Pituitary tissue-specific miR-7a-5p regulates FSH expression in rat anterior adenohypophyseal cells

Animals and ethics

This study was performed with the approval of the Jilin University College of Animal Science. Healthy 8- to 9-week-old sexually mature male Sprague Dawley (SD) rats were used in this study. SD rats were raised in a comfortable room with a 12 h dark/12 h light cycle and free access to food and water.

We cared for the animals and sacrificed the rats in strict accordance with animal welfare laws and regulations and animal welfare ethics requirements. This experiment was approved by the Institutional Animal Care and Use Committee of Jilin University (201705026).

Pituitary extraction and cell culture

Sprague Dawley rats were euthanized by anesthesia and cervical dislocation. Then, we removed the heads with ophthalmic scissors and placed them on gauze disinfected with alcohol-soaked cotton. Next, we separated the rat skin with scissors to expose the rat skull. We opened the skull with tweezers and removed the brain to collect the pituitary gland. Next, the tissues were placed in precooled phosphate-buffered saline (PBS) containing 0.3% bovine serum albumin (BSA) and 1% penicillin/streptomycin. We also separated the neurohypophysis from the pituitary. To ensure aseptic operations during the experiment, we autoclaved all experimental instruments and performed the operations near an alcohol-burning lamp.

After separation, we used PBS (0.3% BSA and 0.1% penicillin/streptomycin) to clean the pituitary glands and wash the blood from the tissue. Next, we placed the samples in one ml Dulbecco’s Modified Eagle’s Medium-F12 (DMEM-F12) containing 2.5% collagenase type I and cut the pituitary into pieces with ophthalmic scissors. We placed the pituitary fragments in a temperature-controlled incubator containing 5% CO₂ at 37 °C for 90 min. After a 90 min incubation, we diluted pituitary cells with PBS (0.3% BSA and 0.1% penicillin/streptomycin) and then filtered the mixture through a 200 mesh (75 μm) cell sieve, which allowed pituitary cells to pass, while cell clusters and unhomogenized tissue could not pass through. The collected cell solution was centrifuged at 200×g for 10 min. After centrifugation, we carefully discarded the supernatant and diluted the cell precipitate with DMEM-F12 containing 15% fetal bovine serum (FBS). Finally, the diluted cell suspension was transferred to six-well plates and cultured in a temperature-controlled incubator with 5% CO₂ at 37 °C. Pituitary cells were monitored for the next few days.

Transfection of miR-7a-5p mimics, inhibitors or siRNA

After cells were cultured in six-well plates for 4 days and in 24-well plates for 1 day, transfection of miR-7a-5p mimics or inhibitors was carried out as FSHb expression and FSH secretion peaked when pituitary cells were cultured for approximately 6 days. We used 30 μl buffer, three μl reagent and 2.5 μl mimics/inhibitors in this transfection. First, we added buffer, reagent and mimics, inhibitors or siRNA to a centrifuge tube. Then, we used a vortex oscillator to centrifuge the sample briefly to ensure homogeneous mixing. Next, we transfected the mixture into 24-well plates filled with pituitary cell suspension. Finally, we placed the 24-well plates into a 5% CO₂ incubator for 24 h to provide adequate time for the reaction. All mimics, inhibitors and siRNAs were purchased from RiboBio (Gouzhou, China), and the transfection method and operation steps were performed in strict accordance with the manufacturer’s recommended protocol. The mimic sequence was the double-stranded sequence of the miRNA mature sequence (sense strand: 5′-UGGAAGACUAGUGAUUUUGUUGU-3′) and its complementary sequence (antisense strand: 3′-ACCUUCUGAUCACUAAAACAACA-5′). The inhibitor was the single-stranded sequence of the reverse complement of the miRNA mature sequence, which underwent full-chain methylated modification (5′-mAmCmAmAmCmAmAmAmAmUmCmAmCmUmAmGmUmCmUmUmCmCmA-3′).

RNA isolation and qRT-PCR

After transfection, we harvested rat primary anterior pituitary cells that were transfected with miR-7a-5p mimics or inhibitors in 24-well plates. We used 350 μl or 600 μl cell lysate RL containing 1% β-mercaptoethanol to cause cell rupture and then extracted RNA according to the instructions of an RNAprep Pure Cell/Bacteria Kit. We subsequently measured the concentration and purity of the RNA with a NanoDrop ND-2000 spectrophotometer (Beijing, China) to verify the operational accuracy and RNA quality with a standard. Total RNA extraction was performed on a clean bench, and the samples were maintained at low temperatures to prevent RNA degradation. Next, we obtained cDNA using a FastQuant RT Kit and acquired raw data via q-PCR with SuperReal PreMix Plus (SYBR Green). These reagent kits were purchased from Tiangen (Beijing, China). The mRNA and miRNA primers used in RT-PCR and qRT-PCR are listed in Table 1.

Detection of cell apoptosis by flow cytometry

Flow cytometry was applied to detect rat adenohypophysis cell apoptosis to evaluate the effect of transfection on cells after 24 h. At an appropriate time point, we used trypsin to digest adhesive cells and transferred them into 10-ml reaction tubes. Then, the cells were centrifuged at 200×g for 5 min and harvested by cell sedimentation. Next, we resuspended cells in 500 μl 1× working fluid by diluting 5× binding buffer with double-distilled water. Then, Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) were added to sample tubes and parameter regulation tubes according to an Annexin V-FITC/PI Apoptosis Kit (Multi Sciences, Hangzhou, China). Finally, we analyzed cell apoptosis via flow cytometry for 1 h.

Construction of the reporter plasmids

The pmiR-FSHb-3′UTR-wild-type (WT) plasmid and pmiR-FSHb-3′UTR-MUT plasmid were constructed to verify the target sites of the FSHb 3′UTR and miR-7a-5p. The PCR primers and mutant sequence of the target site are listed in File S1.

Detection of FSH secretion

After transfection with miR-7a-5p mimics or inhibitors for 24 h, we cultured pituitary cells with serum-free medium instead of DMEM-F12 (15% FBS) because FBS may contain other hormones that influence the results. After 24 h, we collected the culture medium and measured the secretion level of FSH in the culture medium via a Rat FSH enzyme-linked immunosorbent assay (ELISA) Kit (Meilian Biotech Co., Ltd., Shanghai, China).

Statistical analysis

At least three replicates were performed for each experiment. One-way ANOVA and Chi-square tests were performed to evaluate the statistical significance of differences. P < 0.05 was considered statistically significant.

Prediction and verification of the complementary region between miR-7a-5p and the FSHb 3′UTR

First, information on the complementary sequence between miR-7a-5p and the FSHb 3′UTR was acquired via the TargetScan program (http://www.targetscan.org/) (Fig. 1A). Then, to further confirm that miR-7a-5p targets the FSHb 3′UTR, we successfully mutated the target complementary sequence TCTTCCA to AGAAGGT and constructed a FSHb-3′UTR-WT plasmid and FSHb-3′UTR mutated (MT) plasmid (Fig. 1B). Finally, the constructed plasmids were cotransfected with miR-7a-5p mimics into 293T cells. As expected, the luciferase activity was reduced by 36% when we cotransfected the pmiR-FSHb-3′UTR WT plasmid and miR-7a-5p mimics into 293T cells. In contrast, cotransfection of the FSHb-3′UTR MUT plasmid and miR-7a-5p mimics resulted in a 7% reduction in luciferase activity (Fig. 1C). Therefore, we concluded that miR-7a-5p may regulate FSHb expression by directly targeting the FSHb gene.

Expression level of miR-7a-5p in different developmental stages and various rat tissues

To ascertain whether miR-7a-5p expression shows discrepancies, we measured miR-7a-5p levels in different developmental stages and rat tissues. We selected 2-week-old rats as the nonsexually mature group and 4-month-old rats as the sexually mature group. Then, we detected the expression levels of miR-7a-5p by RNA isolation and qRT-PCR and normalized them to the levels in immature animals (by setting this value to 1). MiR-7a-5p expression was downregulated in mature rats compared to that in nonsexually mature rats (Fig. 2A). Next, we collected seven tissues from mature rats and detected the expression level of miR-7a-5p by qRT-PCR. Relative miR-7a-5p expression was extremely high (1,378-fold) in the pituitary gland compared to that in the heart. Furthermore, miR-7a-5p was poorly expressed in the heart, liver, spleen, lung and kidney, although it was slightly expressed in the brain (Fig. 2B). These findings suggested that miR-7a-5p is much more highly expressed in the rat pituitary gland than in other tissues.

Efficiency of transfection and subsequent impact

To detect the efficiency of transfection, we transfected the NC mimic with fluorescent markers into pituitary cells. We detected red fluorescence labeling in the cells. The results showed that the NC mimic was successfully transfected into pituitary cells. The transfection efficiency was approximately 70–80%, as shown in Fig. 3A. Flow cytometry was performed to assess the damage from transfection and certain reagents. No significant differences in cell apoptosis were observed among the four groups, indicating that the impact of transfection was negligible (Fig. 3B). We examined the expression levels of miR-7a-5p after transfection of miR-7a-5p mimics or inhibitors for 24 h to verify whether the mimics and inhibitors were transfected into pituitary cells. The expression levels of miR-7a-5p in cells transfected with mimics were increased, while the expression levels of miR-7a-5p in cells transfected with inhibitors were lower than those in cells transfected with controls (Fig. 3C). In other words, transfection was successful and reliable.

Effects of miR-7a-5p overexpression/blockade on FSH secretion

We examined FSHb mRNA levels and FSH hormone secretion after the transfection of miR-7a-5p mimics, inhibitors and siRNA into rat primary anterior pituitary cells for 24 h to further verify that miR-7a-5p affects FSHb expression and regulates animal reproduction. As a positive control, rat primary pituitary cells were transfected with FSHb siRNA, and we examined the levels of FSHb by quantitative RT-PCR and FSH secretion by ELISA 24 h after transfection. As expected, FSHb levels and FSH secretion significantly decreased (P < 0.001, P = 0.002) (Figs. 4A and 4B). The expression levels of FSHb decreased by 0.60-fold (P = 0.002) after transfection with miR-7a-5p mimics compared to those after transfection with the negative control. In contrast, after transfection with a miR-7a-5p inhibitor, FSHb levels increased by 1.5-fold (P < 0.001) (Fig. 4C). We subsequently measured FSH secretion levels. As expected, FSH secretion showed the same trend as that of FSHb expression. After transfection with miR-7a-5p mimics, the FSH concentration was substantially decreased. Moreover, the FSH concentration increased after transfection with the miR-7a-5p inhibitor (Fig. 4D).

These results indicated that miR-7a-5p can decrease FSHb expression and reduce FSH hormone secretion. Our findings provide additional evidence showing that miRNAs regulate FSH, demonstrating their potential role in the pituitary.