Silencing of MEG3 attenuated the role of lipopolysaccharides by modulating the miR-93-5p/PTEN pathway in Leydig cells

Human Leydig cells were purchased from ScienCell Research Laboratories (ScienCell, San Diego, California, USA) and routinely conserved in our labs and cultured in Dulbecco’s modified Eagle medium (DMEM; GIBCO, NY, USA) supplemented with 10% foetal bovine serum (FBS; Invitrogen, CA, USA), 10 U/mL penicillin and 10 μg/mL streptomycin. Cells were maintained in a 37 °C/5% CO₂ humidified incubator. LPS (Sigma-Aldrich, MO, USA) was dissolved in the medium. The cells were treated with LPS (Sigma-Aldrich, MO, USA) at different concentrations for approximately 48 h. Transfection of small interfering RNA (siRNAs), miR-93 inhibitor, miR-93 mimics and control small RNAs (GenePharma, Shanghai, China) was performed using Lipofectamine 2000 (Thermo Fisher Scientific, CN, USA) over a period of approximately 48 h.

Trizol reagent (Junxin, Suzhou, China) was introduced to extract the total RNA. A NanoDrop 2000 (Thermo Fisher) instrument was used to measure the concentration and purity of total RNA. A reverse transcription kit (Takara, Dalian, China) was applied to synthesize the first-strand cDNA. The 2 × SYBR Green qPCR Mix (Junxin, Suzhou, China) was selected to perform qPCR analysis. The expression levels for lncRNA and mRNA were normalized to β-actin expression, and the expression levels for miRNAs were relative to the U6 expression. The 2^−ΔΔCt method and arbitrary units were introduced to calculate the relative fold change. The primers used in this study are listed in Table 1.

The radio immunoprecipitation assay (RIPA) (Takara, Dalian, China) was introduced to isolate the protein. The BCA protein detection kit (Junxin, Suzhou, China) was applied to measure the concentration of protein. The proteins (30 μg in each lane) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked in 5% fat-free milk and exposed to different primary antibodies at 4 °C overnight. The membranes were incubated with horseradish peroxidase-conjugated IgG. The immunoreactive protein bands were detected with a chemiluminescence (ECL) reagent (Junxin, Suzhou, China) and the Bio-Rad ChemiDocTM XRS system. β-Actin was measured as a loading control. The antibodies used in this study are listed below: The anti-PTEN antibody (Ab267787, Abcam, Cambridge, British), anti-Bcl-2 antibody (Ab32124, Abcam, Cambridge, British), anti-Bax antibody (Ab182734, Abcam, Cambridge, British), anti-β-Actin (Ab207604, Abcam, Cambridge, British) and Goat Anti-Rabbit IgG H&L (HRP) (Ab 6721, Ab207604, Abcam, Cambridge, British) were used according to the protocol.

Cell viability was tested using the CCK-8 assay (Junxin, Suzhou, China). In brief, cells were plated in a 96-well cell culture plate at a concentration of 4 × 10³ cells per well (3 replicates for each experimental condition). After different treatments, 10 μl CCK-8 reagent was added to the cells in each well and incubated for 2 h in a 5% CO₂ humidified incubator at 37 °C. The absorbance of the cells at 450 nm was detected using a microplate reader (Bio-Rad, Hercules, USA).

The cell proliferation potential was assessed using an EdU kit (Junxin, Suzhou, China). In brief, cells were plated in a 48-well cell culture plate at a concentration of 5 × 10⁴ cells per well (3 replicates for each experimental condition). After different treatments, 200 μl EdU reagent was added to the cells in each well and incubated for 2 h in a 5% CO₂ humidified incubator at 37 °C. The EdU analysis was performed according to the manufacturer’s instructions. The images were captured using a fluorescence microscope (Olympus, Tokyo, Japan).

The fragments of MEG3 and PTEN containing the putative miR-93 binding site were amplified and inserted into the psiCheck2 Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) to construct the reporter vectors MEG3-wild-type (MEG3-wt) and PTEN-wild-type (PTEN-wt), respectively. The psiCheck2 vectors cloned with the fragments of MEG3 and PTEN containing the mutant miR-93 binding site were referred to as MEG3-mutant-type (MEG3-mut) and PTEN-mutant-type (PTEN-mut), respectively. These reporters were co-transfected into HEK293 cells with miR-93 mimics using Lipofectamine 2000. The luciferase activities were measured with a Dual-Luciferase® Reporter Assay System Protocol (Promega).

The RIP analysis was performed with the RIP kit (Millipore, MA USA) and anti-Argonaute 2 (Ago2) antibodies (Abcam, USA) according to the protocol. In brief, the cells were lysed in RNA lysis buffer, and the cell lysate was incubated with Protein A/G beads (Biolinkedin, Shanghai, China) and anti-Argonaute 2 (Ago2) antibodies (Abcam, USA) at 4 °C overnight. IgG was added as a negative control. The immunoprecipitated RNA was isolated and reverse transcribed, and qPCR analysis was introduced to measure the enrichment of MEG3 and miR-93.

After different treatments, the culture supernatants were harvested, and the concentrations of the inflammatory cytokines of interleukin-6 (IL6), tumour necrosis factor alpha (TNFα) and testosterone were tested using ELISA kits (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s protocol.

To determine the role of LPS in Leydig cells, we treated Leydig cells with different concentrations of LPS (0, 50, 100, 200, and 400 μg/ml) for 48 h. ELISA analysis demonstrated that LPS induced the secretion of TNFα and IL6 in Leydig cells (Fig. 1a and b). Further research demonstrated that LPS inhibited the testosterone production in Leydig cells (Fig. 1c). The results of CCK8 analysis showed that LPS reduced the cell viability of Leydig cells (Fig. 1d). The results of EdU analysis showed that LPS decreased the cell proliferation of Leydig cells (Fig. 1e). Ultimately, western blot analysis revealed that LPS induced cell apoptosis, decreased Bcl-2 and increased Bax in Leydig cells (Fig. 1f). In summary, these results suggested that LPS induced dysfunction in Leydig cells.

To investigate whether MEG3 was involved in the role of LPS in Leydig cells, we measured the expression of MEG3 in Leydig cells treated with different concentrations of LPS. The results of qPCR analysis demonstrated that LPS increased the expression level of MEG3 in Leydig cells (Fig. 2a). This observation indicated that MEG3 participated in the role of LPS in Leydig cells.

To test whether MEG3 affected the role of LPS in Leydig cells, we first designed and synthesized short-hair RNA specific against MEG3 (shMEG3s) and control short-hair RNA (shNC). We transfected shMEGs and shNC into Leydig cells for 48 h. The results of qPCR analysis demonstrated that both shMEG3s could efficiently downregulate the expression of MEG3 in Leydig cells, and shMEG3–1 was chosen for further experiments (Fig. 2b).

We divided the cells into four groups: shNC, shMEG3, shNC+LPS and shMEG3 + LPS. The ELISA analysis demonstrated that silencing of MEG3 decreased the secretion of TNFα and IL6 and inhibited LPS-induced promotion of TNFα and IL6 secretion in Leydig cells (Fig. 2c). Furthermore, knockdown of MEG3 elevated testosterone production and lowered LPS-induced testosterone production in Leydig cells (Fig. 2d). In addition, suppression of MEG3 enhanced the cell viability and proliferation and attenuated the LPS-induced inhibition of cell viability and proliferation in Leydig cells (Fig. 2e and f).

To dissect the molecular mechanism of MEG3 in LPS in Leydig cells, we used the miRNAs prediction online site to screen the candidate targets of MEG3 and found that miR-93-5p was a candidate of MEG3. We introduced the luciferase reporter system to confirm the interaction between MEG3 and miR-93-5p. First, we cloned MEG3 transcripts containing the predicted binding site of miR-93-5p and the mutant binding site of miR-93-5p into the luciferase reporter vector psiCheck2, referred to as psiCheck2-MEG3 wild type (psiCheck2-MEG3-wt) and psiCheck2-MEG3 mutant type (psiCheck2-MEG3-mut), respectively. We transfected psiCheck2-MEG3-wt and psiCheck2-MEG3-mut together with miR-93-5p mimics into 293 cells. At 48 h, the luciferase activity was measured, and the results demonstrated that miR-93-5p mimics inhibited the luciferase activity of psiCheck2-MEG3-wt, and the luciferase activity was restored in psiCheck2-MEG3-mut (Fig. 3a). Taken together, these results demonstrated that miR-93-5p could bind to the predicted binding site of miR-93-5p on MEG3 transcripts.

To investigate whether miR-93-5p affected the role of LPS on Leydig cells, we first measured the expression of miR-93-5p in Leydig cells treated with different concentrations of LPS, and the results of qPCR analysis demonstrated that LPS decreased the expression level of miR-93-5p in Leydig cells (Fig. 3b). This observation indicated that miR-93-5p participated in the role LPS in Leydig cells. We synthesized the miR-93-5p mimics, mimics NC, miR-93-5p inhibitor and inhibitor NC, and the results of qPCR analysis demonstrated that the miR-93-5p mimics significantly increased the miR-93-5p level and the miR-93-5p inhibitor significantly decreased that in Leydig cells (Fig. 3c).

In addition, we divided the cells into four groups: mimics NC, miR-93-5p mimics, mimics NC + LPS and miR-93-5p mimics+LPS. The ELISA analysis demonstrated that miR-93-5p decreased the secretion of TNFα and IL6 and inhibited the LPS-induced promotion of TNFα and IL6 secretion in Leydig cells (Fig. 3d and e). Furthermore, miR-93-5p increased the testosterone production and decreased the LPS-induced testosterone production of Leydig cells (Fig. 3f). In addition, miR-93-5p enhanced the cell viability and proliferation and attenuated the LPS-induced inhibition of cell viability and proliferation in Leydig cells (Fig. 3g and h). Taken together, these results showed that MEG3 absorbed miR-93-5p and that miR-93-5p alleviated the role of LPS on Leydig cells.

To investigate whether miR-93-5p affected the role of MEG3 in Leydig cells treated with LPS, we divided the cells into six groups: shNC+inhibitor NC, shMEG3 + inhibitor NC, shMEG3+ miR-93-5p inhibitor, shNC+inhibitor NC + LPS, shMEG3 + inhibitor NC + LPS, and shMEG3+ miR-93-5p inhibitor+LPS. The ELISA analysis demonstrated that miR-93-5p inhibitor restored the shMEG3-induced inhibition of TNFα and IL6 secretion of Leydig cells (Fig. 4a and b). Furthermore, the ELISA analysis demonstrated that miR-93-5p inhibitor alleviated the shMEG3-induced elevation of testosterone production of Leydig cells (Fig. 4c). In addition, miR-93-5p inhibitor attenuated the shMEG3-induced enhancement of cell viability and proliferation in Leydig cells (Fig. 4d and e). In summary, these results suggested that inhibition of miR-93-5p alleviated the role of silenced MEG3 in Leydig cells treated with LPS.

To investigate the molecular mechanism of miR-93-5p in Leydig cells, we used the miRNAs prediction online site to screen the candidate target of miR-93-5p and found that PTEN was a candidate of miR-93-5p. To detect whether miR-93-5p could regulate the PTEN expression, we transfected the miR-93-5p mimics, mimics NC, miR-93-5p inhibitor and inhibitor NC into Leydig cells. The results of qPCR and western blots analysis showed that the miR-93-5p mimics inhibited and miR-93-5p inhibitor promoted the PTEN expression in Leydig cells (Fig. 5a and b). To further confirm the interaction between miR-93-5p and PTEN, we applied the luciferase reporter system as a test. First, we cloned the PTEN 3′ UTR containing the predicted binding site of miR-93-5p and the mutant binding site of miR-93-5p into the luciferase reporter vector psiCheck2, known as psiCheck2-PTEN wild type (psiCheck2-PTEN-wt) and psiCheck2-PTEN mutant type (psiCheck2-PTEN-mut), respectively. We transfected psiCheck2-PTEN-wt and psiCheck2-PTEN-mut together with miR-93-5p mimics into 293 cells. At 48 h, the luciferase activity was measured. The results demonstrated that miR-93-5p mimics inhibited the luciferase activity of psiCheck2-PTEN-wt, and the luciferase activity was restored in psiCheck2-PTEN-mut (Fig. 5c and d). Taken together, these results demonstrated that miR-93-5p inhibited PTEN expression in Leydig cells.

To investigate whether PTEN affected the role of miR-93-5p in Leydig cells treated with LPS, we first detected the PTEN expression in Leydig cells treated with different concentrations of LPS. The results of qPCR and western blot analyses showed that LPS elevated the expression level of PTEN in Leydig cells (Fig. 6a), indicating that PTEN was involved in the role of LPS in Leydig cells.

Furthermore, we constructed overexpression clones of PTEN based on the plvx vector (plvx-PTEN), and qPCR and western blot analyses revealed that overexpression clones of PTEN significantly elevated the expression level of PTEN in Leydig cells (Fig. 6b).

We divided the cells into four groups: mimics NC, LPS + mimics NC, LPS+ miR-93-5p mimics and LPS + miR-93-5p mimics+plvx-PTEN. The ELISA analysis demonstrated that plvx-PTEN restored miR-93-5p mimics-induced inhibition of TNFα and IL6 secretion of Leydig cells (Fig. 6c). Furthermore, the ELISA analysis demonstrated that plvx-PTEN alleviated miR-93-5p mimics -induced elevation of testosterone production in Leydig cells (Fig. 6d). In addition, plvx-PTEN attenuated miR-93-5p mimic-induced enhancement of cell viability and proliferation in Leydig cells (Fig. 6e and f). In summary, these observations revealed that upregulation of PTEN restored the role of miR-93-5p in Leydig cells treated with LPS.

To determine whether MEG3 exerted its effect by regulating the miR-93-5p/PTEN pathway in Leydig cells treated with LPS, we first measured whether PTEN was regulated by MEG3/miR-93-5p pathway in Leydig cells treated with LPS. The results demonstrated that inhibition of LPS-induced PTEN by miR-93-5p mimics was restored by overexpressed MEG3 (Fig. 7a). These results demonstrated that MEG3 could alleviate the repression of PTEN by miR-93-5p in Leydig cells treated with LPS.

We designed and synthesized short-hair RNA specific against PTEN (shPTENs) and control short-hair RNA (shNC). We transfected shPTENs and shNC into the Leydig cells for 48 h. The results of qPCR analysis demonstrated that all three shPTENs could efficiently inhibit PTEN expression in Leydig cells, and shPTEN-3 was chosen for further experiments (Fig. 7b).

To determine the role of the MEG3/miR-93-5p/PTEN signalling pathway in Leydig cells treated with LPS, we divided the cells into five groups: mimics NC, LPS + mimics NC, LPS+ miR-93-5p mimics, LPS + miR-93-5p mimics+plvx-MEG3 and LPS + miR-93-5p mimics+plvx-MEG3 + shPTEN. The ELISA, CCK8 and EdU analyses demonstrated that any disturbers of the MEG3/miR-93-5p/PTEN signalling pathway affected the role of LPS in Leydig cells (Fig. 7c-f). In summary, these resulted revealed that LPS played a role via activation of the MEG3/miR-93-5p/PTEN signalling pathway in Leydig cells.