Between January 2023 and February 2023, 10 paired eutopic endometrial (EU) and ectopic endometrial (EC) tissues were derived from 10 adenomyotic patients who received a partial resection of adenomyotic tissues at Shanghai Pudong Hospital (Shanghai, China). The last tissue was collected on February 16, 2023. The preoperative diagnosis of adenomyosis was based on the typical clinical symptoms of dysmenorrhea and/or menorrhagia and pelvic pain, physical examination findings and imaging results, including transvaginal ultrasound and MRI scans. EC tissue, referring to the adenomyosis nidus formed locally by the invasion of the endometrium and stroma into the muscle layer, was collected during laparoscopic surgical procedures, and the paired EU tissue was collected through scraping during surgery. The age of the patients included in the present study ranged between 35 and 45 years, with a mean age of 38.96±5.01 years. All women in the control and adenomyosis groups had regular menstrual cycles (28-35 days). The exclusion criteria were: i) Patients who were diagnosed with uterine fibroids and subsequently received hormonal therapy within the previous 6 months; ii) patients who had reproductive tract infections, immune system disease or endocrine diseases; iii) patients whose preoperative hysteroscopy excluded the diagnosis of endometrial lesions; and iv) patients with comorbidities or major organ dysfunction. Samples were obtained when the women were in the early proliferative phase of the menstrual cycle. Normal endometrial (CE) tissues were obtained between January 2023 and February 2023 from 10 women (age range, 27-41 years; mean age, 34.27±4.62 years) who voluntarily sought placement of an intrauterine device at Shanghai Pudong Hospital (Shanghai, China) and who had no diseases and had not used hormone drugs in the previous 3 months; these patients were considered controls. Written informed consent was obtained from all participating patients. The present study was approved by the Ethics Committee of Shanghai Pudong Hospital (approval no. 2023-WZ-04) and performed in accordance with the tenets and guidelines of the Declaration of Helsinki.

The three types of fresh tissues (CE, EU and EC tissues) were washed three times in Hank's Balanced Salt Solution (Gibco; Thermo Fisher Scientific, Inc.) containing 1,000 U/ml penicillin and 1,000 µg/ml streptomycin. The 10 CE, EU or EC samples were digested together using 0.4% collagenase and 0.05% pancreatic enzyme plus 20 µg/ml DNase I at 37˚C with shaking. After 2-3 h, the digestive solution was replaced with DMEM-F12 (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C with 5% CO₂. CE, EU and EC cells were obtained using a method established in a previous study (18). The primary cells were passaged at a fusion rate of 80% after digestion using 0.25% trypsin. The cells were passaged every 3 days. At 8 days after tissue digestion, CE, EU and EC cells (third passage) were used for the subsequent experiments.

Lentiviruses (including SLC38A2 overexpression and knockdown lentiviruses, constructed into the pLVX-Puro plasmid and pLKO.1 plasmid, respectively) were custom synthesized from Shanghai GeneChem Co., Ltd. Briefly, the SLC38A2 coding sequence was cloned into the pLVX-Puro vector (Clontech; Takara Bio USA, Inc.), and the SLC38A2 short hairpin RNA (shRNA/sh) was cloned into the pLKO.1 vector (Clontech; Takara Bio USA, Inc.). The 293T cells (The Cell Bank of Type Culture Collection of The Chinese Academy of Science) were transfected with the constructed vector, lentiviral packaging and envelope plasmids (psPAX2 and pMD2G; at the ratio of 10:9:1 µg; Addgene, Inc.) using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.). The 293T cells were cultured with DMEM-F12 containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C with 5% CO₂ for 48 h. The lentiviral supernatants were then harvested by centrifugation at 3,000 x g at 4˚C for 10 min. and filtered using a 0.45-µm filter. A MOI of 10 was used to infect cells. The EU/EC cells were infected with lentiviral supernatant, incubated at 37˚C overnight using DMEM-F12 containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C with 5% CO₂, selected with 1.5 µg/ml puromycin (Merck KGaA) for 14 days and maintained with 0.625 µg/ml puromycin for the subsequent experiments. The following three knockdown lentiviruses and a non-targeting control (shNC) were used: shSLC38A2-1, 5'-GCTTTGTTCTTCCTGTTAA-3'; shSLC38A2-2, 5'-GAAGAAGTATGAAACAGAA-3'; shSLC38A2-3, 5'-GCATCTGGATCAATTACAA-3'; and shNC, 5'-GGACGAGCTGTACAAGTAA-3'. The empty pLVX-Puro vector was used as the NC for overexpression, and the cells in the control group were not infected.

The cell experiment was divided into three sections. In section 1, we have assessed the effects of Gln exposure on the cell viability of EU and EC cells. The EU and EC cells were treated with Gln (1.5 mM; G3126; Merck KGaA) and the cells were cultured with DMEM-F12 containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C with 5% CO₂ for 48 h. Finally, cell viability was assessed using a Cell Counting Kit-8 assay. In section 2, the effects of SLC38A2 knockdown on EC cells and SLC38A2 overexpression on EU cells were evaluated. The effectiveness of the vector was detected by RT-qPCR and western blotting. The stable transgenic EC cells and EU cells were cultured with DMEM-F12 containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C with 5% CO₂ for 48 h. The cell viability was assessed at 0, 12, 24 and 48 h. The cellular oxygen consumption rate (OCR), invasion, Gln content and relative protein levels were finally evaluated. In section 3, the SLC38A2-overexpressing EU cells were treated with 10 µM CB-839 (glutaminase inhibitor; Merck KGaA) and cultured with DMEM-F12 containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C with 5% CO₂ for 48 h. The cell viability was assessed at 0, 12, 24 and 48 h. The Gln content and cell invasion were finally evaluated. Three independent experiments were performed in duplicate (six duplications for the OCR assay).

Total RNA was isolated from tissues or treated cells (2x10⁷) using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using a High Capacity cDNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The fluorophore used in qPCR was 2x SYBR Green PCR Master mix (Thermo Fisher Scientific, Inc.), and PCR was performed on an ABI7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Denaturation 95˚C for 5 min, followed by 40 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 20 sec and extension at 72˚C for 20 sec. Relative gene expression was analyzed using the 2^-ΔΔCq method with GAPDH as the internal control (19). The primer sequences are listed in Table I. Three independent experiments were performed in duplicate.

Cells (2x10³ cells per well) were seeded in a 96-well plate with six replicates per group and cultured at 37˚C overnight. Then, cells in each group were treated as indicated. At each time point, the culture medium was replaced with 90 µl DMEM-F12 without FBS and 10 µl Cell Counting Kit-8 (Beyotime Institute of Biotechnology) for 1 h of culture. Finally, cell proliferation was analyzed by measuring the absorbance value at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

The Transwell assay was performed to evaluated the invasion of cells. The Transwell chamber (Corning, Inc.) was pre-coated with 80 µl Matrigel (Corning, Inc.) at 37˚C for 30 min. Subsequently, cells (1x10⁵ cells/ml; 200 µl) were added into the upper chamber of the insert with DMEM-F12 without FBS, and DMEM-F12 with 10% FBS was added to the lower chamber. After 48 h of incubation at 37˚C, the cells in the lower chamber were fixed with 4% formaldehyde solution for 10 min at room temperature and stained with 0.5% crystal violet for 30 min at room temperature. Finally, unmigrated cells in the upper chamber were removed and the migrated cells in the lower chamber were imaged using a light microscope (CX33; Olympus Corporation).

The cellular OCR was analyzed using a Seahorse XF24 analyzer (Agilent Technologies, Inc.) as described in a previous study (20). The OCR experiments were performed in six parallel replicates.

Cells were seeded in a 24-well plate at a density of 1x10⁴ cells/well and treated as indicated. The concentration of Gln was measured using a Glutamine Assay Kit (Colorimetric) (cat. no. ab197011; Abcam) according to the manufacturer's instructions.

The total protein of the cell samples was extracted using protein extraction reagent (RIPA buffer; cat. no. 89901; Thermo Fisher Scientific, Inc.) and the protein concentration was initially measured using the BCA method according to the instructions of the Pierce Rapid Gold BCA assay kit (cat. no. A53226; Thermo Fisher Scientific, Inc.). Thereafter, total protein (25 µg/lane) was fractionated via 10% SDS-PAGE and transferred onto nitrocellulose membranes (MilliporeSigma), which were then blocked with 5% nonfat dry milk at room temperature for 1 h, and incubated with primary antibodies at 4˚C overnight. After washing three times using TBS with Tween-20 (0.1%), the membranes were incubated with horseradish peroxidase-labelled secondary antibody anti-IgG (cat. no. A0208/A0192; 1:1,000; Beyotime Institute of Biotechnology) at 37˚C for 1 h. An enhanced chemiluminescence system (Ranon GIS-2008; Tanon Science and Technology Co., Ltd.) was used to determine the protein levels using ECL-PLUS reagent (Thermo Fisher Scientific, Inc.). The protein bands were analyzed using ImageJ 1.8 (National Institutes of Health). The primary antibodies were: Anti-SLC38A2 (cat. no. bs-12125R; 1:1,000; BIOSS), anti-NADH-ubiquinone oxidoreductase subunit B8 (NDUFB8; cat. no. ab192878; 1:1,000; Abcam), anti-ubiquinol-cytochrome c reductase core protein 2 (UQCRC2; cat. no. ab203832; 1:1,000; Abcam) and anti-β-actin (cat. no. 81115-1-RR; 1:5,000; Proteintech Group, Inc.). β-actin served as the loading control.

The results are presented as the mean ± standard deviation of three or more independent experiments performed in duplicate. GraphPad Prism 7 (Dotmatics) was used for statistical analysis and graph generation. Statistical comparisons between groups were performed using an unpaired Student's t-test, or a one-way ANOVA followed by Tukey's multiple comparisons test, or a two-way ANOVA with Bonferroni as the post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The mRNA expression levels of the SLC38A family, including SLC38A1-11, were examined in CE, EU and EC tissues. As shown in Fig. 1A-C, SLC38A2, SLC38A3, SLC38A4, SLC38A5, SLC38A8, SLC38A10 and SLC38A11 expression was increased in EU and EC tissues. However, only SLC38A2 expression was significantly upregulated in EC samples compared with that in CE samples.

Gln was applied to the cells derived from EU and EC tissues. A cell proliferation assay demonstrated that Gln significantly promoted EC cell proliferation at 48 h after treatment (Fig. 1D). However, no significant increase was observed in EU cells with or without Gln treatment. EC cells with or without Gln treatment exhibited enhanced viability compared with EU cells, indicating that only EC cell proliferation was dependent on Gln.

To explore the function of SLC38A2, lentiviruses targeting SLC38A2 were constructed to knock down SLC38A2 expression. As shown in Fig. 2A, both the protein and mRNA levels of SLC38A2 were markedly decreased following transfection with shSLC38A2 lentiviruses. Cell proliferation was examined and the results showed that SLC38A2 knockdown decreased the proliferation of EC cells at 48 h (Fig. 2B). Similarly, the number of invading cells was significantly decreased in the SLC38A2 knockdown groups compared with that in the NC group, indicating that silencing of SLC38A2 significantly inhibited the invasion of EC cells (Fig. 2C). Furthermore, SLC38A2 knockdown decreased the Gln content in EC cells (Fig. 2D). SLC38A2 knockdown reduced the OCR, and downregulated the protein levels of NDUFB8 and UQCRC2, which are markers of mitochondrial respiration (21) (Fig. 2E-G).

SLC38A2 was overexpressed in EU cells following transfection with SLC38A2 overexpression lentivirus (Fig. 3A). Cell proliferation and invasion were analyzed after SLC38A2 overexpression. As shown in Fig. 3B and C, SLC38A2 overexpression significantly increased the proliferation and invasion of EU cells at 48 h. In addition, SLC38A2 overexpression increased the Gln content in EU cells (Fig. 3D), implying that the amount of Gln was increased. SLC38A2 overexpression increased the OCR, and increased the protein levels of NDUFB8 and UQCRC2 (Fig. 3E-G).

The present study then explored whether the function of SLC38A2 was dependent on the Gln content. Accordingly, 10 µM CB-839, a glutaminase inhibitor, was used to treat EU cells after SLC38A2 was overexpressed. The increase in cell proliferation induced by SLC38A2 overexpression was inhibited by CB-839 at 48 h (Fig. 4A). A higher Gln content was observed in the SLC38A2 overexpression group compared with the vector group. Compared with that in the SLC38A2 overexpression group, the Gln content in the group treated with SLC38A2 overexpression lentivirus and CB-839 was significantly decreased (Fig. 4B). As shown in Fig. 4C, the number of invading cells was increased by SLC38A2 overexpression but decreased by CB-839 treatment compared with the vector group, and CB-839 abolished the effect of SLC38A2 overexpression, indicating that the promoting effect of SLC38A2 overexpression on cell invasion was blocked by CB-839. Additionally, CB-839 treatment alone decreased the cell proliferation at 48 h and invasion rates, and Gln content in EU cells (Fig. 4A-C). Taken together, these results indicated that SLC38A2 functioned by regulating Gln metabolism.