Aberrant DNA methylation of synaptophysin is involved in adrenal cortisol-producing adenoma

Expression of SYP in human adrenocortical adenoma specimens

To identify the expression of SYP in human adrenocortical adenoma, we performed the real time quantitative PCR, western blotting and immunohistochemical staining on 12 adrenocortical adenoma samples from CS and their adjacent normal adrenal cortex tissues.

The qRT-PCR results showed that the expression levels of SYP are significantly higher in the adrenocortical adenoma group than in the control group. Compared to the normal adrenal cortex tissues, the expression level of SYP mRNA was about 12-fold higher in CPAs (Figure 1A). Likewise, the protein expression levels of SYP were also significantly higher in the CPA group compared with the control tissues, as determined by Western blotting (Figure 1B, p<0.05). For the immunostaining, immunoreactivity for SYP was present in both groups. However, the intensity of staining for SYP was significantly stronger in CPAs than in their adjacent normal adrenal cortex tissues (Figure 1C).

Figure 1. The expression of SYP in CPA. (A and B) The relative mRNA levels of SYP determined by real-time PCR (A) and protein levels of SYP determined by Western blotting (B) in CPAs and normal adrenal tissues (n=12). (C) Immunohistochemisty staining determines the expression of SYP in CPAs and normal adrenal tissues. The brown area indicated by arrow is the positive staining of SYP in CPAs. Three independent experiments were performed, and representative data are shown. The data represent the mean ± SD. NC, normal control. **p<001.

Epigenetic mechanism of SYP up-regulation both in vitro and in vivo

Next, we explored the molecular mechanism that mediates the upregulation of SYP in CPA specimens. Previous studies have demonstrated that there is a CpG island located in the promoter of SYP, and the methylation status of the CpG island is related to SYP expression. Recently study reported that SYP is hypermethylated and decreased in human Alzheimer’s Disease brain tissue [21–23]; therefore, we hypothesised that the upregulation of SYP in CPAs is correlated with the promoter of methylation of SYP. We applied methylation PCR assay to detect the methylation status of SYP in CPA samples and the control samples. The results revealed that in the normal adrenal cortex the promoter of the CpG island was hypermethylated and the methylation rate of CpG sites of SYP reached as high as 60.5% whereas the methylation rate of CpG sites in the CPA group was about 34.0% (Figure 2A). To further confirm the promoter hypermethylation in the regulation of SYP expression, we treated cultured H295R cells (the adrenocortical carcinoma cell line, the most commonly cell line used to study adrenal tumours, which possesses a steroid secretion and regulation pattern similar to that of primary adrenal cell cultures.) with the DNA methyltransferase inhibitor 5-aza after identifying that the H295R cells had a hypermethylation status in the SYP promoter. Our results showed that significantly decreased methylation of the SYP promoter (Figure 2B) is associated with increased expression of SYP upon 5-aza treatment as shown by western blotting (Figure 2C, P<0.05). Thus, we reasoned that DNA methylation of the SYP promoter results in different expression of SYP both in vivo and in vitro.

Figure 2. Methylation of the SYP promoter regulates the expression of SYP and cortisol secretion and H295R cells proliferation. (A) SYP methylation levels in CPA and normal adrenal tissues was determined using EpiTect Methyl II PCR Assay (n=12). (B) H295R cells were treated with 5-azaC, SYP methylation levels was determined by EpiTect Methyl II PCR Assay. (C) H295R cells were treated with 5-aza (5 μm) for 48 h the SYP protein level was determined by western blotting. (D) H295R cells were treated with 5-aza (5 μm) for 48 h, then cortisol secretion in H295R cell supernatants were measured in cell supernatants by ELISA. (E and F) Knock-down of SYP by siRNA was confirmed by qRT-PCR (F) and Western blotting (E). (G) The H295R cells was treated with SYP siRNA or control, then cortisol secretion in H295R cell supernatants were measured by ELISA. (H) Knock-down of SYP inhibited H295R cell proliferation measured by CCK assay. Three independent experiments were performed, and representative data are shown. Data are shown as mean ± SD. **p<0.01, compared with normal control. *p<0.05, compared with DMSO control.

DNA methylation of SYP is involved in cortisol secretion and H295R cell proliferation

To determine whether the overexpression of SYP caused by DNA methylation is related to the secretion of cortisol, we measured the secretion of cortisol in cell culture medium after 5-aza treatment in H295R cells. Compared to the control group, the cortisol secretion of the 5-aza treatment group was significantly higher (Figure 2D). To further confirm our speculation that SYP plays a crucial role in regulating the expression of cortisol. We successfully transfected SYP siRNA 2# into H295R cells to knock down the expression of SYP. As shown in Figure 2E, 2FSYP expression was significantly downregulated after SYP siRNA treatment. Not surprisingly, we found that knock-down of SYP results in decreased secretion of cortisol (Figure 2G). Furthermore, the CCK-8 assay showed significant decreases in cell viability with the effective repression of SYP in H295R cells (Figure 2H).

Identification of miRNAs differentially expressed in cortisol-producing adenomas

With the goal of identifying the mechanism involved in dysregulation of SYP through methylation, three adrenocortical adenoma samples associated with CPA and their adjacent normal adrenal cortex tissues were chosen to identify the expression of miRNAs. These tissues were then subjected to miRNA microarray analysis, which identified a large number of miRNAs whose expression changed significantly between cortisol-producing adenomas and normal tissues (Figure 3A). According to the results of the microarray analysis, we chose to further investigate miR-27a-5p. The microarray analysis identified that miR-27a-5p is the most downregulated

Figure 3. Different miRNA expression in CPA and normal adrenal tissues and the function of miR-27a-5p on the proliferation of H295R cells. (A) Hierarchical clustering of differentially expressed miRNAs in CPA samples and normal adrenal tissues. (B) miR-27a-5p is downregulated about 5.4-fold in CPA in comparison with normal adrenal cortex tissues as indicated by microarray analysis. (C) qRT-PCR analysis of miR-27a-5p in CPA samples and their adjacent normal adrenal cortex tissues. Three independent experiments were performed, and representative data are shown. (D) Upregulation of miR-27a-5p using miR-27a-5p mimics, or downregulation of miR-27a-5p using miR-27a-5p inhibitor in H295R cells were confirmed by qRT-PCR. (E) Cell viability in H295R cells transfected with miR-27a-5p mimics and inhibitor were determined by the CCK-8 assay. (F–G) Analysis of EdU staining on miR-27a-5p mimic-treated H295R cells. The EdU incorporation rate was expressed as the ratio of EdU-positive cells to total DAPI positive cells. Red, EDU; Blue, DAPI. Magnification, 400×. Three independent experiments were performed, and representative data are shown. NC, normal control. **p<0.01. *p<0.05. NC, normal control.

miRNA in CPAs; its expression decreased 5.4-fold in CPAs (Figure 3B). As expected, the qRT-PCR analysis confirmed that the expression of miR-27a-5p decreased significantly by 9.7-fold (Figure 3C). According to the results of the microarray analysis and qRT-PCR result, we chose to further investigate miR-27a-5p for further investigation and hypothesised that miR-27a-5p might be involved in the development of CPA.

miR-27a-5p involved in regulating the proliferation of H295R cells

Sharply decreased expression of miR-27a-5p forced us to ask whether miR-27a-5p has a regulatory effect on the proliferation of H295R cells. To identify the role of miR-27a-5p during the proliferation of H295R cells, we used a gain- and loss-of-function approach to overexpress or inhibit expression of miR-27a-5p. Two days after transfection with miR-27a-5p mimics, miR-27a-5p inhibitors, or control oligos, the number of H295R cells were measured by the CCK-8 assay. qRT-PCR results showed that inhibition of miR-27a-5p inhibited miR-27a-5p expression, whereas over-expression induced endogenous miR-27a-5p levels over 184-fold (Figure 3D). The cell viability was significantly reduced by 28% in miR-27a-5p mimic transfected cells compared with NC in the CCK-8 assay, whereas it increased by 31% in miR-27a-5p inhibitor transfected cells compared with NC (Figure 3E). Moreover, we found that the percentage of EDU-positive cells in miR-27a-5p mimic transfected cells decreased significantly and the percentage of EDU-positive cells in miR-27a-5p inhibitor transfected cells showed the opposite results (Figure 3F, 3G). These results indicate that miR-27a-5p plays a negative role in H295R cell proliferation.

TET3 is the direct target gene of miR-27a-5p

miRNAs exert their function by regulating the expression of their downstream target genes [24]. We utilized several bioinformatic target prediction algorithms to search for targets related to miR-27a-5p. TET3 was revealed to be a potential target of miR-27a-5p (Figure 4A). Accordingly, we evaluated the expression levels of TET3 in the tissue samples used for immunohistochemistry. The intensity of staining for TET3 was significantly stronger in the CPAs than in their adjacent normal adrenal cortex tissues (Figure 4B). Furthermore, we detected downregulation of the TET3 protein in miR-27a-5p mimic transfected cells and upregulation of the TET3 protein in miR-27a-5p inhibitor transfected cells by Western blot analysis (Figure 4C).

Figure 4. TET3 is a direct target of miR-27a-5p and TET3 regulated SYP expression and cortisol secretion in H295R cells. (A) RNA22 predicts that TET3 is a potential target of miR-27a-5p. (B) Immunohistochemical staining for TET3 in CPA and normal adrenal tissue. DAB staining showed that the intensity of staining for TET3 was significantly stronger in CPAs than in normal adrenal tissues. The brown area indicated by arrow is the positive staining of TET3 in CPAs. Representative data are shown. (C) TET3 protein levels in H295R cells were assessed by Western blotsin control in miR-27a-5p mimics transfected cells and miR-27a-5p inhibitor transfected cells. (D) Luciferase reporter assays were performed using luciferase constructs carrying a WT or mutant TET3 3_-UTR cotransfected into H295R cells with miR-27a-5p mimics compared with an empty vector control. Firefly luciferase activity was normalized to renilla luciferase activity. (E) Western blot analysis of protein levels in H295R cells transfected with TET3 siRNA (siRNA 1#,2#) or scrambled control for 48h. (F) Methylation level of SYP promoter was analysis by EpiTect Methyl II PCR Assay in H295R cell transfected with TET3 siRNA or control. (G) Cortisol secretion were measured in cell supernatants by ELISA from H295R cell transfected with TET3 siRNA or control. Error bars represent SD. Three independent experiments were performed, and representative data are shown. The data represent the mean SD. NC, normal control. **p<0.01. *p<0.05.

To further determine whether miR-27a-5p directly binds TET3 mRNA and regulates its expression, a luciferase reporter construct containing the wild-type or mutant 3′-UTR coding sequences for TET3 was generated and introduced into miR-27a-5p mimics and H295R cells. Overexpression of miR-27a-5p significantly decreased the relative luciferase activity of the WT-3′-UTR of TET3 reporter plasmids, but when the miR-27a-5p seed sequence in TET3 mRNA 3′-UTR was mutated, the inhibitory effect of miR-27a-5p on the relative luciferase activity was abrogated (Figure 4D). Negative control miR-27a-5p mimics did not affect the wild-type or mutant constructs, confirming the specificity of the action. The results confirmed that TET3 is the direct target of miR-27a-5p.

SYP up-regulation is associated with TET3

The DNA methylation/demethylation status was controlled by the TET family of methylcytosine dioxygenases. We hypothesised that TET3 is associated with SYP hypomethylation in CPAs. Thus, we investigated the effect of TET3 knock-down by siRNA in H295R cells. The western blot results confirmed that TET3 was significantly downregulated when transfected with siRNA 2# (Figure 4E). As expected, SYP was significantly downregulated after being transfected with TET3 siRNA in H295R cells for two days (Figure 4E) and the methylation level of it significantly increased (Figure 4F). Secretion of cortisol was decreased at the same time (Figure 4G).

Tissue samples

Adrenocortical tissue sample collections were approved by the Ethics Committee of the Second Xiangya Hospital, Central South University, complied with the Declaration of Helsinki and written informed consent was obtained from all participants in our experiments.

Twenty-four adrenal tissue samples (12 adrenocortical adenomas from patients with adrenal Cushing’s syndrome, 12 from paired adjacent normal adrenal cortex) were obtained at surgical resection and immediately snap-frozen and stored at −80°C. The diagnosis of CPA was based on a typical clinical manifestation, increased serum cortisol levels with abnormal diurnal rhythm accompanied by decreased serum ACTH levels, increasing 24 h urinary-free cortisol, an un-suppressible result on the dexamethasone challenge test, and computed tomography results revealing unilateral adrenal tumours in all the patients. Moreover, the pathologic study confirmed the diagnosis. The clinical characteristics are summarized in Table 1.

Reagents

Dimethyl sulfoxide (DMSO) and 5-Aza-2′-deoxycytidine (5-Aza) were purchased from Sigma-Aldrich (St Louis, Missouri, USA). DMEM and fetal bovine serum were purchased from Gibco BRL Co. (Grand Island, New York). Lipofectamine 2000 was purchased from Invitrogen Co. (Carlsbad, California, USA). The antibody for β-actin was purchased from Abgent Inc. (San Diego, California, USA). Antibodies for SYP and TET3 were purchased from Abcam (Cambridge, England). Maxima SYBR Green/ROX qPCR Master Mix was purchased from Genecopoeia and all of the primers used in this research were purchased from Genecopoeia. The related secondary antibodies and the ECL detection kit were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA). SYP and TET3 siRNA oligos and control siRNA oligos were purchased from Ribobio (Guangzhou, China). miR-27a-5p mimics, inhibitors and their control oligos were purchased from Ribobio. DAPI was purchased from Solarbia (Beijing, China). The EdU kit was purchased from Ribobio. The CCK8 kit was purchased from 7 sea biotech (Shanghai, China).

Cell culture treatment and transfection

The adrenocortical carcinoma cell line H295R was obtained from the National Platform of Experimental Cell Resources for SciTech (Beijing, China). Cells were maintained in DMEM:F12 medium (Gibco, Life Technologies, Grand Island, New York, USA) supplemented with 2.5% of NuSerum (BD Biosciences, Bedford, Massachusetts, USA), 1% penicillin/
streptomycin (Gibco) and 1% insulin–transferin–selenium culture supplement (BD Biosciences) at 37°C in 5% CO₂ infusion and humidified air and the medium was refreshed every 2 days.

After passaging for 12 hours, cells were treated with 5-aza at 5 uM (Sigma-Aldrich) and vehicle for 24 and 48 hours. The medium was replaced every 24 hours. Total protein and DNA was extracted after 48 hours of treatment and analysed for SYP protein and methylation expression level. The experiments were repeated three times. For transient transfection of SYP siRNA oligos, a combination of oligos (50 nM) and Lipofectamine 2000 was mixed following the manufacturer’s instructions and added to cells in 6-well plates at a density of 2 × 10⁵ cells per well.

Measurement of cortisol

Cortisol secreted into the culture media was measured by competitive ELISA using commercial EIA kits (Cayman Chemical) according to the manufacturer’s instructions.

Western blot analysis

Western blot analysis was carried out for detection of SYP, TET3 or β-actin protein levels as previously described. Briefly, 30 μg of protein from each cell layer extract was loaded onto the same SDS PAGE and then transferred to a PVDF membrane. After blocking with 5% non-fat milk, the membrane was incubated with SYP, TET3 and β-actin overnight at 4°C. The next morning, the membrane was washed with PBS three times every ten minutes. The membrane was then incubated with appropriate secondary antibody at 1:2000 dilution in 2% non-fat milk for 1 h. Blots were processed using an ECL kit, exposed to film and then analysed by densitometry.

miRNA microarray analysis

Total RNA was extracted from cortisol-producing adenoma tissue samples and normal adrenal cortex tissue samples. All samples were assessed for enriched miRNA using an agilent 2100 Bioanalyzer. miRNAs were then labelled using the FlashTag Biotin RNA labelling kit (Affymetrix, Santa Clara, CA, USA) following the manufacturer’s protocol and then hybridized to Affymetrix GeneChip miRNA 1.0 microarrays (Affymetrix). Each miRNA was measured in duplicate.

Gene expression estimated using qRT-PCR

Total RNA was extracted from cortisol-producing adenoma tissue samples and normal adrenal cortex tissue samples, and cDNA was prepared. For analysis of miR-27a-5p expression, reverse transcription and quantitative reverse transcription-polymerase chain reaction (qRT–PCR) were carried out using the primer for human miR-27a-5p (Genecopoiea, MmiRQP0538) and U6 snRNA (Genecopoiea, China) according to the manufacturer’s instructions. SYP (HQP017850) and GAPDH (MQP027158) gene primers were purchased from Genecopoiea, and their mRNA expression was also measured by qRT–PCR amplification. Relative quantification was calculated through the 2^-△△CT method.

Gene specific DNA methylation determination

Total genomic DNA was isolated from cortisol-producing adenoma tissue samples, normal adrenal cortex tissue samples and H295R cells using QIAamp DNA mini kit (Qiagen, Germany). Genomic DNA was isolated using the binding column according to the manufacturer’s instructions. Gene specific DNA methylation was determined by using an EpiTect Methyl II PCR Assay (Qiagen, Germany) and methyl primer synaptophysin (catalog# EPHS115006-1A). We evaluated the methylation level of SYP according to the manufacturer’s instructions.

Immunohistochemistry

Paraffin-embedded 4-mm-thick specimens were dewaxed in turpentine and rehydrate through decreased concentrations of ethanol. Antigen retrieval was not performed. Endogenous peroxidase activity was blocked by using 3% H₂O₂ in methnal for 10min and then soaked with phosphate buffered saline (PBS) (pH 7.2–7.4) three times for 5 min. The sections were then pre-incubated with 0.25% trypsin-EDTA for 10 min to block non-specific antigens. The tissue sections were allowed to react overnight at 4°C with anti-SYP (dilution 1/100; Abcam) TET3 (dilution 1/200, Abcam) antibodies. The slides were then incubated at room temperature for 1h, rinsed with PBS three times and incubated with appropriate biotinylated secondary antibodies for 30 min. For the quantification of immunohistochemistry, 10 fields of each section were viewed and analysed with an image analysis program (Bioquant, Nashville, TN).

Plasmid constructs and luciferase reporter assay

For functional analysis of miR-27a-5p, segments of the TET3 3’-UTR, including the predicted miR-27a-5p binding sites, were amplified using PCR and cloned into the PmeI and XbaI restriction sites of the luciferase reporter vector pmirGLO (Promega, Madison, WI, USA), resulting wild-type TET3 3’UTR (WT-TET3-3’UTR). The TET3 mutants for the miR-27a-5p seed regions were prepared using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA, USA) to get mutant TET3 3’UTR (MUT-TET3-3’UTR). Sequences of the PCR and mutagenic primers are shown in (Table 2). H295R cells were transfected with either WT-TET3-3’UTR or MUT-TET3-3’UTR and miR-27a-5p mimics or control for 48 h. Luciferase activities were detected with the luciferase assay system (Promega).

Statistical analysis

The results of the experiments are presented as means ± SD, and analysis was performed with Statistical Product and Service Solutions (SPSS) software (version 19.0). Comparisons between values of more than two groups were evaluated by an analysis of variance (one-way ANOVA). A level of p<0.05 was considered statistically significant. All experiments were repeated at least three times, and representative experiments are shown in the Figures.