miR-126 significantly decreased the cell proliferate activity at 48 h (p < 0.05) and 72 h ( Figure 2C). Similarly, the EdU incorporation assay results indicated that miR-126 overexpression exhibited higher mitotic activity (p < 0.05), whereas the mitotic activity of the miR-126 inhibitor-transfected cells was significantly impeded when compared with the NC group (p < 0.01) ( Figure 2B,D). Figure 1. miR-126 promotes the cell cycle progression of immature porcine Sertoli cells. The immature porcine Sertoli cells were transfected with miR-126 mimic/mimic NC or miR-126 inhibitor/inhibitor NC respectively. (A,C) The effects of miR-126 overexpression (A) and miR-126 inhibition (C) on the distribution of G1, S and G2 phases in the cell cycle. Cell cycle phase was analyzed 24 h after transfection by a FACSCanto II Flow Cytometer (n = 3). (B,D) The mRNA levels of cell cyclerelated genes, c-MYC, CCNE1, CCND1 and CDK4, were detected in cells transfected with miR-126 mimic (B) or miR-126 inhibitor (D) using a qPCR assay (n = 3). The pig-TBP gene was used as the internal control. All data are shown as the mean ± SEM, * p < 0.05 and ** p < 0.01.
The CCK-8 detection revealed that miR-126 overexpression significantly increased cell proliferation from 48 to 72 h (p < 0.01) (Figure 2A), whereas knockdown of miR-126 significantly decreased the cell proliferate activity at 48 h (p < 0.05) and 72 h ( Figure 2C). Similarly, the EdU incorporation assay results indicated that miR-126 overexpression exhibited higher mitotic activity (p < 0.05), whereas the mitotic activity of the miR-126 inhibitor-transfected cells was significantly impeded when compared with the NC group (p < 0.01) ( Figure 2B,D).

miR-126 Inhibits Immature Porcine Sertoli Cell Apoptosis
We further examined the effect of miR-126 on apoptosis. The results from the Annexin V-FITC/PI staining demonstrated that miR-126 overexpression caused a lower late apoptosis rate (p < 0.05) ( Figure 3A), whereas miR-126 knockdown induced a higher late apoptosis rate than in the control group (p < 0.05) ( Figure 3D). In addition, the relative ATP level was dramatically elevated by miR-126 overexpression (p < 0.01), whereas it was significantly decreased by miR-126 knockdown (p < 0.01) ( Figure 3B,E). miR-126 elevation also promoted BCL2 protein expression and suppressed the protein expression of BAX and Caspase-3 (p < 0.01) ( Figure 3C), whereas miR-126 knockdown resulted in the opposite effect ( Figure 3F).

miR-126 Inhibits Immature Porcine Sertoli Cell Apoptosis
We further examined the effect of miR-126 on apoptosis. The results from the Annexin V-FITC/PI staining demonstrated that miR-126 overexpression caused a lower late apoptosis rate (p < 0.05) ( Figure 3A), whereas miR-126 knockdown induced a higher late apoptosis rate than in the control group (p < 0.05) ( Figure 3D). In addition, the relative ATP level was dramatically elevated by miR-126 overexpression (p < 0.01), whereas it was significantly decreased by miR-126 knockdown (p < 0.01) ( Figure 3B,E). miR-126 elevation also promoted BCL2 protein expression and suppressed the protein expression of BAX and Caspase-3 (p < 0.01) ( Figure 3C), whereas miR-126 knockdown resulted in the opposite effect ( Figure 3F).

miR-126 Directly Targets the PIK3R2 Gene
To uncover the mechanism of miR-126 on cell proliferation and apoptosis, we predicted the potential target genes of miR-126 using the online software TargetScan. An evolutionarily conserved binding site of miR-126 was found in the 3 -UTR of the PIK3R2 gene ( Figure 4A). Then, the psiCHECK-2 vectors (PIK3R2-wt or PIK3R2-mt) were cotransfected into HEK293T cells with miR-126 mimic or mimic NC. The results show that the relative luciferase activity was significantly decreased in the cell group co-transfected with PIK3R2-wt and miR-126 mimic than in the other three co-transfection groups (p < 0.05) ( Figure 4B). In accordance with the luciferase activity results, miR-126 overexpression significantly downregulated the mRNA and protein expression of the PIK3R2 gene, whereas miR-126 knockdown elevated its expression in immature porcine Sertoli cells (p < 0.05) ( Figure 4C,D).

miR-126 Directly Targets the PIK3R2 Gene
To uncover the mechanism of miR-126 on cell proliferation and apoptosis, we predicted the potential target genes of miR-126 using the online software TargetScan. An evolutionarily conserved binding site of miR-126 was found in the 3′-UTR of the PIK3R2 gene ( Figure 4A). Then, the psiCHECK-2 vectors (PIK3R2-wt or PIK3R2-mt) were co-transfected into HEK293T cells with miR-126 mimic or mimic NC. The results show that the relative luciferase activity was significantly decreased in the cell group co-transfected with PIK3R2-wt and miR-126 mimic than in the other three co-transfection groups (p < 0.05) ( Figure 4B). In accordance with the luciferase activity results, miR-126 overexpression significantly downregulated the mRNA and protein expression of the PIK3R2 gene, whereas miR-126 knockdown elevated its expression in immature porcine Sertoli cells (p < 0.05) ( Figure 4C,D).

PIK3R2 Gene Deficiency Promotes Immature Porcine Sertoli Cell Proliferation and Inhibits Apoptosis
Based on these results, we assumed that miR-126 regulated the proliferation and apoptosis of immature porcine Sertoli cells, probably by targeting the PIK3R2 gene. Cell cycle analysis demonstrated that siRNA-induced PIK3R2 knockdown significantly de-

PIK3R2 Gene Deficiency Promotes Immature Porcine Sertoli Cell Proliferation and Inhibits Apoptosis
Based on these results, we assumed that miR-126 regulated the proliferation and apoptosis of immature porcine Sertoli cells, probably by targeting the PIK3R2 gene. Cell cycle analysis demonstrated that siRNA-induced PIK3R2 knockdown significantly decreased the percentage of cells in the G1 phase (p < 0.05) and increased the proportion of cells in the G2 phase (p < 0.01) ( Figure 5A). Similarly, silencing the PIK3R2 gene significantly upregulated the relative expression level of cell cycle-related genes c-MYC, CCNE1, CCND1, and CDK4 (p < 0.01) ( Figure 5B). In addition, the EdU incorporation assay showed that more EdU-positive cells were detected in the PIK3R2 siRNA-transfected cell group than in the control group (p < 0.01) ( Figure 5C). The CCK-8 assay indicated that PIK3R2 knockdown significantly promoted cell proliferation at 24 and 48 h compared with the control (p < 0.05) ( Figure 5D). PIK3R2 knockdown significantly increased the relative ATP level (p < 0.01) ( Figure 5E). Consistently, PIK3R2 knockdown significantly elevated BCL2 protein expression (p < 0.01), whereas it dramatically decreased the protein expression of BAX and Caspase-3 (p < 0.05) ( Figure 5F). The relative ATP levels were measured by an ELISA assay. (F) The protein expression of cell survival-related genes was measured using a Western blot assay. The β-actin gene was used as the internal control. Data are presented as the mean ± SEM, * p < 0.05 and ** p < 0.01.

PIK3R2 Knockdown Attenuated the Regulatory Roles of miR-126 Inhibition
The abovementioned results showed that PIK3R2 knockdown mimicked the regu tory roles of miR-126 overexpression in immature porcine Sertoli cells. Therefore, we f ther determined whether PIK3R2 knockdown could mediate the effects of miR-126. Th co-transfection treatments were conducted in this section, including mimic NC + siRN NC, miR-126 inhibitor + siRNA NC, and miR-126 inhibitor + PIK3R2 siRNA. The CCK assay showed that the inhibition of miR-126 resulting in lower cell proliferation from  D) The effects of PIK3R2 siRNA on cell proliferation was detected using EdU corporation (C) and CCK-8 (D) assays (n = 3). (E) The relative ATP levels were measured by an ELISA assay. (F) The protein expression of cell survival-related genes was measured using a Western blot assay. The β-actin gene was used as the internal control. Data are presented as the mean ± SEM, * p < 0.05 and ** p < 0.01.

PIK3R2 Knockdown Attenuated the Regulatory Roles of miR-126 Inhibition
The abovementioned results showed that PIK3R2 knockdown mimicked the regulatory roles of miR-126 overexpression in immature porcine Sertoli cells. Therefore, we further determined whether PIK3R2 knockdown could mediate the effects of miR-126. Three co-transfection treatments were conducted in this section, including mimic NC + siRNA NC, miR-126 inhibitor + siRNA NC, and miR-126 inhibitor + PIK3R2 siRNA. The CCK-8 assay showed that the inhibition of miR-126 resulting in lower cell proliferation from 24 to 72 h was restrained by the knockdown of the PIK3R2 gene ( Figure 6A). Detection using the EdU incorporation assay showed the same result as cell proliferation ( Figure 6B). In addition, the relative ATP level was down-regulated by miR-126 inhibition, whereas it was antagonized by PIK3R2 knockdown (Figure 6C). effects of PIK3R2 siRNA on cell proliferation was detected using EdU corporation (C) and CCK-8 (D) assays (n = 3). (E) The relative ATP levels were measured by an ELISA assay. (F) The protein expression of cell survival-related genes was measured using a Western blot assay. The β-actin gene was used as the internal control. Data are presented as the mean ± SEM, * p < 0.05 and ** p < 0.01.

PIK3R2 Knockdown Attenuated the Regulatory Roles of miR-126 Inhibition
The abovementioned results showed that PIK3R2 knockdown mimicked the regu tory roles of miR-126 overexpression in immature porcine Sertoli cells. Therefore, we fu ther determined whether PIK3R2 knockdown could mediate the effects of miR-126. Thr co-transfection treatments were conducted in this section, including mimic NC + siRN NC, miR-126 inhibitor + siRNA NC, and miR-126 inhibitor + PIK3R2 siRNA. The CCK assay showed that the inhibition of miR-126 resulting in lower cell proliferation from to 72 h was restrained by the knockdown of the PIK3R2 gene ( Figure 6A). Detection usi the EdU incorporation assay showed the same result as cell proliferation ( Figure 6B). addition, the relative ATP level was down-regulated by miR-126 inhibition, whereas was antagonized by PIK3R2 knockdown (Figure 6C).  3.6. miR-126 Promotes Immature Porcine Sertoli Cell Growth by Activating the PI3K/AKT Signaling Pathway PIK3R2 is a key protein of the PI3K/AKT signaling pathway, which was directly targeted and regulated by miR-126 in the present study. Therefore, we further explored whether miR-126 regulated the PI3K/AKT signaling pathway using a Western blot assay. Our results demonstrated that the phosphorylation of PI3K and AKT was significantly increased by miR-126 overexpression (p < 0.05) ( Figure 7A), whereas their phosphorylation was repressed by the knockdown of miR-126 (p < 0.05) ( Figure 7B). Similarly, siRNAinduced PIK3R2 inhibition significantly elevated the phosphorylation of p-PI3K and p-AKT, which was consistent with miR-126 overexpression (p < 0.05) ( Figure 7C). geted and regulated by miR-126 in the present study. Therefore, we further explored whether miR-126 regulated the PI3K/AKT signaling pathway using a Western blot assay. Our results demonstrated that the phosphorylation of PI3K and AKT was significantly increased by miR-126 overexpression (p < 0.05) ( Figure 7A), whereas their phosphorylation was repressed by the knockdown of miR-126 (p < 0.05) ( Figure 7B). Similarly, siRNAinduced PIK3R2 inhibition significantly elevated the phosphorylation of p-PI3K and p-AKT, which was consistent with miR-126 overexpression (p < 0.05) ( Figure 7C).

Discussion
Recently, hundreds of mature or novel miRNAs have been identified in developing pig testes, and the vast majority of these miRNAs show stage-specific expression [21][22][23][24]. These results provide abundant insights into the activities of miRNAs in pig testis development and spermatogenesis. Studies indicate that miRNAs play key roles in spermatogenesis by regulating genes and/or signaling pathways [25][26][27].
In the present study, miR-126 elevation or PIK3R2 knockdown promoted cell proliferation, and PIK3R2 knockdown abolished the effects of miR-126 inhibition on cell proliferation. c-MYC is a vital promoter in G1/S cell cycle progression by recruiting several genes related to this phase, such as CDKs [28,29]. Activated CDK4 phosphorylates Rb and releases E2F [30]. Then, the E2F further activates the CNNE1 and other genes involved in S phase entry [31,32]. Hence, we found that miR-126 elevation or PIK3R2 knockdown increased the expression of c-MYC, CCNE1, and CDK4 genes, and promoted the cell cycle progression from the G1 to S phase. These results indicate that miR-126 promotes immature porcine Sertoli cell proliferation by controlling the cell cycle-related genes by targeting the PIK3R2 gene. Additionally, the Annexin V-FITC/PI staining assay demonstrated that both miR-126 overexpression and PIK3R2 knockdown inhibited cell apoptosis. Apoptosis is widely mediated by the BCL2 family, caspase protease family, and mitochondrial pathway. In the BCL2 family, BCL2 functions as an antiapoptotic protein, and BAX is a proapoptotic protein. They regulate cell apoptosis by controlling the release of apoptogenic factors from the mitochondrial intermembrane into the cytosol. However, the caspase protease family further cleaves several key cellular proteins. In addition, the ATP level is a major factor in evaluating cell apoptosis as the mitochondria are deeply involved in cell fate decisions. Both miR-126 overexpression and PIK3R2 knockdown promoted BCL2 protein expression and decreased the protein expression of BAX and Caspase-3. Moreover, PIK3R2 knockdown offset the effect of the inhibition of miR-126 on ATP levels. Therefore, we speculate that miR-126 inhibits cell apoptosis by regulating the expression of cell survival-related genes and the function of mitochondria via PIK3R2 depletion.
The PIK3R2 gene was affirmed as a direct target of miR-126 using a bioinformatics algorithm and dual-luciferase reporter gene detection. Additionally, miR-126 negatively regulated PIK3R2 protein expression in immature porcine Sertoli cells. It has been widely reported that miR-126 targets the PIK3R2 gene and represses its expression through a conserved binding site in a diverse group of animal species [13,33,34]. Previous studies have demonstrated that the PIK3R2 gene, a member of the PI3K family, encodes p85β, an enzyme involved in the production of 3-polyphosphoinositides [35]. However, 3-polyphosphoinositides inhibit the phosphorylation of AKT and further repress the activity of the PI3K/AKT signaling pathway [36]. It has been reported that the PI3K/AKT signaling pathway mediates the regulatory roles of the PIK3R2 gene on the proliferation and/or apoptosis of multiple cell types, such as synovial fibroblasts [37], EC109 cells [38], etc. Furthermore, the PI3K/AKT signaling pathway participates in regulating the proliferation and apoptosis of immature porcine Sertoli cells [4,16,39]. For instance, LY294002 induced the inhibition of the PI3K/AKT signaling pathway, represses proliferation, and promotes apoptosis in immature porcine Sertoli cells [40]. In the present study, we found that both miR-126 overexpression and PIK3R2 inhibition elevated the phosphorylation of PI3K and AKT, whereas the miR-126 knockdown showed the opposite result. Therefore, we confirmed that miR-126 regulates the proliferation and apoptosis of immature porcine Sertoli cells partly by activating the PI3K/AKT signaling pathway.
In our previous studies, the results from RNA-seq showed that miR-126 maintains higher expression in both immature and mature porcine testicular tissues than in other developmental stages [22,41]. According to the current study, miR-126 promotes immature porcine Sertoli cell proliferation. However, the potential mechanisms need further validation in the miR-126 knockout model.
Collectively, our study indicated that miR-126 can stimulate cell proliferation and restrain the apoptosis of immature porcine Sertoli cells by targeting the PIK3R2 gene. Through this process, miR-126 further activates the PI3K/AKT signaling pathway. We conjectured that miR-126, PIK3R2, and the PI3K/AKT signaling pathway might play pivotal regulatory roles in porcine spermatogenesis by deciding the destiny of immature Sertoli cells.
Author Contributions: Conceptualization and design, M.R. and B.C.; formal analysis, M.R. and X.T.; investigation, Y.C., H.L., X.T., Q.B., B.W., Y.C., D.C. and A.Y.; writing-original draft preparation, M.R. and X.T. All authors censored and approved the ultimate draft. All authors have read and agreed to the published version of the manuscript.