The study protocol was approved by the Ethics Committee of Affiliated Hospital of Weifang Medical University (approval no. NCT02115879). Oral informed consent was obtained from the participants of the study.

GC samples were collected from patients (31.8-43.3 years old, mean 37.5 years old) (with or without endometriosis) who underwent in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) at the Affiliated Hospital of Weifang Medical University between April 2013 and December 2018. Endometriosis was diagnosed according to laparoscopic or laparotomy findings. Patients with adenomyosis or malignant neoplasm were excluded. Endometriosis stage was assessed in accordance with the revised American Society for Reproductive Medicine criteria (26). Participants that conformed to the following inclusion criteria were included: Basal follicle-stimulating hormone levels of <10 IU/ml, basal estradiol levels of <50 pg/ml, antral follicle count (AFC) of >5 and regular menstrual cycle. At 34-36 h prior to oocyte collection, controlled ovarian stimulation was adopted to perform IVF or ICSI. Basal serum sex hormone levels in patients on days 2-3 of the menstrual cycle was measured using the human follicle stimulating hormone ELISA kit (cat. no. ab108641; Abcam) and estradiol ELISA kit (cat. no. ab108667; Abcam).

After collection, samples were purified as described previously (27). Under transvaginal ultrasound guidance, samples were punctured to collect follicular fluid, and follicles with a diameter of >10 mm were aspirated, followed by centrifugation at 400 x g for 10 min at 4˚C. Using cell pellets, a cell suspension was prepared in a DMEM medium (10569010, Thermo Fisher Scientific, Inc.) and layered over 50% Percoll (P1644; Sigma-Aldrich; Merck KGaA), followed by centrifugation at 400 x g for 20 min at 4˚C. GCs located on the interface were collected and washed in PBS. The resultant pellet was used to perform following analysis.

The KGN cells, derived from human ovarian granulosa-like tumour, were cultured as previously described (28). KGN cells were regularly cultured (37˚C, 5% CO₂) in DMEM (10569010; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (HyClone; Cytiva). For BML-275 treatment, KGN cells were incubated with 5 nM BML-275 for 8 h

MALAT1 knockdown was demonstrated using MALAT1 short hairpin RNA (100 pmol/l, shRNA; sequence: 5'-AATGGAATATGTGTCTGGAGG-3'). The sequence of negative control shRNA is 5'-ACGACGTCAGCTGGTGCATGT-3' (100 pmol/l). At 48 h post transfection, cells were applied for the following experiments. Briefly, GCs were inoculated on six-well plates (4x10⁴ cells/ml) and directly incubated with the MALAT1 or NC shRNA construct. After 2-3 weeks of culture in puromycin, cells with stable MALAT1 knockdown were validated using western blotting.

Cell survival was evaluated using an MTT assay. In brief, after MTT treatment (0.5 mg/ml) at 20 µl, cells were further incubated with 150 µl DMSO until the formazan dye was dissolved. Formazan absorbance was determined at a wavelength of 490 nm using a microplate reader (Tecan Group, Ltd.).

Following inoculation in a 24-well plate (1x10⁴ cells/ml), cells were incubated with an EdU stock solution (Invitrogen; Thermo Fisher Scientific, Inc.) for 2 h at 48 h after transfection. Incubation was terminated by washing the cells in PBS, and cells were fixed in 4% paraformaldehyde for 20 min at 25˚C, permeabilized in 0.3% Triton X-100 for 10 min at 25˚C and blocked in 10% FBS (HyClone; GE Healthcare Life Sciences) for 60 min. Cells were sequentially probed with rabbit anti-EdU primary antibody (cat. no. ab6326; Abcam) at a dilution of 1:200 at 4˚C overnight and with secondary Cy3-conjugated antibody (1:500; cat. no. ab98416; Abcam) for 2 h. Incorporated EdU was detected using a Click-iT^® EdU Kit (Thermo Fisher Scientific, Inc.). Fluorescent signals were detected under a fluorescent microscope (Olympus Corporation). Image analysis was performed using Image-Pro Plus software v6.0 (number of fields=10; magnification, x40) (Media Cybernetics, Inc.).

Apoptosis was assessed using the Annexin V-fluorescein isothiocyanate (FITC)/PI detection kit (cat. no. ab14085; Abcam). The experiment was performed according to the manufacturer's protocol. Briefly, cells (1x10⁴) were sequentially incubated with 20 µl PI binding buffer (50 µg/ml) and 10 µl Annexin V-FITC (10 µg/ml) in the dark. Subsequently, apoptosis was assessed using BD FACSCalibur™ flow cytometer (BD Biosciences) equipped with FACS Diva version 6.0 software (BD Biosciences) to determine early apoptosis cells.

Cell lysate was prepared with RIPA buffer (R0278; Sigma-Aldrich; Merck KGaA). Subsequently, protein concentration was measured using the Bicinchoninic Acid Protein Quantitation kit (cat. no. ab102536; Abcam). A total of 20 µg protein were separated on 10% SDS-PAGE (Bio-Rad Laboratories, Inc.) and electrically transferred onto PVDF membranes (EMD Millipore), followed by blocking in 5% BSA (A4161; Sigma-Aldrich; Merck KGaA) for 1 h at 25˚C. Proteins on PVDF membranes were sequentially probed with primary antibody overnight at 4˚C, followed by secondary antibody incubation for 1 h at 25˚C. The information for antibodies are as follows: Primary antibodies, Bax (1:1,000; cat. no. ab216494; Abcam), cleaved caspase-3 (1:1,000; cat. no. ab2302; Abcam), caspase-3 (1:1,000; cat. no. ab4051; Abcam), β-actin antibody (1:5,000; cat. no. ab8227; Abcam), LC3B (1:500; cat. no. ab51520; Abcam), p62 (1:1,000; cat. no. ab91526; Abcam), AMPK (1:1,000; cat. no. ab3759; Abcam), phosphor-AMPK (1:200; cat. no. ab23875; Abcam) and mTOR (1:1,000; cat. no. ab2732; Abcam); Secondary antibodies, HRP-conjugated rabbit or mouse antibodies (1:1,000; cat. nos. ab6721 and ab6728, respectively; Abcam). β-actin served as the internal reference. Protein bands were developed on the membrane. Protein bands were visualized using an ECL Western Blotting kit (Thermo Fisher Scientific, Inc.), and quantified using the C-DiGit Blot Scanner (LI-COR Biosciences).

RNA from the transfected cells or tissues was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Total RNA (1 µg) was reverse transcribed into cDNA using the miScript Reverse Transcription kit for incubation at 55˚C for 10 min (Qiagen, Inc.), according to the manufacturer's protocol. mRNAs were quantified using SYBR-Green (Roche Applied Science), with GADPH as the internal reference. qPCR was performed on a 20-µl system using the SYBR-Green PCR Master Mix (Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 5 min; 40 cycles of 95˚C for 10 sec, 57˚C for 30 sec and 72˚C for 10 sec. Target values were calculated using the 2^-ΔΔCq method (mean of control samples) (29) and the values were normalized to the negative control groups. The primers used were as follows: MALAT1, forward, 5'-GAATTGCGTCATTTAAAGCCTAGTT-3', and reverse, 5'-GTTTCATCCTACCACTCCCAATTAAT-3'; AMPK, forward, 5'-AGAGCCCCATCTGTCCTCTC-3' and reverse, 5'-ACTGGTAGTCTGCAAAACCAAA-3'; and GAPDH, forward, 5'-GGAGCGAGATCCCTCCAAAAT-3' and reverse, 5'-GGCTGTTGTCATACTTCTCATGG-3'.

Regarding the microtubule-associated proteins 1A/1B light chain 3B (LC3B) puncta assay, the KGN cells in a 24-well plate (1x10⁴ cells/ml), were transfected with green fluorescent protein (GFP)-LC3B expression constructs (Addgene, Inc.). Images were captured using a fluorescent microscope (Olympus Corporation; magnification, x400) (30). Moreover, the GFP puncta in 10 different microscopic fields was assessed.

Following the aforementioned transfections, AMPK activity was measured using a kinase assay buffer (V9101; Promega Corporation) with the AMP-[γ-^32P] ATP mixture and SAMS peptide (ab120182; Abcam). The reaction was terminated using a phosphocellulose paper, and AMPK activity was measured. Briefly, samples were incubated with SAMS peptide at 30˚C for 20 min and then spotted with 15 µl of samples on a disc of P81 Whatman phosphocellulose paper and allowed to absorb (1-3 sec). Subsequently, the discs were washed in a stirring bath containing 200 ml 1% phosphoric acid, acetone and petroleum ether. The AMPK activity was counted using Cerenkov counting (31).

Data was presented as the mean ± standard deviation. Comparison between multiple groups or two groups was performed using one-way ANOVA with Tukey's post-hoc test or Student's t-test, respectively. P<0.05 was considered to indicate a statistically significant difference.

Compared with normal GCs, MALAT1 expression significantly increased in GCs in endometriosis (Fig. 1A). MALAT1 expression obtained from patients with stage III/IV endometriosis was significantly higher compared with patients with stage I/II endometriosis (Fig. 1B). In addition, as a GC tumor-derived cell line, KGN is commonly used to study the molecular mechanism underlying endometriosis (18,32,33); thus, MALAT1 expression was compared between KGN cells and normal GCs. MALAT1 expression was significantly upregulated in KGN cells compared with normal GCs (Fig. 1C); therefore, MALAT1 may be involved in GC activity in endometriosis.

Following transfection with MALAT1, MALAT1 expression significantly decreased compared with the NC group (Fig. 2A). MTT assay indicated that at 24-96 h after transfection, KGN cell proliferation significantly decreased compared with the NC group (Fig. 2B). EdU staining assay demonstrated that MALAT1 knockdown significantly reduced EdU-positive cell levels compared with that the NC group (Fig. 2C). Therefore, MALAT1 may play a role in GC cell proliferation. In addition, flow cytometry revealed that MALAT1 knockdown led to an increase in the apoptotic cell number, levels of Bax, total caspase-3, and cleaved caspase-3, which are biomarkers of apoptosis (Fig. 3A and B). Additionally, whether MALAT1 knockdown caused the autophagy of KGN cells was also investigated. The location of LC3B protein following MALAT1 knockdown was traced using IFA. LC3B (green) accumulated after MALAT1 shRNA transfection (Fig. 4A). MALAT1 knockdown enhanced the autophagy rate of KGN cells, as indicated by increased LC3B biosynthesis and processing (reduced LC3B I and II levels) and p62 degradation (Fig. 4B). Therefore, MALAT1 may inhibit autophagy of KGN cells, which is partially responsible for its facilitative effect on GC proliferation.

MALAT1 targets the AMPK-mTOR pathway (18,34); thus, the AMPK levels in GCs obtained from patients with endometriosis and in KGN cells were determined. AMPK mRNA levels were significantly decreased in GCs and KGN cells compared with normal GCs (Fig. 5A and B). In addition, the effect of MALAT1 knockdown on AMPK1 expression in KGN cells was analysed using western blotting. Compared with the NC group, the expression levels of total and phosphorylated AMPK1 were significantly upregulated by MALAT1 knockdown, but its downstream target mTOR was significantly downregulated (Fig. 5C). In addition, MALAT1 knockdown significantly increased AMPK1 activity (Fig. 5D). Therefore, AMPK1 expression increased following MALAT1 knockdown.

To determine whether AMPK1 deactivation suppresses the influence of MALAT1 on KGN cell viability, apoptosis and autophagy, AMPK1 activity in KGN cells was inhibited by BML-275 treatment. AMPK1 activity detection assay was conducted to confirm AMPK1 deactivation (Fig. 6A). Western blotting demonstrated that compared with the knockdown (KD)/vehicle (0.1% DMSO) group, phosphorylated AMPK1 levels significantly downregulated, however, mTOR expression significantly upregulated due to BML-275 treatment of MALAT1-knockdown KGN cells (Fig. 6B). In addition, with the inhibition of AMPK1, KGN cell proliferation (which was prevented by MALAT1 knockdown) recovered, as demonstrated by EdU staining assay (Fig. 6C).

To evaluate the role of AMPK1 in apoptosis, KGN apoptosis was assessed by flow cytometry. Compared with the KD/Vehicle group, BML-275 treatment significantly decreased the number of apoptotic KGN cells that underwent transfection with MALAT1 knockdown (Fig. 6D). In addition, BML-275 treatment downregulated autophagy, as evidenced by the attenuated LC3B expression (Fig. 6E). Therefore, AMPK1 deactivation may restore proliferation, which was inhibited by MALAT1 knockdown via apoptosis and autophagy.