LncRNA NEAT1 promotes endometrial cancer cell proliferation, migration and invasion by regulating the miR-144-3p/EZH2 axis

The human endometrial cancer cell lines, HEC-1-A, HEC-1B, I shikawa (type I), RL-95-2, and JEC and human normal endometrial stromal cells (hESCs) were maintained in DMEM (Gibco, USA) with 10% foetal bovine serum (FBS, Gibco, USA). The cells were cultured at 37ºC with 5% CO₂.

All the EC and adjacent normal endometrial tissues were collected from EC patients at the First Hospital of Lanzhou University. The EC tissues (n = 36) and tumour adjacent tissues (n = 36) were collected during surgery and then rapidly frozen with liquid nitrogen and stored at -80°C. This study was approved by the Ethics Committee of the Reproductive Medicine Special Hospital of the First Hospital of Lanzhou University.

Total RNA extraction was performed using Trizol reagent (Invitrogen USA), and a TaqMan Reverse Transcription Kit (Thermo Fisher, USA) was used for reverse transcription into cDNA according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (qPCR) was used to quantify the expression level of NEAT1 in cultured cells using an Applied Biosystems 7500 Fast Dx Real-Time PCR instrument (Thermo Fisher, USA). SYBR Green reagent was used for qPCR in this study. GAPDH was used as an internal reference to normalize NEAT1 and EZH2 values. U6 was used as an internal reference to normalize miR-144-3p values. The following primers were used for analysis:

NEAT1, 5’-TTGGGACAGTFFACGTGTGG-3’ (forward), and 5’-TCAAGTCCAGCAGAGCA-3’ (reverse);

miR- 144-3p,

5’-GGCCGGCGTACAGTATAGATGA-3’ (forward), and 5’- GTGCAGGGTCCGAGGT-3’ (reverse);

EZH2, 5’-AAGCACAGTGCAACACCAAG-3’ (forward), and 5’-

CAGATGGTGCCAGCAATAGA-3’ (reverse);

GAPDH, 5’-TGACGTGCCGCCTGGAGAAC-3’ (forward), and

5’-CCGGCATCGAAGGTGGAAGAG-3’ (reverse);

U6, 5’-CTCGCTTCGGCAGCACA-3’ (forward), and 5’- AACGCTTCACGAATTTGCGT-3’(reverse). Relative RNA levels were calculated using the 2^-ΔΔCt method.

Small hairpin RNA (shRNA) for NEAT1 or its respective negative control were designed, synthesized and cloned into the shRNA vector U6/GFP/ Neo plasmid (GenePharma, Shanghai, China). A miR-144-3p mimic and its control were designed and synthesized by Sangon (Shanghai, China). The validated and purified reconstructed vectors were transfected into the indicated cells by Lipofectamine 3000 (Thermo Fisher, USA) according to the manufacturer’s guidelines. Briefly, 0.2 μg indicated plasmid and 0.4 μL Lipofectamine 3000 were mixed with 5 μL Opti-MEM medium (Thermo Fisher, USA), and then, Opti-MEM medium containing plasmid was added to the other Opti-MEM medium containing Lipofectamine 3000. Subsequently, this mixture was added to cells whose density was approximately 70%.

To verify the binding ability of NEAT1 to miR-144-3p and miR-144-3p to EZH2, we performed a dual-luciferase reporter assay using a Promega dual-luciferase reporter assay kit (Madison, USA). Briefly, we constructed a pmiR-RB-REPORT™ (Ribobio) plasmid containing the exact sites for wild type NEAT1 and EZH2 3’-UTRs and the corresponding mutated sequences. The cells seeded in 96-well plates were co-transfected with the indicated plasmids and miR-144-3p mimic or NC duplex (Gene Pharma). After 48 h, the cells were harvested, and luciferase activity was measured using a dual-luciferase reporter assay kit (Promega Corporation) and a multi-plate reader (Synergy 2, Bio Tek).

An MTT assay was performed to measure cell proliferation. Cells were cultured in 96-well plates (1 × 10⁴ per well). MTT (0.5 mg/mL, Gibco, USA) was added to the medium. Then, the plate was incubated at 37°C for 4 h and the supernatant was discarded. Next, 150 μL DMSO (Thermo Fisher, USA) was added to each well and the OD490 nm value was measured immediately using a microplate reader (3100, Thermo Fisher, USA).

A colony formation assay was used to determine cell proliferation. HEC-1-A and Ishikawa cells in logarithmic growth were digested by trypsin (Gibco, USA) and then resuspended in DMEM containing 10% FBS. Cells were seeded in culture dishes at a density of 500 cells per dish. Then, the cells were cultured at 37°C and 5% CO₂ until visible colonies were formed. The culture medium was discarded, and the cells were fixed using methanol and stained with a crystal violet solution (Gibco, USA). The number of colonies was counted.

The migration and invasion assays were performed using transwells. For this assay, 24-well plates with 8-μm pore polycarbonate membranes (BD Biosciences, USA) were used. The upper side of the membranes was coated with Matrigel (20 μg/ well, BD Biosciences, USA) and then air-dried for 1 h at 37°C. EC cells (2×105) in 200 μL of FBS-free medium were placed in the upper chamber, which was uncoated (migration assay) or coated (invasion assay). The lower chamber was filled with medium with 10% FBS as a chemoattractant. After 48 h of incubation, the cells on the upper surface of the membrane were removed by gentle scrubbing with a cotton swab. The membranes were fixed in a stationary liquid of 95% ethanol and 5% acetic acid for 30 min and stained with a crystal violet solution. The number of cells on the lower surface of the membrane in 5 random visual fields (magnification, ×200) was then counted using an Eclipse TE2000-U inverted microscope.

A protein detection kit (Thermo Fisher, USA) was used to measure the amount of protein. Equivalent amounts of protein samples were separated on 8-10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF, Thermo Fisher, USA). After blocking with 0.1% Tween 20 and 5% skimmed milk, the protein-containing PVDF membranes were incubated for 12 h in solutions containing primary antibodies obtained from Abcam (anti-EZH2, ab186006; anti-GAPDH, ab9485) at 4°C. Then, secondary antibodies were incubated for 2 h. A western blotting kit (Thermo Fisher Scientific, USA) was used to detect the signals.

Statistical analysis was performed using GraphPad Prism. The continuous variables are shown as the mean ± standard deviation (SD), and their differences were analysed by Student’s t test or ANOVA. P values less than 0.05 predicted the statistical significance of the results. Each assay was performed in triplicate.

We first evaluated the expression of NEAT1 in cancer tissues and adjacent tissues derived from EC

patients by qRT-PCR. As shown in Figure 1A, we found that the expression of NEAT1 was prominently higher in EC tissues than in adjacent tissues (P<0.05, indicated a significant difference). To determine whether NEAT1 is highly expressed in various EC-related cell lines, we performed qRT-PCR to detect its expression in hESCs and endo-metrial cancer cell lines. Interestingly, we found that the transcription levels of NEAT1 were significantly higher in EC cell lines, including HEC-1A, HEC-1B, Ishikawa, RL-95-2, and JEC, than in hESCs (Figure 1B). Among these cell lines, HEC-1A and Ishikawa cells had the highest and lowest expression of NEAT1, respectively, and they were selected for the following experiments.

Figure 1

To investigate whether endogenous NEAT1 plays a role in EC, we constructed a NEAT1-shRNA plasmid that efficiently downregulated NEAT1 expression after transfection into HEC-1A and Ishikawa cells (Figure 2A). MTT and colony formation assays were performed to measure the EC cell proliferation ability. According to the MTT assay, knockdown of NEAT1 significantly inhibited cell growth compared with the NC and control conditions in HEC-1A (Figure 2 B) and Ishikawa cells (Figure 2 C). Furthermore, colony formation assays indicated that the suppression of NEAT1 reduced colony formation in HEC-1A and Ishikawa cells (Figure 2 D, E). Taken together, the MTT assay and colony formation assays results indicated that knockdown of NEAT1 inhibited EC cell proliferation.

Figure 2

To determine whether endogenous NEAT1 affects EC cell migration and invasion, we performed a transwell assay to detect cell migration and invasion abilities. We found that knockdown of NEAT1 attenuated the migration of HEC-1A and Ishikawa cells (Figure 2 F, G). Consistently, we also found that knockdown of NEAT1 inhibited the invasion of HEC-1A and Ishikawa cells (Figure 2 H, I). Taken together, these data suggest that NEAT1 promotes EC cell proliferation, migration, and invasion.

LncRNAs may act as sponges for direct binding to miRNA. In our previous work, we predicted that NEAT1 may target miR-144-3p using Starbase (http://starbase.sysu.edu.cn/) for bioinformatics prediction (Figure 3 A). The miR-144-3p level was much lower in EC tissues than in adjacent tissues according to qRT-PCR. MiR-144-3p was also expressed at lower level in EC cells than in hESCs (Figure 3 B, C). Next, we performed qRT-PCR to detect whether NEAT1 targets miR-144-3p to promote EC progression. As shown in Figure 3D, we found that knockdown of NEAT1 increased the level of miR-144-3p. To determine whether NEAT1 binds directly to miR-144-3p, we performed a dual luciferase activity assay to evaluate the binding ability by designing wild-type NEAT1 (WT-NEAT1) and a mutant (MUT-NEAT1). The luciferase signal will decrease when the lncRNA binds to its target miRNA. We found that the luciferase activity was decreased in the WT-NEAT1 and miR-144-3p mimic co-transfected group, while no significant difference was observed between the MUT-NEAT1 group and he mimic NC group (Figure 3E). These data suggested that NEAT1 could bind to miR-144-3p.

Figure 3

We designed experiments to reveal the function of miR-144-3p in EC cells. First, miR-144-3p was overexpressed in EC cell lines by miR-144-3p mimic transfection (Figure 4 A). Next, EC cell proliferation was examined via MTT and colony formation methods. In HEC-1A and Ishikawa cells, the MTT assay showed that cell proliferation was significantly inhibited in the miR-144-3p mimic group compared with that in the control and NC groups (Figure 4 B, C). Furthermore, colony formation assays showed that the miR-144-3p mimic markedly attenuated the cell proliferation of HEC-1A and Ishikawa cells compared with the control and NC groups (Figure 4 D, E). Finally, to determine whether miR-144-3p affects EC cell invasion and migration, we performed transwell assays to measure the migration and invasion abilities of EC cells. We found that the miR-144-3p mimic inhibited the migration of HEC-1A and Ishikawa cells (Figure 4 F, G). Consistently, we also found that the miR-144-3p mimic attenuated invasion of HEC-1A and Ishikawa cells (Figure 4 H, I). Taken together, these data indicated that miR-144-3p inhibited EC cell proliferation, migration, and invasion.

Figure 4

We found that knockdown of NEAT1 suppressed the transcription of the EZH2 gene in HEC-1A and Ishikawa cells (Figure 5 A). Consistently, Western blot analysis showed that knockdown of NEAT1 decreased the expression of EZH2 in HEC-1A and Ishikawa cells (Figure 5 B, C). However, whether miR-144-3p regulates EZH2 in EC cells was unknown. To verify the relationship between miR-144-3p and EZH2 in EC cells, we overexpressed miR-144-3p in HEC-1A and Ishikawa cells by miR-144-3p mimic transfection and evaluated the expression of EZH2 by qRT-PCR and Western blotting. The qRT-PCR results showed that the transcription of the EZH2 gene was significantly lower in the miR-144-3p mimic group than in the control and NC groups in HEC-1A and Ishikawa cells (Figure 5 D). Consistently, Western blot analysis showed that the miR-144-3p mimic decreased the expression of EZH2 in HEC-1A and Ishikawa cells (Figure 5 E). To verify whether miR-144-3p decreases EZH2 expression by binding to its 3’-UTR site, we performed a dual luciferase activity assay to evaluate the binding ability and designed wild-type EZH2 (WT-EZH2) and its mutant (MUT-EZH2) sequences (Figure 5 F). The dual-luciferase activity assay results showed that the luciferase activity was lower in the miR-144-3p mimic group than in the NC WT-EZH2 group, while there was no difference in the MUT-EZH2 groups (Figure 5 G). Taken together, these data suggested that miR-144-3p could directly target the 3’-UTR region of EZH2. These data suggested that NEAT1 may act as a ceRNA of miR-144-3p, leading to increased the expression of EZH2 which is a target gene of miR-144-3p.

Figure 5