Clinical samples of pituitary tumors tissues and paired normal tissues from patients at Shanghai Changzheng Hospital (Shanghai, China) were collected after surgery with written informed consents from all patients and in compliance with the Medical Ethics Committee of Shanghai Changzheng Hospital under the approval no. 2021SLYS5. A total of 20 patients with a median age of 38.3 years were included in the present study. Patients without any treatment prior to surgery were enrolled, and samples were obtained from January 2018 to December 2019.

AtT-20 (cat. no. TCM 1) and 293T (Cat. no. GNHu17) were purchased from the Cell Bank of Chinese Academy of Sciences, Shanghai, China. GT1-1 (cat. no. 1101MOU-PUMC000009) was purchased from the National Infrastructure of Cell Line Resource. AtT-20 was cultured in RPMI-1640 medium (cat. no. L210KJ; Shanghai BasalMedia Technologies Co., Ltd.) supplemented with 10% fetal bovine serum (FBS) (product code 04-001-1ACS; Wolcavi Biotech), while 293T and GT1-1 were cultured in Dulbecco's modified Eagle's medium (DMEM) (cat. no. L110KJ; Shanghai BasalMedia Technologies Co., Ltd.) supplemented with 10% FBS. All culture medium contained penicillin and streptomycin (both were 100 U/ml; cat. no. P1400; Beijing Solarbio Science & Technology Co., Ltd.), and was conditioned at 37°C with 5% CO₂ in an incubator (STERI-CYCLE i160; Thermo Fisher Scientific, Inc.).

Unless otherwise stated, all drugs were obtained from MedChemExpress, and incubations were performed with full medium in the incubator for 24 h. Nicotinamide (cat. no. HY-B0150) was used at 5 mM, resveratrol (cat. no. HY-16561) was used at 10 µM, PJ34 (cat. no. HY-13688A) and DPQ (cat. no. T19849; TargetMol Chemicals, Inc.) were used at 10 µM, and JQ1 (cat. no. HY-112789) was used at 0.5 µM. Insulin (cat. no. 11038-HNAY; SinoBiological, Inc.) was used at 20 ng/ml and IGF-1 (cat. no. 50437-MNAY; SinoBiological Inc.) was used at 100 ng/ml; both growth factors were incubated with cells for 1 h in the incubator.

Total RNA was extracted from pituitary tumor and adjacent normal tissues by TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and quantified by NanoDrop 2000c (Thermo Fisher Scientific, Inc.). A total of 2 µg RNA was reverse transcribed according to the manufacturer's instructions using Reverse Transcription System (cat. no. A3500; Promega Corporation). For real-time PCR, 2X SYBR Premix Taq system (Yeasen Biotechnology Co., Ltd.) was used and for detection on PikoReal Real-Time PCR system (Thermo Fisher Scientific, Inc.) was used. Thermocycling conditions were as follows: Initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec, extension at 72°C for 20 sec. The method of quantification used was 2^−ΔΔCq (12). Primers used to detect SIRT1 were as follows: Forward primer, 5′-GGCGGCTTGATGGTAATCAG-3′ and reverse primer, 5′-AGTCCCAAATCCAGCTCCTC-3′. Primers used to detect GAPDH were as follows: Forward primer, 5′-CCATGGGGAAGGTGAAGGTC-3′ and reverse primer, 5′-GGGATCTCGCTCCTGGAAGA-3′.

Immunostaining of SIRT1 (cat. no. 13161-1-AP; diluted 1:100) and interleukin (IL)-6 (cat. no. 21865-1-AP; diluted 1:100; both from ProteinTech Group, Inc.) was performed with paraffin-embedded pituitary tumor sections [first fixed in 4% paraformaldehyde (cat. no. P1110; Beijing Solarbio Science & Technology Co., Ltd.) for 24 h in a cold room] of 5-µm thickness. Essentially, sections were first deparaffinized at 65°C for 30 min, and incubated with antibodies overnight in a cold room (4–8°C), followed by incubation with an HRP-conjugated secondary antibody at room temperature for 1 h (product no. 7074; diluted 1:200; Cell Signaling Technology, Inc.). The signal was obtained with DAB Substrate Kit (product no. 8059; Cell Signaling Technology). Images were obtained at ×40 and ×200 magnifications with a light microscope (BX61; Olympus Corporation).

Western blotting was performed traditionally with slight modifications. Briefly, cells were seeded at 3×10⁵ cells/well of a 6-well plate, and were harvested 48 h later, followed by lysing in RIPA buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) supplied with protease inhibitor (cat. no. B14002; Bimake.cn). Protein concentration was determined with Bradford reagent (Bio-Rad Laboratories, Inc.). Unless otherwise stated 30 µg protein samples were added per lane of 10% SDS-PAGE gel. A total of 5% BSA dissolved in TBST was used to block the PVDF membrane at room temperature for 1 h, and to dilute primary and secondary antibodies. Incubation of the primary antibodies was performed in a cold room overnight, and secondary antibody incubation at room temperature for 1.5 h. ECL (cat. no. sb-wb012; http://www.share-bio.com/) was used to detect HRP signal intensity. Chemiluminescent signals were recorded by CCD camera (Tanon system; Tanon Science & Technology Co., Ltd.). Primary antibodies used in this experiment included (unless otherwise specified all antibodies were purchased from Affinity Biosciences): anti-SIRT1 (cat. no. DF6033), anti-β-actin (cat. no. AF7018), anti-CD38 (cat. no. DF6551), anti-PTTG1 (cat. no. AF0354), pan-AKT (cat. no. AF6261), phosphorylated (p)-AKT (Thr308) (cat. no. AF3262), anti-GCN5L2 (cat. no. DF3383). The secondary antibody used was goat anti-rabbit (H + L) linked with HRP (cat. no. S0001; Affinity Biosciences).

Stable cell lines were constructed via lentivirus-mediated gene delivery. pEF1a-MCS-IRES-GFP and pLKO.1-TRC were used to overexpress and knockdown target genes respectively. SIRT1 H363Y mutation was obtained by site-directed mutagenesis, and primers used were as follows: Forward primer, 5′-aggataattcagtgttatggttcct-3′ and reverse primer, 5′-gttgcaaaggaaccataacactga-3′. To produce lentiviruses, 293T cells were transfected with the aforementioned core plasmids, along with the packaging plasmids psPAX2 and pMD2.G (cat. no. 12260 and 12259, respectively; Addgene, Inc.) at a ratio of 12:8:4 µg and Lipofectamine^® 2000 transfection reagent (used according to the manufacturer's instructions; refreshing the medium 6 h post-transfection; Thermo Fisher Scientific, Inc.). Viruses were harvested at 72-h post-transfection (the medium was refreshed every 24 h). Transfection of GT1-1 and AtT20 cells was performed in 6-well plates with polybrene at a final concentration of 2.5 µg/ml for 24 h and at an MOI of 0.5. Cells were sorted for GFP signals by FACS using FACSAria (Beckman Coulter, Inc.) or selected with puromycin at 4 µg/ml for 3 days or longer. Overexpression and knockdown efficacies were demonstrated by western blot analysis. Short hairpin (sh)RNA primers used were as follows: shSIRT1-1 forward primer, 5′-CCGGAGTGAGACCAGTAGCACTAATCTCGAGATTAGTGCTACTGGTCTCACTTTTTTG-3′ and reverse primer: 5′-AATTCAAAAAAGTGAGACCAGTAGCACTAATCTCGAGATTAGTGCTACTGGTCTCACT-3′; shSIRT1-2 forward primer, 5′-CCGGCCTCGAACAATTCTTAAAGATCTCGAGATCTTTAAGAATTGTTCGAGGTTTTTG-3′ and reverse primer, 5′-AATTCAAAAACCTCGAACAATTCTTAAAGATCTCGAGATCTTTAAGAATTGTTCGAGG-3′; shGCN5 forward primer, 5′-CCGGGCTACCTACAAAGTCAATTATCTCGAGATAATTGACTTTGTAGGTAGCTTTTTG-3′ and reverse primer, 5′-AATTCAAAAAGCTACCTACAAAGTCAATTATCTCGAGATAATTGACTTTGTAGGTAGC-3′; shBRD4-1 forward primer, 5′-CCGGTGAACCTCCCTGATTACTATACTCGAGTATAGTAATCAGGGAGGTTCATTTTTG-3′ and reverse primer, 5′-AATTCAAAAATGAACCTCCCTGATTACTATACTCGAGTATAGTAATCAGGGAGGTTCA-3′; shBRD4-2 forward primer, 5′-CCGGGCGGCAGCTAAGTCTAGATATCTCGAGATATCTAGACTTAGCTGCCGCTTTTTG-3′ and reverse primer, 5′-AATTCAAAAAGCGGCAGCTAAGTCTAGATATCTCGAGATATCTAGACTTAGCTGCCGC-3′.

MTT assays were performed to determine the cell proliferation rate. GT1-1 cells were dissociated from a 10-cm plate, AtT-20 cells were pelleted down, and the cell concentration was determined by autonomous cell counter. Cells (1×10³) were added into each well of a 96-well plate. After 24 h of seeding in the incubator, MTT was added into the 96-well plate for 4 h, followed by aspiration to remove the media. DMSO was then used to dissolve cellular debris and the OD570 was measured and recorded. These steps were repeated for the next 4 days. All data was combined and analyzed using GraphPad Prism 6.0 software (GraphPad Software, Inc.).

Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. Cells were seeded at 1×10⁴ cells/well of a 96-well plate. After 24 h of seeding, drugs indicated in the specific assays were added. After another 24 h, 20 µl CCK-8 was added, and incubated for 3 h. Following incubation, the absorbance at 450 nm was determined using a microplate reader (PerkinElmer Enspire; PerkinElmer, Inc.). Data was processed using GraphPad Prism 6.0 software.

The invasion assay was performed using a Transwell chamber (8 µM pore size; product no. 3422; Corning, Inc.). A total of 1×10⁶ cells resuspended in 200 µl DMEM or RPMI-1640 medium were seeded into each Transwell with 50 µl Matrigel (precoated at 37°C for 30 min and diluted 1:2) while 700 µl full medium was added to the lower compartment. Following incubation for 48 h in the incubator, cells on the outer membrane were stained at room temperature with 0.05% crystal violet containing 20% methanol for 10 min. Images were obtained with a light microscope (RVL-100-G; ECHO).

ChIP-qPCR assays were used to determine the binding of SIRT1 and H3K9ac to PTTG1 promoter. Cells were first cross-linked with 1% formaldehyde, followed by scraping into ice-cold PBS with protease inhibitors. Then cells were resuspended in lysis buffer (20 mM HEPES, pH=7.9, 420 mM NaCl, 0.2 mM EDTA, 0.5% NP-40, 25% glycerol and 1.5 mM MgCl₂), followed by several brief periods of sonication. One-third of the cell extract was kept as the input sample, and two-thirds of the cell extract was used as the substrate for immunoprecipitation with anti-SIRT1 and anti-H3K9ac antibodies. A total of 5 M NaCl was used to reverse the cross-linking after which the eluted DNA was extracted for PCR analysis. The primer sequences for the PTTG1 promoter are listed as follows: forward primer, 5′-ttcgtgaaagagtaatatgg3′ and reverse primer, 5′-atcctcaatacttcaggctaag-3′.

All data analyzed is presented as the mean ± standard deviation (SD), and was performed using GraphPad Prism 6. Paired Student's t-test was used to compare variants in two groups. For each Student's t-test, each group contained more than 3 replicates. P<0.05 was considered to indicate a statistically significant difference.

The expression pattern of SIRT1 was assessed in 20 pairs of clinical pituitary tumors by RT-qPCR. The results demonstrated that SIRT1 expression was lower in tumor tissue compared with paired normal tissue in 15 (75% of total samples examined) patients (Fig. 1A). Consistently, immunohistochemical staining of SIRT1 also revealed that SIRT1 expression was decreased in tumors compared with normal tissue (Fig. 1B). To determine the function of SIRT1 in pituitary tumors, the endogenous expression of SIRT1 was knocked down in AtT20 cells (a mouse pituitary tumor cell line) (Fig. 1C). Subsequently, cellular growth rate and invasion were assessed. The results revealed that the growth rate of tumor cells was accelerated, while AtT20 cell invasion was also increased, as a result of SIRT1 knockdown (KD) (Fig. 1C and D). The aforementioned results were also observed in the GT1-1 cell line (Fig. 1E and F). Furthermore, SIRT1 was exogenously expressed in both AtT20 and GT1-1 cells (Fig. 1G and I). To this end, SIRT1 overexpression in both cell lines inhibited cell growth and invasion (Fig. 1G-J). Collectively, SIRT1 was downregulated in pituitary tumors and its expression reduced pituitary tumor cell growth and invasion.

Due to the fact that resveratrol is an activator of SIRT1 (10), the present study aimed to determine whether resveratrol mimicked the tumor suppressive effect of SIRT1 overexpression in pituitary tumor cells. The results revealed that resveratrol induced AtT20 and GT1-1 cell death in a concentration-dependent manner (Fig. 2A). The invasion of AtT20 and GT1-1 cells was also inhibited (Fig. 2B). While these results indicated that the promotion of SIRT1 activity inhibited pituitary tumor cell growth and invasion, whether SIRT1 inhibition could increase tumor cell growth and invasion required further elucidation. As nicotinamide is a known antagonist of SIRT1 (10), the present study treated pituitary cells with nicotinamide. The results demonstrated that nicotinamide promoted cell growth (Fig. 2C) and invasion (Fig. 2D).

It has been demonstrated that a mutation of histidine to tyrosine in the SIRT1 protein at position 363 abrogates the enzymatic activity of SIRT1 (13). To further verify that SIRT1 activity regulated pituitary tumor cell growth and invasion, wild-type (WT) SIRT1 and H363Y mutant SIRT1 was expressed in AtT20 cells (Fig. 2E). While WT SIRT1 inhibited cell growth, H363Y mutant SIRT1 exerted no effect (Fig. 2F). Further treatment with resveratrol induced the robust inhibition of cell growth in AtT20 cells expressing WT SIRT1; however, this effect was not observed in H363Y SIRT1-expressed cells (Fig. 2G). The endogenous activity of SIRT1 is mainly regulated by NAD⁺ levels, indicating that upregulating cellular NAD⁺ levels may promote SIRT1 activity. PJ34 and DPQ are two chemical compounds that are known to inhibit PARP function and upregulate cellular NAD⁺ levels (14). In the present study, treatment of control AtT20 cells with PJ34 and DPQ inhibited cell growth, while SIRT1 KD abolished this effect (Fig. 2H). To decrease cellular NAD⁺ levels, CD38 was overexpressed, as it is an NADase enzyme. In AtT20 cells, the cell growth assay revealed that CD38 overexpression promoted cell growth, while SIRT1 KD inhibited this effect (Fig. 2I). Thus, the enzymatic activity of SIRT1 regulated pituitary tumor cell growth and invasion.

PTTG1 is an oncogene that is expressed in pituitary tumors but not in normal pituitary glands. As SIRT1 is mainly localized to the nucleus (10), the present study aimed to determine whether PTTG1 expression was regulated by SIRT1. The results demonstrated that PTTG1 mRNA and protein levels were increased upon SIRT1 inhibition (Fig. 3A). Furthermore, SIRT1 overexpression in AtT20 cells led to decreased levels of PTTG1 mRNA and protein expression (Fig. 3B). Moreover, SIRT1 enzymatic activation by resveratrol decreased PTTG1 expression, while its inhibition by nicotinamide increased PTTG1 expression (Fig. 3C and D). This trend was also observed in clinical pituitary tumor tissues (Fig. 3E). As insulin and insulin-like growth factor 1 (IGF-1) activate PTTG1 mRNA transcription (15), the present study aimed to elucidate whether SIRT1 was involved in this process. As presented in Fig. 3F, insulin and IGF-1 treatment increased PTTG1 expression, while resveratrol attenuated PTTG1 expression. The aforementioned results demonstrated that SIRT1 regulated PTTG1 expression.

To gain insight into the mechanism underlying PTTG1 regulation by SIRT1, the present study hypothesized that SIRT1 may deacetylate H3K9 to suppress PTTG1 transcription. The results demonstrated that SIRT1 was localized to the PTTG1 promoter region, and its inhibition enriched H3K9ac at the PTTG1 promoter (Fig. 4A). Furthermore, resveratrol treatment, which activated SIRT1, decreased H3K9ac levels at the PTTG1 promoter (Fig. 4B). To confirm that H3K9ac levels regulated PTTG1 expression, the expression of GCN5 was knocked down, as GCN5 is known to modify H3K9 with acetyl moiety (16). The results of the chromatin immunoprecipitation assay revealed that H3K9ac levels were decreased at the PTTG1 promoter (Fig. 4C). Furthermore, GCN5 inhibition resulted in the downregulation of PTTG1 mRNA (Fig. 4D). As GCN5 inhibition decreased H3K9ac levels at the PTTG1 promoter, the present study aimed to determine whether SIRT1 regulated PTTG1 expression independently of H3K9ac. As presented in Fig. 4E, while SIRT1 KD upregulated PTTG1, this regulation did not occur in GCN5 KD cells. The results suggested that SIRT1 deacetylated the H3K9 to regulate PTTG1 expression.

To corroborate that H3K9ac mediated the regulation of PTTG1 expression by SIRT1, the expression of BRD4 was knocked down. BRD4 is an H3K9ac reader that when coupled to this, causes downstream RNA synthesis (17). The results revealed that BRD4 KD reduced PTTG1 expression (Fig. 5A and B). JQ1 has been reported to be an inhibitor of the BRD protein family (18), thus indicating that JQ1 treatment may mimic the effect of BRD4 KD. As presented in Fig. 5C and D, JQ1 treatment led to a decreased expression of PTTG1 in a concentration-dependent manner. Furthermore, treatment of JQ1 abolished the PTTG1 expression that was induced by SIRT1 KD (Fig. 5E). The aforementioned results revealed that PTTG1 expression was regulated by H3K9ac, and that this modification could be divided into two aspects: Processes determining the abundance of H3K9ac and cellular ability to decode H3K9ac. While SIRT1 reduced the abundance of H3K9ac, BRD4 KD attenuated the ability of cells to decode the H3K9ac signal. To assess whether SIRT1 activation and BRD4 inhibition would synergically inhibit PTTG1 expression, AtT20 and GT1-1 cells were treated with resveratrol and JQ1. It was demonstrated that resveratrol treatment alone inhibited cell growth, and that the concurrent treatment of resveratrol and JQ1 significantly reduced cell growth (Fig. 5F and G). The results suggested that H3K9ac mediated PTTG1 expression via SIRT1.