In the study, we retrospectively reviewed 48 patients who had undergone pituitary surgery at Beijing Tiantan Hospital between 2008 and 2012. All patients had plasma prolactin (PRL) levels >200 ng/ml, and positive immunostaining for PRL. Medical therapy was interrupted at least 2 months before surgery. Tumor size was determined by MRI, and tumors were classified as microadenomas (<1cm diameter), macroadenomas (>1 cm and <4 cm), and giant adenomas (>4 cm). The mean postoperative follow-up was 4.8 years (range: 2.5–7 years). Recurrence-free survival was measured from the date of surgery to the date of tumor recurrence. Patients were censored at the date of the last neuroimaging follow-up. Normal human anterior pituitaries of people who died of non-neurological or non-endocrine diseases were obtained from a donation program. The Ethics Committee of Beijing Tiantan Hospital study approved the protocol, and informed consent was obtained from all patients. The patient characteristics are summarized in Table 1.
Tumor samples and tissue microarray construction
Formalin-fixed paraffin-embedded tissue blocks were sectioned and stained with hematoxylin and eosin (H&E). Three 2.0 mm diameter core biopsies were selected from the paraffin-embedded tissue blocks and transferred to tissue microarrays (TMAs) using a Mini core Tissue Arrayer (Mitogen, UK). Tissue microarrays were cut into 4 μm sections using a serial microtome and samples were randomly ordered and anonymized on the TMA slides. To minimize loss of antigenicity, the microarray slides were processed within 1 week of cutting.
IHC techniques and antibodies
In advance of IHC, TMA slides were stained with H&E and evaluated for quality and tumor content. TMAs were processed in a Leica BOND-III (Leica Biosystems, Germany) automated, random, and continuous-access slide staining system that simultaneously performed several IHC assays. A Bond Polymer Refine Detection System (Leica Biosystems, Germany) was used for the detection of primary antibodies. Appropriate positive and negative controls were used for each antibody, and TMAs were stained for each antibody in the same run to avoid interassay variability. The immunostained slides were examined for expression using an Aperio AT2 digital scanner (Leica Biosystems, Germany). Primary antibodies anti-pSer126-Pit-1 (4 μg/ml, Abmart), was commercially developed using standard methods by injection of specific Pit-1 -phosphothreonine peptide Ac-VVL(pS) PSHGIE-amide into a rabbit at the Abmart antibody production facility, Shanghai, China. The optimal titer of primary antibodies had been determined in previous experiments. The percentage of immunostaining and the staining intensity (0, negative; 1+, weak; 2+, moderate; and 3+, strong) were recorded and an H-score was calculated as follows:
H-score = (% cells 1+) + 2(% cells 2+) + 3(% cells 3+).Based on the H-score, pSer126-Pit-1 staining in the tissue sections was categorized as low (H-score of ≤168), or high (H-score >168).
Rat pituitary cells (GH3) were obtained from the China Infrastructure of Cell Line Resources (Beijing, China) and cultured in 35 mm dishes, we use ATCC‐formulated F‐12K medium (Invitrogen) containing 2.5% fetal bovine serum (Gibco) and 15% horse serum (Gibco) in a 37°C incubator with a humidified atmosphere of 95% air and 5% CO2. The culture medium was replaced every other day.
Plasmid construction and CDK5 inhibitor
A CDK5 siRNA (SR507441) construct was purchased from OriGene Technologies (Rockville, MD, USA). Mutant Pit-1 (GFP-S126A-Pit-1) and Pit-1 were generated by GenScript Biotech (Nanjing, China). All constructs were confirmed by DNA sequencing (Shanghai Shenggong Bio, China). Roscovitine was obtained from Sigma-Aldrich (R7772; St. Louis, MO, USA).
Cell counting kit-8 (CCK-8) assay
Cells were seeded in 96-well plates at the density of 1 ×104 cells/well in 100 μl of cell culture medium for 24 hours and were then transiently transfected with the indicated plasmids and short interfering RNA Cell viability was measured using the CCK-8 assay kit (Dojindo, Japan). Following incubation, 10 μl of CCK-8 solution was added to each well of the 96-well plate and cultured for 3 h in an incubator. The optical density was measured at 450 nm and a proliferation curve based on time and absorbance was generated.
Colony formation test
The treated cell lines were seeded into six-well plates at 1000 cells per well and incubated for 2 weeks. After incubation, the cells were fixed in 4% paraformaldehyde for 15 minutes and stained in 1 mL of a 0.1% crystal violet solution for 30 minutes. Photograph the culture plate. Visible colonies in each well were quantified by Image J software.
PRL protein levels were determined using a rat PRL ELISA kit from BioVision (K4688‐100) according to the manufacturer's instructions. GH3 cells were harvested 72 hr after treatment with plasmid. The total protein content of the cells was determined for standardization of PRL production with a BCA protein assay kit (Pierce Biotechnology). The culture supernatants were collected and normalized to the cell numbers.
Cell apoptosis assay
cell apoptosis was determined using Annexin V-FITC/PI kits (BD Biosciences, Franklin Lakes, NJ). Cells were seeded for 48h after transiently transfected with the indicated plasmids. Then cells were harvested and stained with annexin V-FITC and PI according to the instructions of the manufacturer. Cells were analyzed using BD Accuri™C6 (BD Biosciences). Data analysis was performed using CFlow® software (BD Biosciences).
Electrophoretic mobility shift assay (EMSA)
We used a 5'-biotinylated oligonucleotide as a probe. The probes were incubated with the recombinant protein at room temperature for 30 min. The entire reaction mixture was run on a nondenaturing 0.5×TBE 6% polyacrylamide gel at 60 V for 1 h at 4°C, and then the mix was transferred onto Biodyne® B nylon membranes (Pall Corporation). Signals were visualized with reagents included in the kit and with a ChemiDoc XRS system (Bio-Rad Laboratories, UAS).
Luciferase reporter assay
GH3 cells were cultured at a density of 2 × 104 cells/well in 96-well culture plates. The cells were transfected with 0.2 μg of dual-luciferase reporterconstruct p1, or they were cotransfected with 0.2 μg of the luciferase reporter construct p2 and the internal control vector pRL-TK, pRL-SV40, or pRL-CMV (Promega, Madison, WI) at a ratio of 20:1 (reporter construct: control vector); transfections were performed using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Five hours posttransfection, the transfection medium was removed and replaced with medium containing 6 μM curcumin (Sigma-Aldrich, St. Louis, MO) solubilized in 100% dimethyl sulfoxide (Sigma). Forty-eight hours posttransfection, luciferase activity was measured using a Dual-Luciferase® Reporter Assay System (Promega). Renilla luciferase activity was normalized to firefly luciferase activity in cells transfected with the dual-luciferase reporter construct p1, and firefly luciferase activity was normalized to Renilla luciferase activity in cells cotransfected with the reporter construct p2 and the control vector.
Protein extraction and Western blot analysisand antibodies
Collected cells were washed with 1× PBS buffer, prepared with RIPA buffer supplemented with protease/phosphatase inhibitor cocktail, and centrifuged at 12,000 r/min for 5 min at 4°C to yield the total protein extract in the supernatants. The protein concentration was measured with a BCA assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Equal amounts of protein were separated by 8% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% nonfat milk in Tris‐buffered saline with Tween®20 (TBST) for approximately 1 hr, followed by incubation with anti-β-actin (1:5000; A1978, Sigma‐Aldrich), and rabbit polyclonal anti-CDK5 (ab40773, 1/200) overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) at room temperature for 1 hr. ImageJ (NIH) was used to quantify the protein band densities. Primary antibodies anti-CDK5 (ab40773, 1/200) were obtained from Abcam (Cambridge, MA, USA). Anti-Pit-1 (sc-393943, 1/100) was sourced from Santa Cruz Biotechnology (Dallas, TX, USA). The optimal titer of primary antibodies had been determined in previous experiments.
All statistical analyses used GraphPad Prism 7.00 statistical software. Experimental data are reported as the mean ± SD (standard error of measurement) of at least three independent experiments, as indicated in the respective figure legends and methods. Statistical analysis was performed by one-way ANOVA or Student’s t-test. A P-value＜0.05 was considered statistically significant.