Snap-frozen pituitary tumor tissues were obtained from 50 pituitary tumor patients (age range, 45–68 years; mean age, 58.4 years; female:male ratio, 1:1.32) between October 2013 and August 2015. Patients who had received chemotherapy or radiotherapy were excluded. The pituitary samples from patients without a diagnosis of pituitary cancer were included as the non-tumor control group to compare the expression of miR-1 in cancer tissues (n=50; mean age, 55.8 years; age range, 41–69 years; female:male ratio, 1:1.27). These tissues were used for analyzing the expression of miR-1 in cancer, and for evaluating the association between the expression of miR-1 and the metastasis and tumor size of the patients. Another 120 patients, who were used for analyzing the association between the expression of miR-1 and the 5-year overall survival rate, were enrolled between April 2008 and July 2011 (n=120; mean age, 60.2 years; female:male ratio, 1.18).

All the tissue samples were obtained from the People's Hospital of Yichang City Center affiliated to China Three Gorges University (Yichang, Hubei, China). Patients were not subjected to radiotherapy or chemotherapy prior to the surgical resection. All tissues were stored in liquid nitrogen until use. Written informed consent was provided by all participants and the study was approved by the Ethical Committee of China Three Gorges University. The clinicopathological parameters, including age, gender, differentiation, tumor size, lymph node metastasis and clinical stage, of all 170 patients are summarized in Table I.

The human pituitary adenoma HP75 cell line and the rat pituitary gland neoplasm MMQ cell line were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 12.5% horse serum and 2.5% fetal bovine serum (all Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), with 5 U/ml penicillin and 5 g/ml streptomycin (Beyotime Institute of Biotechnology, Shanghai, China) in an atmosphere with 5% CO₂ at 37°C. For the cell transfection, MMQ and HP75 cells were seeded in 6-well plates and when the cell confluence reached 70–80%, miR-1 mimics (5′-UGGAAUGUAAAGAACU-3′) or negative control miRNA (5′-UUUGUACUACACAAAAGUACUG-3′) were transfected into the cells at the final concentration of 20 nM for 48 h with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols.

The full length of G6PD was amplified by PCR (as described below) and inserted into the pcDNA3.0-Flag vector. To investigate the effect of G6PD on the growth of pituitary cancer cells, Flag-G6PD was transfected into the cells with Lipofectamine 2000.

Total RNA was extracted from pituitary tissues and cell lines with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The purity and quality of RNA were determined with the NanoDrop-2000 (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). cDNA was obtained with the Omniscript Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) by reverse transcription with 0.5 µg RNA. Using the cDNA as template, the relative expression of miR-1 was determined with SYBR green mix (Tiangen Biotech Co., Ltd., Beijing, China) on the ABI7500 quantitative PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) in a 10-µl reaction system. The primers of miR-1 and U6 were designed as below: miR-1 forward, 5′-CAGTGCGTGTCGTGGAGT-3′ and reverse, 5′-GGCCTGGATGTAAAGAAGT-3′; and U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′. The reaction conditions were set as: 95°C for 5 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Data were analyzed with the 2^−ΔΔCq method (41). The level of miR-1 was normalized using U6 RNA as the internal reference.

The wild-type or mutant 3′-UTR sequences of G6PD containing the putative binding sites of miR-1 were cloned into the pmirGLO reporter vector (Promega Corporation, Madison, WI, USA), respectively. MMQ and HP75 cells were co-transfected with this luciferase reporter vector and the indicated miRNA using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Subsequent to transfection for 48 h, the cells were harvested and the luciferase activity was determined with the Dual-luciferase Reporter assay system (cat. no. E1910; Promega Corporation). Renilla luciferase activity was measured as the method of endogenous normalization.

MMQ and HP75 cells were transfected with miR-1 mimics or control miRNA using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Subsequent to transfection for 48 h, the cells were collected and lyzed with the NP-40 lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was determined with the bicinchoninic acid assay (BCA) kit (Beyotime Institute of Biotechnology). Protein from each sample (20 µg) was loaded and separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. The membranes were blocked with 5% skimmed milk in PBS at room temperature for 1 h and then incubated with anti-G6PD antibody (1:3,000 dilution; cat. no. PA5-18734; Thermo Fisher Scientific, Inc.) at room temperature for 2 h. The membranes were washed twice with Tris-buffered saline plus Tween-20 and incubated with goat anti-mouse horseradish-peroxidase-conjugated secondary antibody (1:5,000 dilution; cat. no. AS003; ABclonal Biotech Co., Ltd., Woburn, MA, USA) for 1 h at room temperature. The expression of β-actin (1:3,000; cat. no. AA128; Beyotime Institute of Biotechnology) was detected as the loading control. The protein bands were visualized with electrochemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.) using the typhoon scanner (GE Healthcare, Chicago, IL, USA).

The growth of the cells was evaluated using the CellTiter 96 AQueous One Solution Cell Proliferation assay kit (Promega Corporation) according to the manufacturer's protocols. Briefly, the cells transfected with miR-1 mimics or control miRNA were re-seeded into a 96-well plate at a density of 1,000 cells/well. Subsequent to being cultured for 24 h, 20 µl MTT was added once into the DMEM at the time point of 1, 2, 3, 4 or 5 days and incubated at 37°C for 3 h. The absorbance (formazan was dissolved in DMSO) of each well at 480 nm was measured with a microplate reader. The experiment was performed in triplicate.

The cell apoptosis percentage of the pituitary tumor cells was determined with the Annexin V-Fluorescein Isothiocyanate Apoptosis Detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Briefly, cells were transfected with miR-1 mimics or control miRNA. Subsequent to transfection for 48 h, the cells were stained with Annexin V-FITC and propidium iodide working solution for 15 min in the dark at room temperature. The cell apoptosis rate was analyzed using flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA).

Pituitary tumor cells transfected with the indicated miRNA were seeded in the 6-well plate at 1,000 cells/well. Subsequent to being cultured for 2 weeks, the cells were washed twice with PBS and fixed with methanol for 15 min at room temperature. The cell colonies were stained with 1% crystal violet for 10 min at room temperature. The colonies were counted by light microscopy.

The downstream targets of miR-1 were found using the TargetScan database (http://www.targetscan.org/mamm_31/), with the species set as human and the conserved microRNA family as miR-1/206 HMRDC. Upon submitting the search request, the putative downstream targets of miR-1 were summarized in a table, which included the gene name and the binding sites in the 3′-UTR.

Pituitary tumor cells were transfected with miR-1 or control miRNA using Lipofectamine 2000 (Invitrogen: Thermo Fisher Scientific, Inc.). When the cell confluence reached 60–70%, the cells were washed with fresh DMEM without glucose and incubated with medium containing 10 mM [2-¹³C] glucose for 10 h. The medium from the experimental and control groups was analyzed in a 20-nm nuclear magnetic resonance tube with a 9.4T spectrometer at 100.66 MHz. Incorporation of ¹³C in the carbon 2 and 3 of the lactate represented the glucose metabolism from glycolysis and the PPP, respectively. The oxidative PPP flux was determined by the ratio of ¹³C in carbon 2 (glycolysis) and carbon 3 (oxidative PPP flux) of lactate, as well as the rate of glucose uptake. The number of cells that were transfected with miR-1 or control miRNA was compared to eliminate the influence caused by the variation of cell amounts.

Pituitary tumor cells were transfected with miR-1 mimics or control miRNA. Subsequent to transfection for 48 h, the cells were lysed with buffer containing 0.1 M Tris-HCl (pH 8.0), 0.05% Triton X-100 and 0.01 M EDTA. The lysates were sonicated and centrifuged at 3,000 × g for 10 min at 4°C. The level of NADPH was detected for the absorbance at the wavelength of 341 nm by spectrometry. The protein concentration was determined using the BCA kit (Beyotime Institute of Biotechnology) to normalize the influence of the cell number.

The data from three independent experiments are presented as the mean ± standard error. The statistical analysis was performed using the SPSS software (13.0, IBM Corp., Armonk, NY, USA). The difference between two groups was analyzed with Student's t-test. The differences between more than two groups were detected by one-way analysis of variance followed by Dunnett's post hoc test. Kaplan-Meier survival analysis was performed to compare the overall survival rates of patients with pituitary tumors grouped by high and low miR-1 expression levels (the mean value of miR-1expression in pituitary cancer tissues was considered as the cut-off). The log-rank test was used to analyze the statistical significance. Spearman's rank correlation test was performed to analyze the correlation between the expression of miR-1 and G6PD in the pituitary tumor tissues. P<0.05 was considered to indicate a statistically significant difference.

To understand the function of miR-1 in pituitary tumors, the expression of miR-1 in pituitary tumor tissues and adjacent normal pituitary tissues was evaluated by RT-qPCR analysis. As shown in Fig. 1A, the expression of miR-1 was significantly decreased in pituitary tumor tissues compared with that in the control group. The decreased expression of miR-1 in pituitary tumor tissues motivated investigation of the association between the level of miR-1 and the clinicopathological factors of patients with pituitary cancer. The analysis data showed that among the 50 patients, the expression of miR-1 was significantly lower in the patients with lymph metastasis compared with that in the patients without metastasis (Fig. 1B). However, there was no significant difference in the expression of miR-1 with regard to the size of the tumors (Fig. 1C). Based on these data, Kaplan-Meier survival analysis was performed to investigate the association between the expression of miR-1 and the 5-year overall survival of patients was analyzed in another 120 pituitary tumor patients. As shown in Fig. 1D, low expression of miR-1 was significantly associated with a worse prognosis in the patients. These results suggested that decreased abundance of miR-1 was a possible biomarker for predicting the prognosis of patients with pituitary cancer.

Given the decreased expression of miR-1 in pituitary tumors, the influence of miR-1 on the growth of pituitary tumor cells was then investigated. MMQ and HP75 cells were transfected with miR-1 mimics or control miRNA. The expression of miR-1 was confirmed by RT-qPCR analysis, as presented in Fig. 2A. An MTT assay was performed to detect the proliferation rate of pituitary tumor cells expressing miR-1 mimics or control miRNA. The results showed that upregulation of miR-1 significantly decreased the proliferation of the MMQ and HP75 cells (Fig. 2B and C). To further characterize the inhibitory effect of miR-1 on pituitary tumor cells, an in vitro colony formation assay was performed with MMQ and HP75 cells harboring overexpressed miR-1 or control miRNA. The data indicated that in the MMQ and HP75 cells, highly expressed miR-1 significantly suppressed the colony formation of the pituitary tumor cells (Fig. 2D). The cell apoptosis of MMQ and HP75 cells with overexpressed miR-1 was also evaluated. As illustrated in Fig. 2E, a significantly increased cell apoptosis rate was observed in the pituitary tumor cells with highly expressed miR-1. These results demonstrated that overexpression of miR-1 inhibited the growth of pituitary tumor cells.

To understand the functional mechanism of miR-1 in pituitary cancer, the downstream targets of miR-1 were predicted by the TargetScan database, and ~440 possible conserved targets of miR-1 were found. These targets mainly function in regulating the proliferation and differentiation of cells. G6PD ranked top among all the predicted target candidates of miR-1. The putative binding sites of miR-1 at the 3′-UTR of G6PD are shown in Fig. 3A. To test this observation, a luciferase reporter assay was performed by co-transfecting miR-1 mimics with the wild-type or mutant 3′-UTR of G6PD into the pituitary tumor cells. The data showed that overexpression of miR-1 significantly decreased the luciferase activity of the wild-type but not the mutant 3′-UTR of G6PD in the MMQ and HP75 cells (Fig. 3B and C). To further confirm this result, the protein level of G6PD in pituitary tumor cells expressing miR-1 mimics or control miRNA was detected by western blotting with anti-G6PD antibody. A decreased protein level of G6PD was observed with the high expression of miR-1 in the MMQ and HP75 cells (Fig. 3D). At the same time, the mRNA level of G6PD was also evaluated with overexpressed miR-1. However, the results showed that no significance was obtained for the mRNA abundance of G6PD in the presence of miR-1 compared with that of the control cells (Fig. 3E). These results demonstrated that G6PD was a target of miR-1 and negatively regulated the protein expression of G6PD in pituitary tumor cells.

It has been well documented that the PPP generates NADPH and ribose-5-phosphate to facilitate the biosynthesis of nucleotides and glycolysis, respectively, which maintains the rapid growth of cancer cells (42). G6PD has been demonstrated as the first and rate-limiting enzyme of the PPP (42). Due to the negative regulation of miR-1 on G6PD, an assessment of whether overexpression of miR-1 modulated the PPP in pituitary tumor cells was performed. The oxidative PPP flux (flux through the oxidative branch of PPP and glycolysis), glucose consumption and lactate production of pituitary tumor cells transfected with overexpressed miR-1 were detected. As shown in Fig. 4A, the ectopic expression of miR-1 resulted in a significant decrease in the oxidative PPP flux. Since PPP serves important roles in the production of cellular NADPH, the influence of miR-1 on the generation of NADPH was also measured. The data showed that overexpression of miR-1 suppressed the level of NADPH compared with that of the control group (Fig. 4B). The glucose uptake and lactate production were also significantly decreased in the MMQ and HP75 cells expressing miR-1 mimics (Fig. 4C and D). These results indicated that miR-1 suppressed the PPP and glucose metabolism of the pituitary tumor cells.

To further characterize the association between miR-1 and G6PD, the expression of G6PD in paired pituitary tumor tissues and adjacent normal tissues was detected by RT-qPCR. The data showed that the mRNA level of G6PD was significantly increased in the pituitary tumor tissues compared with that in the corresponding normal tissues (Fig. 5A). Spearman's rank correlation test indicated that the expression of miR-1 and G6PD in pituitary tumor tissues was significantly inversely correlated (Fig. 5B). To confirm that the inhibitory effect of miR-1 on the growth of pituitary tumor cells was through the regulation of G6PD, MMQ and HP75 cells expressing miR-1 mimics were transfected to ectopically express G6PD. As shown in Fig. 5C and D, the MTT assay showed that highly expressed miR-1 decreased the cell proliferation, while restoring the expression of G6PD significantly inhibited the suppressive function of miR-1 on the growth of pituitary tumor cells. To further confirm this observation, the colony formation of pituitary tumor cells that were transfected with miR-1 or control miRNA with G6PD was performed. The data showed that in comparison with the cells expressing miR-1, the overexpression of G6PD significantly promoted the colony formation of the MMQ and HP75 cells (Fig. 5E). The fluorescence-activated cell sorting (FACS) analysis showed that overexpression of G6PD attenuated the cell apoptosis of pituitary tumor cells that were induced with the transfection of miR-1 (Fig. 5F). These results suggested that G6PD serves important roles for mediating the inhibitory effect of miR-1 on the growth of pituitary tumor cells.