miR-34c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene

RAN Mao-liang, WENG Bo, CAO Rong, PENG Fu-zhi, LUO Hui, GAO Hu, CHEN Bin

College of Animal Science and Technology/Hunan Provincial Key Laboratory for Genetic Improvement of Domestic Animal, Hunan Agricultural University, Changsha 410128, P.R.China

Abstract MicroRNAs (miRNAs) are implicated in swine spermatogenesis via their regulations of cell proliferation, apoptosis, and differentiation. Recent studies indicated that miR-34c is indispensable in the late steps of spermatogenesis. However,whether miR-34c plays similar important roles in immature porcine Sertoli cells remain unknown. In the present study, we conducted two experiments using a completely randomised design to study the function roles of miR-34c. The results from experiment I demonstrated that the relative expression level of miR-34c in swine testicular tissues increased (P=0.0017)quadratically with increasing age, while the relative expression level of SMAD family member 7 (SMAD7) decreased(P=0.0009) with curve. Furthermore, miR-34c expression levels showed a significant negative correlation (P=0.013) with SMAD7 gene expression levels. The results from experiment II indicated that miR-34c directly targets the SMAD7 gene using a luciferase reporter assay, and suppresses (P＜0.05) SMAD7 mRNA and protein expressions in immature porcine Sertoli cells. Overexpression of miR-34c inhibited (P＜0.05) proliferation and enhanced (P＜0.05) apoptosis in the immature porcine Sertoli cells, which was supported by the results from the Cell Counting Kit-8 (CCK-8) assay, the 5-Ethynyl-2´-deoxyuridine(EdU) assay, and the Annexin V-FITC/PI staining assay. Furthermore, knockdown of SMAD7 via small interfering RNA(siRNA) gave a similar result. It is concluded that miR-34c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene.

Keywords: immature porcine Sertoli cell, cell proliferation, apoptosis, SMAD7, miR-34c

1. Introduction

Sertoli cell, the first cell to differentiate into the initially bipotential genital ridge, plays crucial regulatory roles in spermatogenesis by forming the blood-testis barrier,secreting the androgen binding protein, and also by providing nutrients and regulatory factors for the developing germ cells. However, Sertoli cell numbers are generally stable in adult testicular tissue as they lose the ability to proliferate in adults once mature numbers have been reached.Each Sertoli cell supports only a limited number of germ cells, as evidenced by the fact that the number of Sertoli cells are related to the daily sperm production per testis(Johnsonet al. 2008). MicroRNAs (miRNAs) are implicated in Sertoli cell proliferation and apoptosis. miR-133b,miR-762, miR-1285, miR-301b-3p, and miR-3584-5p promote proliferation and inhibit apoptosis in Sertoli cells,but miR-638 and miR-26a have the opposite effects (Jiaoet al. 2015; Maet al.2016; Yaoet al.2016; Huet al.2017; Yinet al.2017). However, many other miRNAs that regulate Sertoli cell proliferation and apoptosis are yet to be identi fied.

We previously identi fied 354 miRNAs in developing swine testicular tissues. We also found that miR-34c showed much higher expression in testicular tissue samples taken from animals between puberty and maturity than in juvenile animals (Ranet al.2015). Other studies have shown that miR-34c is indispensable in the late steps of spermatogenesis in chickens and mice (Bouhallieret al.2010; Wuet al.2014;Yuanet al.2015). Sperm-borne miR-34c is also important for the first cell divisionviaits regulation of B-cell lymphoma-2(BCL2) expression (Liuet al.2012). Remarkably, miR-34c enhances murine male germ cell apoptosis, and promotes the differentiation of mouse spermatogonial stem cells into male germ cells and mouse embryonic stem cells into male germ-like cells (Lianget al.2012; Zhanget al.2012; Yuet al.2014). These evidences suggest that miR-34c plays key roles in spermatogenesis by regulating proliferation and apoptosis in male reproduction-related cells. However, the regulatory roles of miR-34c in porcine Sertoli cell proliferation and apoptosis are still unknown.

The SMAD family member 7 (SMAD7) geneis expressed in a higher levels from 0 to 15 days of age than in adult mouse Sertoli cells (Itmanand Loveland 2008).SMAD7is a key negative regulator of transforming growth factor-β (TGF-β) signaling, and is an important crosstalk mediator between the TGF-β signaling pathway and other signaling pathways (Yanet al.2009; Yanand Chen 2011). It has been reported that TGF-βs can regulate the opening and closing of the blood-testis barrier, which is mainly constructed by Sertoli cells (Luiet al.2003a, b).However, the function role ofSMAD7in Sertoli cells was not well characterized.

Based on the above-mentioned clues, we designed the present study to characterize the regulatory effects of miR-34c on proliferation and apoptosis in immature porcine Sertoli cells. Our results indicated that miR-34c inhibits proliferation and promotes apoptosis in immature porcine Sertoli cells through its targeting of theSMAD7gene.

2. Materials and methods

2.1. Ethics statement

All animal work and experimental procedures in the present study were reviewed and approved by the Ethics Committee of the Animal Science and Technology College of Hunan Agricultural University, China.

2.2. Experimental design and treatments

A completely randomised design was used in the present study. In experiment I, there were a total of 7 treatments including 1, 30, 60, 90, 120, 150, and 180 days of age,respectively. In experiment II, there were a total of 6 cell transfection treatments including miR-34c mimic, mimic negative control (NC), miR-34c inhibitor, inhibitor NC,SMAD7small interfering RNA (siRNA), and siRNA NC,respectively.

2.3. Animals, feeding and sample collection (experiment I)

Shaziling pigs (Sus scrofa), an indigenous Chinese pig breed, were provided by the Xiangtan Bureau of Animal Husbandry and Aquatic Products (Xiangtan, China) and fed as the feeding standard of Shaziling pig. A total of 21 Shaziling boars (1 day of age, body weight: (0.91±0.09) kg)were randomly allotted to 1 of 7 treatments with 3 independent biological replicates of 1 boar per replicate for each treatment based on the body weight. Boars in these 7 treatments were castrated at 1 day of age (body weight:(0.89±0.06) kg), 30 days of age ((4.26±0.16) kg), 60 days of age ((10.78±0.44) kg), 90 days of age ((15.49±0.96) kg), 120 days of age ((20.52±1.27) kg), 150 days of age ((28.36±1.72)kg), and 180 days of age ((35.20±2.15) kg), respectively.All Shaziling boars were given a general anesthetic (Zoletil 50, Virbac Co., France) before sampling. Testicular tissue samples were immediately flash frozen in liquid nitrogen and stored at –80°C prior to use.

2.4. Cell culture and transfection (experiment II)

Swine testes cells (ATCC®CRL-1746™, USA) were purchased through the BOSTER Company (Wuhan, China)and identi fied as immature porcine Sertoli cells with a purity of nearly 100% (Maet al.2016a, b). The immature porcine Sertoli cells were cultured in Dulbecco’s modi fied Eagle medium (HyClone, USA) containing 10% fetal bovine serum at 37°C with a 5% CO2atmosphere. We then seeded the cells in 6- or 96-well plates (Invitrogen, Carlsbad, CA,USA) for the subsequent studies. Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) was used to transfect 100 μmol of the miR-34c mimic, the mimic NC, the miR-34c inhibitor, the inhibitor NC,SMAD7siRNA, or siRNA NC (Ribobio, Guangzhou, China) into immature porcine Sertoli cells with 3 independent biological replicates of 1 well per replicate for each treatment when the cells had reached approximately 80% con fluence. All of these abovementioned experimental procedures were conducted with 3 independent biological replicates.

2.5. Dual luciferase activity assay (experiment II)

The 3´-untranslated region (3´UTR) of theSMAD7gene(500 bp) with the predicted target site (position 943–948 bp)and a mutantSMAD73´UTR (500 bp) with a 2-base mutation in the predicted target site were synthesized by Biosune Co., Ltd., (Shanghai, China). These 2 fragments were then subcloned into a psi-CHECK-2 dual-luciferase reporter vector (Promega, USA) and co-transfected with miR-34c or the mimic NC into the immature porcine Sertoli cells. A total of 48 h after transfection, the luciferase activity of each treatment was measured using a Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA). Renilla luciferase(Promega) activity was used as an internal control. At least 3 independent biological replicates were used.

2.6. Western blotting (experiment II)

Total protein was extracted from Sertoli cells using RIPA lysis buffer (Beyotime, China). The concentration of each total protein sample was determined using a BCA Protein Assay Kit (Beyotime, China). Equal amounts (40 μg) of each protein sample were subjected to SDS-PAGE and the fractionated proteins were transferred onto a PVDF membrane (Beyotime, China). The PVDF membrane was then blocked with skim milk and probed with the SMAD7 antibody (1:1 000; Proteintech Group, Rosemont, IL USA),the Bcl2 antibody (1:1 000; Proteintech Group), the BAX antibody (1:2 000; Proteintech Group), the Caspase-3 antibody (1:100; Abcam), and the β-actin antibody (1:5 000;Proteintech Group). Immunoblotting was performed with secondary antibodies diluted 1:5 000. The signal intensities of the individual protein bands were captured using an ECL Advanced Western Blotting Detection Kit (Beyotime, China).

2.7. Cell proliferation analysis (experiment II)

Immature porcine Sertoli cells were seeded into 6-well plates for the cell cycle assay and into 96-well plates for the Cell Counting Kit-8 (CCK-8) (Multiscience, China) and 5-Ethynyl-2´-deoxyuridine (EdU) (RioboBio, China) assays.Immature porcine Sertoli cells were transfected with the miR-34c mimic, the mimic NC, theSMAD7siRNA, or the siRNA NC, and allowed to grow for 24 h. To measure the cell cycle distribution, the cells were harvested into 1.5-mL centrifuge tube and fixed in 70% ethanol overnight at –20°C. The fixed cells were then analyzed with a Cell Cycle Detection Kit using a FACSCanto II Flow Cytometer(Becton Dickinson, USA). A total of 10 μL of the CCK-8 reagent was added to each well and incubated for 4 h at 37°C. The absorbance was measured at 450 nm using an ELISA Plate Reader (Molecular Devices, USA). A total of 100 μL of EdU 33342 medium (50 μmol) was added to each well, and the cells were incubated for 2 h at 37°C.The preceding tests were performed according to the manufacturers’ protocols. The cells were examined under a fluorescence microscope at 20×.

2.8. Cell apoptosis analysis (experiment II)

Sertoli cells transfected with the miR-34c mimic, the mimic NC,SMAD7siRNA, or the siRNA NC were analyzed for cell apoptosis using the Annexin V-FITC/PI Staining Assay (Key GEN BioTECH, Nanjing, China). Cells were harvested into 1.5-mL centrifuge tubes and washed with PBS buffer. Following centrifugation at 2 000 r min–1for 5 min, the cells were resuspended in 500 μL binding buffer and were subsequently analyzed using a FACSCanto II Flow Cytometer (Becton Dickinson, USA). We also measured the expression levels of 3 cell survival-related genes (BCL2,BCL2 associated X protein(BAX), andCaspase-3) in each harvested cell group using quantitative real-time PCR(qRT-PCR).

2.9. qRT-PCR

Total RNA was isolated from testicular tissues in experiment I and Sertoli cells in experiment II using TRIzol®Reagent(Invitrogen, Carlsbad, CA, USA) according to the protocols provided by the manufacturer. The miRNA-specific stemloop primers for miR-34c and the primers for protein-coding genes were designed using Oligo 7.0 (Appendix A) and were synthesized by Sangon Bio. (Shanghai, China). Total RNA(1 μg) from each sample was reverse transcribed using a reverse transcriptase kit (TaKaRa, Dalian) with a miRNA-specific stem-loop primers (for miR-34c) or random primer(for protein-coding genes). Each qRT-PCR reaction mixture(25 μL) reaction contained 2.0 μL cDNA, 1 μL sense primer(10 μmol), 1 μL antisense primer (10 μmol), 8.5 μL ddH2O,and 12.5 μL SYBR PremixEx TaqII (TaKaRa, Dalian). qRTPCR amplifications were performed on a Thermo Scientific PIKO REAL 96 Real-Time PCR System. The thermal cycling program used for miR-34c quantification consisted of 2 initial steps of 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The qRT-PCR procedure for protein-coding genes quantification consisted of an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 10 s and 59°C for 50 s.U6andβ-actinwere used for normalization of the expression of miR-34c and protein-coding genes, respectively. The 2–ΔΔCTmethod was used to measure the relative changes in expression(Livakand Schmittgen 2001). At least three independent biological replicates were used for each assay.

2.10. Statistical analysis

Data were analyzed by one-way ANOVA using the SAS System (release 9.4; SAS Inst. Inc., Cary, NC, USA). In experiment I, Duncan’s multiple comparison was employed to test the differences among different treatments. In experiment II, theT-test was used to test the differences between the mimic NC and miR-34c mimic treatments,inhibitor NC and miR-34c inhibitor treatments, as well as siRNA NC andSMAD7siRNA treatments. Orthogonal comparisons in experiment I were applied to test linear and quadratic responses of miR-34c andSMAD7mRNA expressions in developing swine testicular tissues with increasing ages. The CORR procedure of SAS System was used in experiment I to do regression analyses between miR-34c andSMAD7mRNA expressions in swine testicular tissues and increasing ages. Each replicate served as an experiment unit. The results are shown as the mean±SE.TheP＜0.05 was considered statistically significant.

3. Results

3.1. Expression of miR-34c and SMAD7 in developing porcine testicular tissue (experiment I)

We previously reported thatSMAD7gene is a potential target gene of miR-34c based on a bioinformatic analysis of developing pig testicular tissues (Ranet al.2015). To validate this result, we measured the expressions of miR-34c andSMAD7gene in the same pig testicular tissue samples using the qRT-PCR method. We found that miR-34c is expressed with lower levels in testicular tissue samples from 1 to 30 days of age animals, after which expression of miR-34c increased sharply (P＜0.05) from 30 days of age and persisted from 30 to 180 days of age in testicular tissue samples (Fig. 1-A). The relative expression level of miR-34c in swine testicular tissues increased quadratically (y=0.979x2–0.004x–10.502,R2=0.835,P=0.0017; Appendix B)with increasing age. The pattern ofSMAD7gene expression showed an opposite trend to the expression of miR-34c in these testicular tissue samples (Fig. 1-B and C). As age increased, the relative expression level ofSMAD7decreased with quadratic (y=1.202x–0.651,R2=0.826,P=0.0009;Appendix B) in swine testicular tissues. Furthermore,miR-34c expression levels showed a significant negative correlation (P=0.013) withSMAD7gene expression levels(Fig. 1-C).

Fig. 1 The SMAD7 gene is a direct target of miR-34c. A and B, miR-34c and SMAD7 mRNA levels were measured in the developing swine testicular tissues using qRT-PCR (experiment I). β-Actin was used as the internal control. C, in experiment I,miR-34c expression showed a significant negative correlation with SMAD7 expression in the developing swine testicular tissues.D, in experiment II, luciferase reporter assay results were quanti fied after co-transfection of the miR-34c/mimic negative control(NC) and psi-CHECK-2-3´UTR-WT/MT into immature porcine Sertoli cells. The relative luciferase activity was evaluated 48 h later.Renilla luciferase (Promega) activity was used as an internal control. Data are shown as the mean±SE (n=3). Different letters mean values within each section were significantly different. ＊＊ P＜0.01.

3.2. SMAD7 gene is a direct target of miR-34c (experiment II)

A luciferase reporter assay was performed to validate the target association between miR-34c andSMAD7.Wild-type and mutantSMAD73´UTRs were subcloned separately into the psi-CHECK-2 vector. The inserts in these 2 resulting vectors were veri fied by Sanger DNA sequencing to con firm their identities (Sangeret al.1977; Walkerand Lorsch 2013). The results of the assay indicated that co-transfection of miR-34c with the wildtypeSMAD73´UTR psi-CHECK-2 vector significantly suppressed (P＜0.01) luciferase activity more than in the other three treatments (Fig. 1-D).

In addition, expressions of bothSMAD7mRNA and protein were also examined in miR-34c mimic- or the miR-34c inhibitor-transfected immature porcine Sertoli cells. Transfection ef ficiency of miR-34c mimic and inhibitor were evaluated by qRT-PCR assay. The results showed that the miR-34c mimic significantly increased (P＜0.01)miR-34c expression, while miR-34c expression was significantly suppressed (P＜0.05) by the miR-34c inhibitor in the immature Sertoli cells (Fig. 2-A). qRT-PCR assays and Western blotting results demonstrated thatSMAD7mRNA and protein levels decreased significantly (P＜0.05)in response to overexpression of miR-34c, while both mRNA and protein expression were significantly increased in response to knockdown of miR-34c (Fig. 2-B and C). These results showed that miR-34c directly targets theSMAD73´UTR and represses its expression.

3.3. Overexpression of miR-34c inhibits proliferation and promotes apoptosis in immature porcine Sertoli cells (experiment II)

In order to decipher the functional role of miR-34c on Sertoli cell proliferation, the miR-34c mimic or mimic NC was transfected into immature porcine Sertoli cells. The results of cell cycle analysis indicated that the percentage of cells in G1 phase significantly increased (P＜0.05) in the miR-34c mimic-transfected cells compared with the mimic NC. Fewer (P＜0.05) miR-34c mimic-transfected cells were identi fied in S phase than in the mimic NC-transfected cells(Fig. 3-A). Overexpression of miR-34c caused the Sertoli cells to arrest in G1 phase. The CCK-8 assay results showed that Sertoli cell proliferation at 0, 24, and 48 h was significantly decreased (P＜0.01) in cells transfected with miR-34c mimic compared with mimic NC-transfected cells (Fig. 3-B). However, proliferation of the mimic NC-transfected Sertoli cells at 72 h was significantly lower(P＜0.01) than proliferation at 48 h in the limited space of the 96-well plates (Fig. 3-B). Furthermore, the EdU assay also demonstrated that the number of EdU-positive cells in the miR-34c mimic-transfected cell group was significantly lower (P＜0.05) than in the Sertoli cells transfected with the mimic NC (Fig. 3-C and D).miR-34c mimic-transfected cells had a higher (P＜0.05)apoptosis rate compared with the mimic NC-transfected group (Fig. 4-A-C). In addition, we found that mimic-induced miR-34c overexpression led to an up-regulation (P＜0.05)ofBAXandCaspase-3mRNA and protein levels, as well as a down-regulation (P＜0.05) ofBCL2mRNA and protein levels in the immature porcine Sertoli cells (Fig. 4-D–F).Taken together, these results indicated that miR-34c inhibits proliferation and promotes apoptosis in immature porcine Sertoli cells.

3.4. SMAD7 deficiency inhibits proliferation and promotes apoptosis in Sertoli cells (experiment II)

Fig. 3 Overexpression of miR-34c represses proliferation in immature porcine Sertoli cells (experiment II). A, cell cycle analysis showed that overexpression of miR-34c arrested the immature porcine Sertoli cells in the G1 phase. B, the Cell Counting Kit (CCK)assay was used to measure the proliferation of immature porcine Sertoli cells transfected with the miR-34c mimic and the mimic negative control (NC). C, ratio of 5-Ethynyl-2´-deoxyuridine (EdU)-positive cells in immature porcine Sertoli cells transfected with the miR-34c mimic and the mimic NC. D, representative images of EdU staining of immature porcine Sertoli cells transfected with the miR-34c mimic and the mimic NC. Proliferating cells are stained red. The cell nuclei stained in blue with Hoechst 33342 dye represent the total cell population. Scale bar=50 μm. Data are shown as the mean±SE (n=3). ＊ P＜0.05 and ＊＊ P＜0.01, respectively.

Based on the above-mentioned results, we further examined the regulatory role ofSMAD7in immature porcine Sertoli cell proliferation and apoptosis through following several lines of experimentations. ThreeSMAD7siRNAs were designed and transfected into Sertoli cells, respectively.qRT-PCR and Western blotting results indicated thatSMAD7siRNA effectively suppressed (P＜0.05)SMAD7mRNA and protein expression levels (Fig. 5-A and B).Flow cytometry assay showed that fewer (P＜0.05)SMAD7siRNA-transfected cells were observed in the G1 phase, but more cells (P＜0.05) were detected in the S phase compared with the NC (Fig. 6-A). The CCK-8 assay results showed that knockdownofSMAD7gene expression significantly inhibited (P＜0.05) cell proliferation from 0 to 72 h (Fig. 6-B).Similarly, fewer EdU-positive cells (P＜0.05) were detected in cells transfected withSMAD7siRNA compared with the NC (Fig. 6-C and D).

Fig. 4 Overexpression of miR-34c promotes apoptosis in immature porcine Sertoli cells (experiment II). A and B, cell apoptosis induced by transfection with the miR-34c mimic and the mimic negative control (NC) was measured using the Annexin V-FITC/PI staining assay. The flow cytometry images are representative of the results obtained. C, overexpression of miR-34c promotes apoptosis in immature porcine Sertoli cells. D, relative mRNA expression levels of BCL2, BAX, and Caspase-3 were assayed in immature porcine Sertoli cells transfected with miR-34c mimic and mimic NC using qRT-PCR. E and F, protein expression levels of BCL2, BAX, and Caspase-3 were assayed in miR-34c mimic-/mimic NC-transfected immature porcine Sertoli cells using Western blotting. β-Actin was used as the internal gene expression control. Data are shown as the mean±SE (n=3). ＊ P＜0.05 and ＊＊ P＜0.01, respectively.

Fig. 5 SMAD7-specific small interfering RNA (siRNAs) can repress SMAD7 mRNA (A) and protein (B) levels (experiment II).Immature porcine Sertoli cells were transfected with SMAD7 siRNA (siSMAD7) and the negative control (NC). β-Actin was used as the internal control. Data are shown as the mean±SE (n=3). ＊ P＜0.05.

Fig. 6 SMAD7 deficiency inhibits proliferation in immature porcine Sertoli cells (experiment II). A, flow cytometry was used to determine the percentage of immature porcine Sertoli cells transfected with miR-34c mimic or mimic negative control (NC) in the G1, S, and G2 phases of the cell cycle. B, the Cell Counting Kit-8 (CCK-8) assay was used to measure the proliferation of SMAD7 siRNA–/siRNA NC-transfected immature porcine Sertoli cells. C, 5-Ethynyl-2´-deoxyuridine (EdU) assay results shown that SMAD7 deficiency inhibits proliferation in immature porcine Sertoli cells. D, representative images of EdU staining of immature porcine Sertoli cells transfected with siSMAD7 or the NC. Proliferating cells are labeled red. The cell nuclei stained blue (Hoechst 33342 dye)represent the total cell population. Scale bar=50 μm. Data are shown as the mean±SE (n=3). ＊ P＜0.05 and ＊＊ P＜0.01, respectively.

We also investigated the effects ofSMAD7siRNA on immature porcine Sertoli cell apoptosis by flow cytometry(Fig. 7-A and B).SMAD7siRNA significantly increased(P＜0.05) the rate of apoptosis in the immature porcine Sertoli cells (Fig. 7-C). Furthermore,BAXandCaspase-3mRNA and protein levels were significantly increased (P＜0.01),whileBCL2mRNA and protein levels were significantly reduced (P＜0.05) by knocking down theSMAD7gene in immature porcine Sertoli cells (Fig. 7-D–F). These results strongly suggested that miR-34c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting theSMAD7gene.

4. Discussion

Sertoli cells play crucial roles in regulating spermatogenesis.There is increasing evidence to suggest that miRNAs widely regulate proliferation and apoptosis in immature porcine Sertoli cell. miR-762 has been shown to promote proliferation and inhibit apoptosis in immature porcine Sertoli cell by targeting thering finger protein 4gene (Maet al.2016b). miR-638 inhibits proliferation and promotes apoptosis in immature porcine Sertoli cells by targetingsperm-associated antigen 1which indirectly inactivates the PI3K/AKT pathway (Huet al.2017). 17β-Estradiol reduces proliferation in immature porcine Sertoli cells by inhibiting miR-1285 and thus activating adenosine monophosphateactivated protein kinase (Jiaoet al.2015). In the present study, we have demonstrated that miR-34c inhibits proliferation and promotes apoptosis in immature porcine Sertoli cells by targeting theSMAD7gene.

Fig. 7 SMAD7 deficiency promotes apoptosis in immature porcine Sertoli cells (experiment II). A and B, the Annexin V-FITC/PI staining assay was used to determine cell apoptosis phase distributions after transfection of immature porcine Sertoli cells with SMAD7 siRNA and siRNA negative control (NC). The flow cytometry images are representative of the results obtained. C, siRNA-induced SMAD7 knockdown promotes apoptosis in immature porcine Sertoli cells. D, relative expression levels of the BCL2,BAX, and Caspase-3 genes were measured in immature porcine Sertoli cells transfected with SMAD7 siRNA and the siRNA NC using qRT-PCR. E and F, protein expression levels of BCL2, BAX, and Caspase-3 were assayed immature porcine Sertoli cells transfected with SMAD7 siRNA and the siRNA NC using Western blotting. β-Actin was used as the internal gene expression control. Data are shown as the mean±SE (n=3). ＊ P＜0.05 and ＊＊ P＜0.01, respectively.

miR-34c is a member of the miR-34 miRNA family.Recent studies have demonstrated that miR-34c regulates proliferation in multiple cell types, including colorectal cancer cells (Yanget al.2015), glioma cells (Wuet al.2013),hepatocellular carcinoma cells (Songet al.2015), vascular smooth muscle cells (Choeet al.2015), and mouse C2C12 myoblast cells (Wanget al.2017). As mentioned previously,miR-34c also plays key regulatory roles in spermatogenesisrelated cells (Lianget al.2012; Liuet al.2012; Liet al.2013;Yuet al.2014). Overexpression of miR-34c induced cell cycle arrest at the G1 phase and inhibited proliferation in immature porcine Sertoli cells (Fig. 3). This might at least partially explain the fundamental role played by miR-34c in modulating proliferation and apoptosis in swine immature Sertoli cells.

SMAD7has been widely reported to regulate proliferation in multiple cell types. Overexpression ofSMAD7promotes beta-cell proliferation by increasing CyclinD1 and CyclinD2 expression and also by inducing nuclear exclusion of p27(Xiaoet al.2014). In HaCaT cells,SMAD7knockdown suppresses cell growth by promoting the accumulation of cells in the S-phase of the cell cycle (Di Fuscoet al.2017).SMAD7(–/–) mice has reduced muscle mass, and hypotrophy and hypoplasia of muscle fibers as a result of decreased proliferation, activation and differentiation of satellite cells (Cohenet al.2015). In addition, a series of miRNAs regulate cell proliferation by targeting theSMAD7gene; examples are miR-17-5p in femoral head mesenchymal stem cells (Jiaet al.2014), miR-497 in breast cancer cells (Liuet al.2016), and miR-590 in human umbilical cord mesenchymal stem cells (Liuet al.2017). In the present study,SMAD7was con firmed to be the target gene of miR-34c in immature porcine Sertoli cells based on results of the luciferase reporter assay(Fig. 1-D).SMAD7gene expression was also found to be regulated by overexpression or down-regulation of miR-34c(Fig. 2). siRNA-inducedSMAD7knockdown promotes the accumulation of immature porcine Sertoli cells in G1 phase and inhibits proliferation (Fig. 6).

Furthermore, we tested apoptosis after the cell transfected with miR-34c orSMAD7siRNAs. Flow cytometry indicated that Sertoli cells transfected with the miR-34c mimic orSMAD7siRNAs had a lower apoptosis rate compared with the control group (Figs. 4 and 7). In addition, we also measured the expression levels of 3 cell survival-related genes, includingBCL2,BAX, andCaspase-3. Previous researches have shown thatBCL2andBAX, members of theBCL2gene family, are apoptotic regulators that participate in a complex network of heterodimeric interactions, whileBCL2is an anti-apoptotic gene andBAXis a pro-apoptotic effector gene (Kvansakuland Hinds 2013; Mignardet al.2014;Volkmannet al.2014).Caspase-3, a member of caspase protease family, is a crucial mediator of programmed cell death (apoptosis), and can catalyze the specific cleavage of many key cellular proteins which leads to apoptosis. The mRNA and protein levels of bothBAXandCaspase-3were significantly increased, whileBCL2gene expression was significantly reduced, by both knockdown ofSMAD7and overexpression of miR-34c in swine immature Sertoli cells.

5. Conclusion

Our study provides evidence that miR-34c inhibits proliferation and promotes apoptosis in immature porcine Sertoli cells by targeting theSMAD7gene and regulating its expression. miR-34c could be a potential candidate for regulating spermatogenesis in pigs by determining the fate of immature Sertoli cells.

Acknowledgements

This work was financially supported by the earmarked fund for China Agriculture Research System (CARS-36),the Hunan Provincial Natural Science Foundation of China (2018JJ3219 and 2018JJ2176), and the Excellent Doctoral Dissertation Cultivating Fund of Hunan Agricultural University, China (YB2015001). Authors would like to thank Yang Guan, Vanderbilt University, USA, for critical reading of the manuscript.

Appendicesassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm