The study was performed in the Neuroscience Institute, Lithuanian University of Health Sciences between 2010 and 2016. After surgical resection tumor tissues were frozen in nitrogen liquid and the following clinical characteristics were gathered: gender, age at the time of the operation, relapse, tumor size, PA activity, invasiveness, hypersecretion of PRL, GH, IGF-1, ACTH or more than one hormone and diagnoses of Cushing syndrome, acromegaly or prolactinoma.
Study participants comprised of 102 patients (56.86% women and 43.14% men, 60.78% older than 60 years and 39.22% younger than 60 years). Clinical findings showed that the endocrinological features were: 54 functioning and 48 non-functioning adenomas. Functioning adenomas were: 4 GH-secreting adenomas, 2 IGF-1-secreting adenomas, 36 PRL-secreting adenomas, 1 ACTH-secreting adenoma and 11 adenomas secreting more than one hormone. Also, magnetic resonance imaging findings and the Hardy classification, modified by Wilson was used to quantify the sphenoid sinus invasion and suprasellar extension . In connection with the fact that not for all patients were able to receive data, relapse was established in 14 and invasiveness - in 88 patients. From them, it was found 58 invasive and 30 non-invasive PAs. It is important to mention that all PA tumors were macroadenomas.
Methylation-specific polymerase chain reaction (MS-PCR)
For the DNA methylation studies, 102 frozen PA tissues were used. First, DNA was extracted by 10% “UtraPure™ ”SDS (Invitrogen, USA), proteinase K (Carl Roth® GmbH, Germany), followed by phenol–chloroform (Carl Roth® GmbH, Germany) and 96% ethanol (Stumbras, Lithuania) precipitation. Subsequently, according to the manufacturer’s instructions, genomic DNA was modified with sodium bisulfite using EZ DNA methylation kit™ (Zymo Research, USA) and eluted in 40 μL of nuclease-free water.
The MS-PCR was performed in 15 μl of a mixture containing 10 pmol of each primer (Metabion International AG, Germany), 7.5 μL Maxima® Hot Start PCR Master Mix (ThermoFisher Scientific, USA) with Hot Start Taq DNA polymerase and nuclease-free water. MethPrimer online tool  was used to design primers for methylated STAT3 allele: 5′-TTTTGGGTGGTCGAACG-3′ (forward), 5′-AAAAACAACGCCAAACCG-3′ (reverse), resulting in a 222 bp PCR amplicon and for non-methylated allele: 5′-ATTTTTGGGTGGTTGAATG-3′ (forward), 5′-AAAAAAAACAACACCAAACC-3′ (reverse), resulting in a 225 bp PCR amplicon. The reaction was hot started at 95 °C for 5 min and MS-PCR conditions for all of the reactions were as follows: denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 15 s, for 38 cycles, and final 5 min extension at 72 °C. In each set of methylation-specific PCR reactions three controls were included: normal human blood lymphocyte DNA treated with bisulfite (non-methylated control), the Bisulfite-Converted Universal Methylated Human DNA Standard (Zymo Research, USA) (methylated control) and nuclease-free water (negative control).
The MS-PCR products were analyzed on a 2% agarose gel with ethidium bromide (10 mg/ml, Invitrogen, USA) by horizontal electrophoresis.
Quantitative real-time PCR (qRT-PCR)
For the STAT3 gene mRNA expression analysis, total RNA was extracted from the same set of PA samples (102 samples) that has been used for STAT3 gene methylation analysis with Trizol reagent (Ambion, Life Technologies, USA), according to the manufacturer’s instructions and stored at −80 °C. RevertAid H Minus M-MuLV Reverse Transcriptase (ThermoFisher Scientific, USA) and random hexamer primers (ThermoFisher Scientific, USA) were used to produce the first-strand cDNA. Total reaction volume was 20 μl. Negative controls without reverse transcriptase were prepared as above.
Quantitative real-time PCR for STAT3 and β-actin was performed in a Real-Time PCR System “Applied Biosystems 7500 Fast”(Applied Biosystems, USA) with SYBR Green chemistry. The qRT-PCR was carried out in a 12 μl of mixture which consisted of 6 μl Maxima SYBR Green/ROX qPCR Master Mix (2X) (ThermoFisher Scientific, USA), 15 ng of the cDNA, nuclease-free water and STAT3 gene-specific primers that were designed according to the published data : 5′-CATATGCGGCCAGCAAAGAA-3′ (forward), 5′-ATACCTGCTCTGAAGAAACT-3′ (reverse), resulting in a 152 bp PCR amplicon to a total concentration of 0.3 μM. The housekeeping gene β-actin with primers designed according to the published data : 5′- AGAGCTACGAGCTGCCTGAC-3′ (forward), 5′-AGCACTGTGTTGGCGTACAG-3′ (reverse), resulting in a 184 bp PCR amplicon to a total concentration of 0.1 μM, was used as an internal control. The PCR amplification cycles were as follows: the denaturation step at 95 °C for 10 min and then 92 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s for 40 cycles and a final step for the generation of a melting curve. Also, GAPDH TaqMan assay (Applied Biosystems, Hs02758991_g1, cat. No. 4331182) was used to verify the expression of another endogenous control in PAs. The PCR amplification was performed according to the manufacturer’s protocol. All sample measurements were performed in triplicate. Healthy human brain RNA sample (RHB) “FirstChoice Human Brain Reference RNA” (Ambion, Life Technologies, USA, cat. No. AM6050) was used for standard curve design and the parameters were: for STAT3: efficiency 92.68%, R2 = 0.99, slope − 3.51; for β-actin: efficiency 100.08%, R2 = 0.99, slope − 3.32.
Finally, results were presented as 2-ΔΔCt calculations, where ΔΔCt = (Ct,
)Sample x - (Ct,
Statistical analyses were conducted with the SPSS Statistics 19 (SPSS Inc., Chicago, IL) and GraphPad Prism 7 (California, USA) software packages. The Mann-Whitney test was used to find the associations between STAT3 gene mRNA expression and the methylation status, also the clinical factors: gender, age, relapse, Cushing syndrome, acromegaly, prolactinoma, invasiveness, secreting and non-secreting PAs and hormone groups. The associations among STAT3 gene promoter methylation and clinical characteristics of PA patients were assessed with Chi-square test and p < 0.05 values were considered statistically significant.