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. 2024 Feb 14;76(2):231–246. doi: 10.1007/s10616-023-00612-z

NEAT1 promotes the progression of prostate cancer by targeting the miR-582-5p/EZH2 regulatory axis

Weiqiang Xu 1,3,✉, Yu Wu 3, Guoxi Zhang 2,✉
PMCID: PMC10940559  PMID: 38495291

Abstract

In several forms of malignant tumors, nuclear enriched abundant transcript 1 (NEAT1), a lncRNA, has been identified to play an important role. NEAT1’s regulation patterns in prostate cancer (PCa) are, however, mainly unknown. This study was aimed to evaluate and study the roles and regulatory mechanisms of NEAT1 in PCa. NEAT1, miR-582-5p, and enhancer of zeste homolog 2 (EZH2) expression were detected by qRT-PCR. The PCa cells’ invasive, migrative, and proliferative activities in vitro were assessed using transwell migration and invasion, wound-healing, cloning creation, and CCK-8 assays. In the present study, impaired proliferative, migrative, and invasive capacities were observed in the NEAT1-deficient PCa (PC3 and LNCaP) cells. Further mechanistic studies found that NEAT1 performs its function through sponging miR-582-5p. Furthermore, EZH2 was confirmed to be the downstream target gene of miRNA-582-5p. The impaired progression caused by NEAT1 deficiency in PCa cells was significantly restored by the inhibition of miR-582-5p, while these effects were largely abolished by the deletion of EZH2. Finally, the xenograft nude mouse model showed that knocking down the expression of NEAT1 suppressed the growth of PCa. In conclusion, NEAT1 promotes the progression of PCa by controlling the miR-582-5p and miR-582-5p-mediated EZH2.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10616-023-00612-z.

Keywords: Nuclear enriched abundant transcript 1, miR-582-5p, Enhancer of zeste homolog 2, Prostate cancer, Epithelial-to-mesenchymal transition

Introduction

Prostate cancer (PCa) is the leading malignant tumor in men and the second leading cause of death in the United States (Siegel et al. 2021). In China, approximately 115,000 people were newly diagnosed with PCa in 2020, making it the ninth most common type of cancer (Qiu et al. 2021). Primary PCa is mostly hormone-dependent, and androgen deprivation therapy significantly is effective for high-risk intermediate and advanced PCa. However, after 6 to 23 months of sensitivity to castration therapy, almost all patients develop castration-resistant PCa, especially in patients with metastases. The survival is usually less than two years, and there is no effective treatment in the long term (Halabi et al. 2003).

Long noncoding RNAs (lncRNAs) have a role in chromatin remodeling, chromosomal inactivation, genomic imprinting, and transcriptional activation/interference, among other biological activities (Spizzo et al. 2012). The promoter region of lncRNA is widely conserved, and its transcriptional regulation depends on the state of chromatin and histone (Chandra Gupta and Nandan Tripathi 2017). The most common degradation pathways of lncRNAs include exonuclease degradation or exosomal digestion (Flippot et al. 2019). In the past years, there has been a lot of research about lncRNAs in malignant tumors (Bhan et al. 2017; Alvarez-Dominguez and Lodish 2017; Bach and Lee 2018).

Nuclear enriched abundant transcript 1 (NEAT1) is a newly discovered lncRNA in recent years, and it is an important part of paraspeckles in the nucleus. It mainly participates in gene expression regulation by maintaining mRNA stability in the nucleus (Clemson et al. 2009). In recent years, significant associations of NEAT1 with the occurrence and progression of malignant tumors have been demonstrated in several studies. A previous study has found that NEAT1 expression in the blood of patients with colon cancer and lung cancer is significantly upregulated (Wu et al. 2015). NEAT1 expression has also been observed to be significantly enhanced in esophageal cancer, glioma, liver cancer, and PCa tissues in other investigations. Moreover, it is significantly correlated with lymph node metastasis, clinical stage, and the survival time of patients (Chakravarty et al. 2014; He et al. 2016). These findings indicated that NEAT1 is a central indicator of poor prognosis. Furthermore, Chakravarty et al. (Chakravarty et al. 2014) demonstrated that NEAT1 can serve as the target gene of estrogen receptor α to promote the malignant development of prostate cancer.

MicroRNA (miRNA) is a small RNA that does not have a coding function, and it can regulate gene expression in eukaryotes and prokaryotes (Ambros 2004). At present, there are different views on the mechanism of action of miRNA in organisms. Previous research has found that when miRNA suppresses mRNA expression, activated polyribosomes develop after it is translated, and then polyribosomes are connected by mRNA, implying that miRNA inhibition does not occur at the start of the translation process but rather later on (Chen et al. 2020). The regulation of mRNA expression by miRNA is of great physiological significance. For example, miRNA can regulate the physiological activities of tumor cells, including cell proliferation and apoptosis, by regulating their target genes. The participation of miRNAs in PCa pathogenesis was also observed previously (Abramovic et al. 2020; Jeon et al. 2020; Wang et al. 2021). Additionally, significant associations between miRNA-582-5p with the clinicopathologic features and prognosis of PCa have been observed (Li et al. 2022, p. 1).

The carcinogenic effect of lncRNA NEAT1 in various tumors has been reported. However, the relationship between NEAT1 and miR-582-5p, as well as the downstream miRNA genes controlled by NEAT1, is still unknown. Therefore, this study aims to clarify the role and mechanism of NEAT1 regulating miR-582-5p and downstream target genes in the progression of PCa.

Materials and methods

Cells, cell culture, and cell transfection

LNCaP, P4E6, RWPE-1, PC3, and DU145 cells were obtained from the Chinese Academy of Sciences. DMEM/RPMI1640 (Hyclone, AB216694) medium containing FBS (10%, GIBCO, 10270-106) was used for cell culture. The cells were maintained at 37 °C in a humidified incubator with 5% CO2.

shRNAs (NEAT1 shRNA-1: 5′-TCATGGACCGTGGTTTGTTACTATAGTGT-3′; NEAT1 shRNA-2: 5′-CACCTGTTTGCCTGCCTTCTT-3′; NEAT1 shRNA-3: 5′-ACGCAGCAGATCAGCATCCTT-3′) was used to knock down NEAT1 expression. An shRNA (5′-GGATGGTACTTTCATTGAAGA-3′) was used to knock down EZH2 expression. In addition, a control shRNA (5′-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTG-3′) was employed as a negative control. The cells were transfected with the miR-582-5p mimics (RiBo-Bio, China, miR10003247-1-5), inhibitors (RiBo-Bio, China, miR20003247-1-5) and corresponding controls (RiBo-Bio, China, miR1N0000001-1-5 as NC mimic, miR2N0000001-1-5 as NC inhibitor) per the manufacturer’s instructions, respectively. The sequences of miR-582-5p mimic, NC mimic, miR-582-5p inhibitor and NC inhibitor are shown in Table S2. Transfection assays were conducted using the Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, USA).

Clinical tissue samples

Between January 2020 and January 2022, the Urology Department of the Second Affiliated Hospital of Bengbu Medical College gathered clinical data on 30 patients with PCa, including PCa (n = 30), and matched neighboring non-tumor (n = 30) tissues. Table S1 shows the PCa patient’s clinicopathological characteristics (n = 30). All specimens were confirmed by pathological diagnosis. All patients agreed by consent written to use all samples for scientific research. The Ethics Committee of the Second Affiliated Hospital of Bengbu Medical College reviewed and approved our study protocol and informed consent documents.

qRT-PCR

Trizol (Solarbio, Beijing, China) was employed to extract the total RNA from indicated cells and tissues, and the purity of RNA was determined. Then the RNA was reverse transcribed using PrimeScript RT Master Mix (YEASEN, Shanghai, China). Finally, fluorescent quantitative PCR amplification experiments were performed using SuperReal PreMix Plus (YEASEN, Shanghai, China). Table 1 shows the primers for our qRT-PCR assays.

Table 1.

The sequences of all primers used in qRT-PCR

Gene name Primer sequence (5′-3′)
NEAT1 Forward: 5′-ACATTGTACACAGCGAGGCA-3′
Reverse: 5′-CATTTGCCTTTGGGGTCAGC-3′
miR-582-5p Forward: 5′-TTGAACAACTGAACCCAA-3′
Reverse: 5′-GTTTCTACTTTGCACCCT-3′
EZH2 Forward: 5′-TTCTCAAGATGAAGCTGACAGAAGAGGG-3′
Reverse: 5′-TGAAGCTAAGGCAGCTGTTTCAGAGG-3′
β-actin Forward: 5′-CGGTCAGGTCATCACTATCGG-3′
Reverse: 5′-CACAGGATTCCATACCCAGGA-3′
U6 Forward: 5′-CAGCACATATACTAAAATTGGAACG-3′
Reverse: 5′-ACGAATTTGCGTGTCATCC-3′
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qRT-PCR, quantitative real-time quantitative PCR; NEAT1, nuclear enriched abundant transcript 1; EZH2, enhancer of zeste homolog 2

Western blotting

Proteins were extracted from cells using RIPA lysate (Best biological co., LTD, Nanjing, China), and the concentration of protein was determined using a BCA quantification kit (Beyotime, Shanghai, China). The protein samples were electrophoresed, separated, and transferred. Incubation of primary antibodies was then performed, antibodies against E-cadherin (1:1000, Abcam, ab1416), vimentin (1:1000, Abcam, ab92547), matrix metallopeptidase 2 (MMP2; 1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab76003), EZH2 (1:1000, Abcam, ab191080) and α-SMA (1:500, Abcam, ab5831), overnight at 4 °C. After that, the secondary antibody (1:4000, Santa Cruz Biotechnology, sc-2357 or sc-516,102) was incubated for 1.5 h on a room temperature shaker. Finally, the protein-antibody complex was identified using the ECL Plus chemiluminescence kit (YEASEN, Shanghai, China). As an internal reference, Abcam’s actin (1:2000) was employed.

CCK-8 detection

CCK-8 experiments were conducted to measure the proliferative potential of PCa cells according to the manufacturer’s instructions. We measured the absorbance value at 450 nm, which represents the proliferative capacity of PCa cells.

Plate clone-forming assay

After 24-hour incubation (200 cells/well in a 6-well plate), the sh-RNA or miRNA mimic or inhibitor was added, and the culture was continued. Until small visible clones (cell clones with diameters greater than 1 mm, about 50 cells/clone) were grown, the amounts of the clones were counted after crystal violet (Servicebio, Wuhan, China) staining.

Wound-healing assay

Cells were seeded into 6-well plates at a density of 5 × 105 cells per well. After a confluent monolayer formation, the wound was conducted using a sterile pipette tip in the monolayer cells. The cells were washed with phosphate-buffered saline (PBS, Beyotime, Shanghai, China) three times to remove cell debris, then the cells were cultured. 48 h later, we observed and analyzed the scratched wounds under a microscope. After imaging the scratched wounds, we added lines to delineate the margins used for measurements in the images. Then we measured the distance of the scratch. The extent to which the wound had closed over 48 h was calculated and expressed as a percentage of the difference between 0 and 48 h.

Transwell migration and invasion assays

To test the PCa cell’s invasive or migratory capabilities, researchers used Transwell chambers covered with or without Matrigel (BD). Cells (5 × 104) were seeded in Transwell chambers that had been pre-coated with or without Matrigel. For migration assays, cells were seeded in Transwell chambers without Matrigel. For invasion assays, cells were seeded in Transwell chambers with Matrigel. As a chemoattractant, a medium containing 10% FBS was utilized. The migratory and invasive cells were fixed with 4% formaldehyde and stained with crystal violet after a 24-hour incubation period. The stained cells were counted and then evaluated.

Luciferase reporter assays

Full-length EZH2 and NEAT1 were amplified and introduced into the pMiRGLO vector to create wild-type EZH2 (EZH2-WT) and NEAT1 (NEAT1-WT) plasmids. GenePharma (Shanghai, China) provided the mutant types of EZH2 (EZH2-Mut) and NEAT1 (NEAT1-Mut). Plasmids were transfected or co-transfected into PC3 and LNCaP cells using the Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, USA). After 48 h of growth, the cells were collected and lysed, and the luciferase activities were measured using a dual-luciferase reporting system (Promega, Madison, USA).

Mouse prostate cancer xenograft model

Male nude mice (18–20 g weight, 5–8 weeks old) were purchased from Shanghai Lab Animal Research Center (Shanghai, China). The mouse PCa xenograft model was performed in accordance with the ARRIVE guidelines. A total of 6 mice were utilized, with 3 mice in sh-NEAT1group and 3 mice in sh-NC group. The health and behaviour of mice were monitored every 12 h after being inoculated with PCa cells. The mice were anesthetized by 2% isoflurane inhalation, and 50% Basement Membrane Matrix suspended PC3 cells (2 × 106) were subcutaneously injected into the dorsal flank on the right side of each mouse. Five weeks after tumor injection, all mice were euthanized by using pentobarbital (200 mg/kg; Euthasol, Virbac Animal Health, Westlake, USA) by intraperitoneal injection. The mice were confirmed dead by observing no signs of life (no fluctuation in the chest, white in the eyelids, and no visual reaction). Then the xenograft tumors were removed, photographed, and weighed. Then we measured the length and width of tumors, and the tumor volumes were calculated as = 0.5 × length × width2. No mice died were observed earlier than 5 weeks in each group. n = 3 per group. Animal care and conditions followed institutional protocols and guidelines, and all studies were approved by Bengbu Medical College Animal Ethics Committee.

HE staining and IHC staining assays

After fixation using 4% neutral paraformaldehyde (PFA, Beyotime, Shanghai, China) and embedding with paraffin, the tissue sections were stained in hematoxylin (Servicebio, Wuhan, China) for 5 min. After washing away the hematoxylin solution with running water, the tissue slices were stained for 2 min in eosin (Servicebio, Wuhan, China). For immunohistochemistry (IHC) staining, the primary antibody of ki67 (Abcam, ab15580) was used at a dilution of 1:200 for staining. The tissue slices were stained with DAB reagents (Beyotime, Shanghai, China) at room temperature before being counterstained with hematoxylin. The slides were documented using light microscopy and photography.

TUNEL assay

The cell apoptosis in the xenograft tumors was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (Roche) following the manufacturer’s instructions.

Statistical analysis

The overall survival and disease-free survival rates of PCa patients (n = 30) were calculated and compared by using Kaplan–Meier analysis and the log-rank test. The Chi square test was used to compare the differences of categorical variables. The numerical variables were represented by the mean ± SD. Experiments were repeated three times. Two-sided Student’s t-test was used to analyze the differences between two groups, differences among ≥ 3 groups were analyzed using one-way analysis of variance (ANOVA), Tukey’s test was used for the post hoc analysis. For correlation analysis between two continuous variables, Pearson’s correlation analysis was used, and p value was calculated by Pearson’s correlation test. The statistical cutoff value for the relative expression of NEAT1 was defined by the highest Youden index obtained from the receiver operator characteristic (ROC) curves. Then, a final expression of NEAT1 ≥ 2.5805 was used to define tissues with high NEAT1 expression, and an expression of NEAT1 < 2.5805 was defined as low NEAT1 expression. All statistical analyses were accomplished with SPSS 22.0 (IBM Corporation, Armonk, USA) and Prism 8.0 software (GraphPad). A p < 0.05 was considered statistically significant.

Results

NEAT1 is abnormally overexpressed in PCa

We found that NEAT1 was significantly overexpressed in PCa tissues (Fig. 1A), and NEAT1 expression was dramatically enhanced in advanced PCa (Fig. 1B), suggesting that NEAT1 may be an oncogenic gene. Additionally, compared to RWPE-1, DU145, LNCaP, and PC3 PCa cells exhibited significantly high NEAT1 expression (Fig. 1C). Using survival analysis (Fig. 1D and E), high overall survival and disease-free survival rates were observed in the NEAT1 low expressed patients compared to those high NEAT1 expressed patients.

Fig. 1.

Fig. 1

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NEAT1 is abnormally overexpressed in PCa. A NEAT1 mRNA levels analyzed using qRT-PCR were significantly overexpressed in PCa tissues than corresponding non-tumors, data expressed as a mean ± SD, paired t test. B NEAT1 expression is elevated in advanced PCa, data expressed as a mean ± SD, unpaired t test. C The expression level of NEAT1 in PCa cells (P4E6, PC3, LNCaP and DU145) was significantly higher than that in prostate epithelial cells (RWPE-1), data expressed as a mean ± SD, one-way ANOVA followed by Tukey’s test (RWPE-1 as control). D PCa patients with high expression of NEAT1 (high expression group, n=16) have lower overall survival than that with low expression of NEAT1 (low expression group, n=14), log-rank test. E PCa patients with high expression of NEAT1 (high expression group, n=16) have lower disease-free survival than that with low expression of NEAT1 (low expression group, n=14), log-rank test. ***p<0.001. PCa, prostate cancer; NEAT1, nuclear enriched abundant transcript 1; qRT-PCR, quantitative real-time quantitative PCR; SD, standard deviation; ANOVA, analysis of variance

Suppression of NEAT1 suppressed the proliferation, migration, invasion capabilities and induced changes in EMT-associated proteins in PCa

We further used the specific sh-RNA to knock down NEAT1 in PCa cell lines (PC3 and LNCaP). In this assay, three different sequences of NEAT1 shRNAs (shRNA-1, shRNA-2, and shRNA-3) were used, we found that sh-NEAT1 significantly reduced the RNA expression of NEAT1 in PC3 and LNCaP cells (Fig. 2A). Since shRNA-1 has the best knockdown efficiency, shRNA-1 was chosen as the best shRNA for further experiments. Additionally, the capacity of PC3 and LNCaP cells to proliferate was also significantly reduced after NEAT1 was deleted (Fig. 2B). The results of the plate clone-forming assay showed that the cloning formation ability of PC3 and LNCaP cells was significantly reduced after knocking down NEAT1 (Fig. 2C). In addition, NEAT1 knockdown PC3 and LNCaP cells exhibited obviously low migrative (Fig. 2D) and invasive (Fig. 2E) ability compared to the negative control cells. These observations indicated that NEAT1 expression in PCa cells was associated with their proliferative, migrative, and invasive capacities. Moreover, suppression of NEAT1 induced changes in epithelial-to-mesenchymal transition (EMT)-related protein and MMP2, MMP9 expression in PC3 and LNCaP cells, respectively. We found that in PC3 and LNCaP cells, suppression of NEAT1 improved E-cadherin expression and significantly decreased the expression of Vimentin, α-SMA, MMP2 and MMP9 (Fig. 2F).

Fig. 2.

Fig. 2

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Suppression of NEAT1 suppressed the proliferation, migration, invasion capabilities and induces changes in EMT-associated proteins in PCa. A qRT-PCR showed that NEAT1 mRNA level was significantly decreased in the NEAT1 shRNA-transfected PC3 and LNCaP cells compared to the NC shRNA-transfected PC3 and LNCaP cells, data expressed as a mean ± SD, one-way ANOVA followed by Tukey’s test (the sh-NC group as control). B The proliferation capabilities of PCa cells were detected by the CCK-8 assay, knocking down NEAT1 prominently reduced the proliferation ability of PC3 and LNCaP cells, data expressed as a mean ± SD, unpaired t test. C The clonal formation ability of PC3 and LNCaP cells was significantly decreased after knock-down of NEAT1, data expressed as a mean ± SD, unpaired t test. D The migration ability of PC3 and LNCaP cells was significantly decreased after knock-down of NEAT1 by using the wound-healing assay, data expressed as a mean ± SD, unpaired t test. E The migration abilities of the PC3 and LNCaP cells were dramatically reduced after transfection with NEAT1 shRNAs by using the transwell migration assays. The invasion potential of PCa cells was detected by a transwell invasion assay, and the invasion abilities of the PC3 and LNCaP cells were dramatically decreased after transfection with NEAT1 shRNAs, data expressed as a mean ± SD, unpaired t test. F Western blot analysis of EMT-related protein (E-cadherin, Vimentin and α-SMA), MMP2 and MMP9 expressions in PC3 and LNCaP cells, data expressed as a mean ± SD, unpaired t test. **p<0.01, ***p<0.001. NC, negative control; EMT: epithelial-to-mesenchymal transition

NEAT1 performs its function through sponging miRNA-582-5p

A previous study (Zhao et al. 2022) has reported that NEAT1 can function by sponging miRNAs in PCa cells. The StarBase database found promising binding sites between miR-582-5p and NEAT1 (Fig. 3A). We also found that miR-582-5p expression was notably increased after treatment with a miR-582-5p mimic in PC3 and LNCaP cells (Fig. 3B). Next, to verify the regulation of NEAT1 on miR-582-5p, a luciferase reporter assay was conducted in LNCaP and PC3 cells. After adding miR-582-5p mimic, reduced luciferase activity was observed in the NEAT1-WT-transfected cells but not in NEAT1-Mut-transfected cells (Fig. 3C). Additionally, compared to the normal tissues, PCa tissues exhibited significantly reduced miR-582-5p expression (Fig. 3D). This observation follows the findings from PCa cell lines (Fig. 3E). Moreover, we also observed remarkably enhanced miR-582-5p expression in the NEAT1 deficient PC3 and LNCaP cells (Fig. 3F). We also found that there was a negative relationship between NEAT1 and miR-582-5p in PCa tissues (Fig. 3G).

Fig. 3.

Fig. 3

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NEAT1 performs its function through sponging miRNA-582-5p. A The binding sites between NEAT1 and miR-582-5p were predicted by StarBase. B The expression of miR-582-5p was treated by NC mimic or miR-582-5p mimic in PC3 and LNCaP cells, data expressed as a mean ± SD, unpaired t test. C The relationship between NEAT1 and miR-582-5p was testified by the luciferase reporter assay in PC3 and LNCaP cells, data expressed as a mean ± SD, unpaired t test. D The level of miR-582-5p in PCa tissues vs. non-tumor tissues, data expressed as a mean ± SD, unpaired t test. E The level of miR-582-5p in PCa cell lines (P4E6, PC3, LNCaP and DU145) vs. prostate epithelial cells (RWPE-1), data expressed as a mean ± SD, one-way ANOVA followed by Tukey’s test (RWPE-1 as control). F The level of miR-582-5p in PC3 and LNCaP cells treated by sh-NC or sh-NEAT1, data expressed as a mean ± SD, unpaired t test. G Pearson’s correlation analysis demonstrated the correlation of NEAT1 expression with miR-582-5p, r represented Pearson’s correlation coefficient, and p value was calculated by Pearson’s correlation test. ***p<0.001.

Overexpression of miRNA-582-5p inhibited the proliferation, migration, and invasion capabilities of PCa cells

Next, in PC3 and LNCaP cells, an miR-582-5p mimic was used to overexpress miR-582-5p. The proliferation capabilities of PC3 and LNCaP cells were inhibited after overexpression of miRNA-582-5p (Fig. 4A and B). miR-582-5p upregulation significantly inhibited the migrative and invasive abilities of PC3 and LNCaP cells (Fig. 4C and D). Furthermore, overexpression of miR-582-5p in PC3 and LNCaP cells reduced α-SMA, Vimentin, MMP2 and MMP9 expression while increasing E-cadherin expression (Fig. 4E).

Fig. 4.

Fig. 4

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Overexpression of miRNA-582-5p inhibited the proliferation, migrationand invasion capabilities in PCa cells. A CCK-8 assay showed the proliferation capabilities of PC3 and LNCaP cells treated by NC mimic or miR-582-5p mimic. B The cloning formation ability of PC3 and LNCaP cells treated by NC mimic or miR-582-5p mimic. C The wound-healing assay showed the migration ability of PC3 and LNCaP cells treated by NC mimic or miR-582-5p mimic. D The transwell migration and invasion assays showed the migration and invasion abilities of PC3 and LNCaP cells treated by NC mimic or miR-582-5p mimic. E Western blot analysis of EMT-related protein (E-cadherin, Vimentin and α-SMA), MMP2 and MMP9 expressions in PC3 and LNCaP cells treated by NC mimic or miR-582-5p mimic. Data expressed as a mean ± SD, unpaired t test. **p<0.01, ***p<0.001

EZH2 was a target of miRNA-582-5p

To further identify an miR-582-5p target, we used the StarBase database (https://starbase.sysu.edu.cn/) to predict that EZH2 might be the downstream target gene of miR-582-5p (Fig. 5A). After miR-582-5p mimic transfection, reduced luciferase activity was seen in the EZH2-WT containing LNCaP and PC3 cells but not in the EZH2-Mut containing cells (Fig. 5B). In contrast to non-tumor tissues, PCa tissues had much higher EZH2 expression (Fig. 5C), which was consistent with the findings from PCa cell lines (Fig. 5D). Moreover, after treatment with miR-582-5p mimic, the mRNA and protein level of EZH2 was significantly decreased in PC3 and LNCaP cells (Fig. 5E and F). The miR-582-5p inhibitor significantly reduced the expression of miR-582-5p in PC3 and LNCaP cells (Fig. S2A). We also found that the mRNA and protein level of EZH2 was significantly increased in PC3 and LNCaP cells (Fig. 5G and H) after treatment of miR-582-5p inhibitor. Additionally, after treatment of miR-582-5p mimic, reduced EZH2 expression was seen in the NEAT1-WT containing LNCaP and PC3 cells but not in the NEAT1-Mut containing cells (Fig. S1).

Fig. 5.

Fig. 5

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EZH2 was a target gene of miRNA-582-5p. A The binding sites between miR-582-5p and EZH2 predicted by StarBase. B The interaction between miR-582-5p and EZH2 testified by the luciferase reporter assay, data expressed as a mean ± SD, unpaired t test. C The mRNA level of EZH2 in PCa tissues vs. non-tumor tissues tested by qRT-PCR, data expressed as a mean ± SD, unpaired t test. D The mRNA level of EZH2 in PCa cell lines (P4E6, PC3, LNCaP and DU145) vs. prostate epithelial cells (RWPE-1) tested by qRT-PCR, data expressed as a mean ± SD, one-way ANOVA followed by Tukey’s test (RWPE-1 as control). E The mRNA level of EZH2 in PC3 and LNCaP cells treated by NC mimic or miR-582-5p mimic, data expressed as a mean ± SD, unpaired t test. F Western blot analysis of EZH2 protein expression in PC3 and LNCaP cells treated by NC mimic or miR-582-5p mimic, data expressed as a mean ± SD, unpaired t test. G The mRNA level of EZH2 in PC3 and LNCaP cells treated by NC inhibitor or miR-582-5p inhibitor, data expressed as a mean ± SD, unpaired t test. F Western blot analysis of EZH2 protein expression in PC3 and LNCaP cells treated by NC inhibitor or miR-582-5p inhibitor, data expressed as a mean ± SD, unpaired t test. **p<0.01, ***p<0.001. EZH2, enhancer of zeste homolog 2

NEAT1 regulated PCa progression through the miRNA-582-5p/EZH2 axis in vitro

Next, rescue assays were performed in NEAT1-downregulated PC3 cells treated with sh-NEAT1. We found that sh-EZH2 significantly reduced the RNA expression of EZH2 in PC3 and LNCaP cells (Fig. S2B). We utilized a miRNA-582-5p inhibitor to reduce miRNA-582-5p expression and sh-EZH2 to knock down EZH2 expression. After miRNA-582-5p was downregulated, the proliferation ability of NEAT1-depleted PC3 cells improved. However, shutting down the expression of EZH2 reversed this behavior (Fig. 6A and B). Meanwhile, the impaired migrative and invasive capacities induced by NEAT1 deficiency were also significantly increased by miR-582-5p knockdown. However, EZH2 suppression abrogated these effects (Fig. 6C and D). In PC3 cells, miR-582-5p knocking down restored the repressed Vimentin, MMP2, MMP9 and increased E-cadherin expressions induced by NEAT1 deficiency; however, EZH2 suppression abrogated all of these effects (Fig. 6E). The above findings indicated that NEAT1 promotes PCa progression by the miRNA-582-5p/EZH2 axis in vitro.

Fig. 6.

Fig. 6

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NEAT1 regulated PCa progression by the miRNA-582-5p/EZH2 axis in vitro. A CCK-8 assay showed the proliferation capabilities of PC3 cells treated by sh-NC+NC inhibitor, sh-NEAT1+NC inhibitor, sh-NEAT1+miR-582-5p inhibitor, or sh-NEAT1+miR-582-5p inhibitor+sh-EZH2. B The clonal formation ability of PC3 cells treated by sh-NC+NC inhibitor, sh-NEAT1+NC inhibitor, sh-NEAT1+miR-582-5p inhibitor, or sh-NEAT1+miR-582-5p inhibitor+sh-EZH2. C The wound-healing assay showed the migration ability of PC3 cells treated by sh-NC+NC inhibitor, sh-NEAT1+NC inhibitor, sh-NEAT1+miR-582-5p inhibitor, or sh-NEAT1+miR-582-5p inhibitor+sh-EZH2. D The migration and invasion abilities of PC3 cells treated by sh-NC+NC inhibitor, sh-NEAT1+NC inhibitor, sh-NEAT1+miR-582-5p inhibitor, or sh-NEAT1+miR-582-5p inhibitor+sh-EZH2 were detected by the transwell migration and invasion assays. E Western blot analysis of E-cadherin, Vimentin, MMP2 and MMP9 protein expressions in PC3 cells treated by sh-NC+NC inhibitor, sh-NEAT1+NC inhibitor, sh-NEAT1+miR-582-5p inhibitor, or sh-NEAT1+miR-582-5p inhibitor+sh-EZH2. Data expressed as a mean ± SD, one-way ANOVA followed by Tukey’s test. ***p<0.001, compared with the sh-NC+NC inhibitor group; ##p<0.01, ###p<0.001, compared with the sh-NEAT1+NC inhibitor group; $p<0.05, $$p<0.01, $$$p<0.001, compared with the sh-NEAT1+miR-582-5p inhibitor group

Knockdown of NEAT1 suppressed the growth of PCa in vivo

Using a xenograft nude mouse model, we found that tumors in the sh-NEAT1 group had lower volumes and weights than those in sh-NC tumors (Fig. 7A-C). In addition, IHC staining showed that NEAT1-deficient cells exhibited less Ki-67 expression and more cell apoptosis. The HE staining supported these findings (Fig. 7D). Furthermore, we discovered that in sh-NC tumors, NEAT1 and EZH2 expression was dramatically reduced, whereas miR-582-5p expression was clearly enhanced (Fig. 7E). Overall, these findings indicated that knockdown of NEAT1 inhibited prostate carcinogenesis in vivo.

Fig. 7.

Fig. 7

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Knockdown of NEAT1 suppressed the growth of PCa in vivo. A Images of xenograft tumors in sh-NC and sh-NEAT1 groups. B Weight of xenograft tumors in sh-NC and sh-NEAT1 groups. C The volume of xenograft tumors in sh-NC and sh-NEAT1 groups. D Representative HE staining, IHC staining of Ki-67, and TUNEL staining in tumor xenografts. E The mRNA level of NEAT1, miR-582-5p and EZH2 were tested by qRT-PCR in tumor xenografts. Data expressed as a mean ± SD, unpaired t test. n=3 per group; ***p<0.001.HE, hematoxylin and eosin; IHC, immunohistochemistry; TUNEL, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling

Discussion

NEAT1 is significantly overexpressed in PCa cells and tissues, according to our findings. The expression of NEAT1 was increased in advanced PCa. Xia et al. (Xia et al. 2022) used in situ hybridization to identify NEAT1 expression in PCa and paracarcinomatous clinical samples and found that PCa tissues had higher NEAT1 expression than normal tissues. In cutaneous squamous cell carcinoma tissues, NEAT1 lncRNA was expressed at high levels and associated with the lymph node metastasis and TNM stages (Gong et al. 2022). In patients with hepatocellular carcinoma, higher NEAT1 expression was linked to advanced TNM stages and metastasis, and patients with elevated NEAT1 had a worse prognosis (Zhang et al. 2020). These results indicate that NEAT1 is a potential prognostic biomarker for malignant tumors.

We found that NEAT1-deficient PCa cells had poor proliferative, migrative, and invasive abilities, as well as increased E-cadherin expression and decreased Vimentin and α-SMA expression in vitro. Further mechanistic studies revealed that NEAT1 performed its function through sponging miRNA-582-5p. Moreover, overexpression of miRNA-582-5p inhibited the proliferation, migration, and invasion capabilities of PCa cells. A previous study (Huang et al. 2019) has found that miRNA-582-5p expression was associated with PCa bone metastasis. Increasing the expression of miR-582-5p not only suppressed the invasion and migration abilities of PCa cells in vitro but also inhibited bone metastasis in vivo. Recently, correlations between miR-582-5p with the clinicopathologic features and prognosis in prostate cancer were identified (Li et al. 2022, p. 1). Consistent with these findings, we also found that PCa tissues and cells (P4E6, PC3, LNCaP, and DU145) exhibited significantly reduced miR-582-5p expression compared to their controls. Meanwhile, using the StarBase database, potential binding sites between miR-582-5p and NEAT1 were found; hence, we postulated that NEAT1 promotes cancer by competing for interactions with miR-582-5p. After performing the luciferase reporter assays and cellular functional experiments, we confirmed that NEAT1 promotes the progression of PCa through sponging miRNA-582-5p.

EZH2 plays a vital role in embryo development (Steele et al. 2006). Some miRNAs, such as miR-101, miR-26a, and miR-214, can regulate the function of EZH2. They also participated in regulating tumor occurrence and progression by controlling EZH2 and other target gene expressions (Derfoul et al. 2011; Jansen et al. 2012). Upregulation of EZH2 in tumor cells was linked to increased proliferative, migratory, invasive, and metastatic abilities (Liu et al. 2019). Furthermore, overexpression and negative regulation of EZH2 on the interferon-stimulated genes (ISGs) in PCa were identified; meanwhile, inhibition of EZH2 was also significantly involved in the regulation of double-stranded RNA–STING–ISG stress response activation and immune-related gene expressions (Morel et al. 2021). This evidence demonstrated that EZH2 played an important role in the antitumor immunity of PCa. In our present study, EZH2 was identified as a miRNA-582-5p downstream target. The rescue assays demonstrated that impaired progression of PCa induced by NEAT1 deficiency was significantly restored by miR-582-5p knocking down, while these effects were largely abolished by suppression of EZH2. Moreover, the xenograft nude mouse model suppressed PCa growth by NEAT1 knockdown in vivo. Li et al. (Li et al. 2018) proposed that NEAT1 played an essential role in maintaining PCa cell growth and preventing DNA damage via direct binding with cell division cycle 5-like protein. However, this study has not investigated the downstream miRNA genes controlled by NEAT1. Another study (Bai and Huang 2020, p. 1) investigated the expression of NEAT1 in PCa tissues and the clinical significance of NEAT1 in predicting the prognosis of patients with PCa. This study has not investigated the specific mechanism of NEAT1 in PCa. In lung cancer, it was reported that NEAT1 interacted with miR-582-5p, by releasing hypoxia-inducible factor-1α played the pro-tumor effects (Jiang et al. 2021). However, in this study, no animal experiments were performed to strengthen the in vitro data. In our work, EZH2 was identified as a miRNA-582-5p downstream target. The NEAT1/miR-582-5p/EZH2 axis was revealed to be significant in playing the pro-tumor effects in PCa. Moreover, animal experiments have indicated that knockdown of NEAT1 inhibited prostate carcinogenesis in vivo.

EMT is involved in PCa metastatic progression (Mancini et al. 2021), and previous studies (Lo et al. 2017; Liu et al. 2020; Mehra et al. 2021) have reported that EMT is a crucial stage during PCa cell migration and invasion processes. As a result, we investigated whether NEAT1/miR-582-5p/EZH2 axis can cause EMT in PCa cells. We observed that NEAT1 reduced α-SMA and Vimentin expression but increased E-cadherin expression in PCa cells, which were the same as those after miR-582-5p overexpression. However, when EZH2 was knocked out, the above effects were abolished. These findings indicated that overexpression of NEAT1 triggered metastatic phenotypes (induced EMT leading to enhanced cell proliferation, motility, and invasion), and the NEAT1/miR-582-5p/EZH2 axis played a key role in inducing EMT in PCa.

In the current work, the NEAT1/miR-582-5p/EZH2 axis was revealed to be significant in controlling the proliferative, migratory, and invasive abilities of PCa cells. Nevertheless, the limitations of our study should be considered. The mechanism of in vivo experiments is not thoroughly studied. Additional thorough studies are required to clarify the NEAT1/miR-582-5p/EZH2 axis in regulating the progression of PCa in vivo in the future.

Conclusions

In conclusion, the significant findings of our study were that NEAT1 overexpression was associated with the progression of PCa. Mechanistically, NEAT1 promotes the growth and progression of PCa cells via regulating the miR-582-5p-mediated EZH2 expression. The NEAT1/miR-582-5p/EZH2 regulatory axis may provide a theoretical basis for targeted therapy of PCa in the future.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 1049 kb) (1MB, docx)

Acknowledgements

The authors thank the patients for their contributions to sample collection. We thank all authors for their contributions to finish this article.

Abbreviations

PCa

Prostate cancer

lncRNA

Long noncoding RNA

NEAT1

Nuclear enriched abundant transcript 1

EZH2

Enhancer of zeste homolog 2

miRNA

microRNA

WT

Wild-type

Mut

Mutant

IHC

Immunohistochemistry

TUNEL

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

ANOVA

Analysis of variance

ROC

Receiver operator characteristic

EMT

Epithelial-to-mesenchymal transition

SD

Standard deviation

qRT-PCR

Quantitative real-time quantitative PCR

NC

Negative control

HE

Hematoxylin and eosin

Author contributions

All authors contributed to the study conception and design. XW and ZG contributed to the design and writing of the study. XW and WY performed the experiments and contributed to the data analysis. XW and ZG contributed to drafting the manuscript, critically modifying important content, and approving the version to be published. All authors read and approved the final manuscript.

Funding

This work was supported by the Natural science Key project of Bengbu Medical College (2022byzd081).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare they have no competing interests.

Ethical approval

The Ethics Committee of the Second Affiliated Hospital of Bengbu Medical College reviewed and approved our study protocol and informed consent documents. According to the ethical guidelines of the Helsinki Declaration, the experimental protocol in our study was established, and it was approved by the Animal Ethics Committee of Bengbu Medical College.

Informed consent

All patients agreed by consent written to use all samples for scientific research.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Weiqiang Xu, Email: sincere0731@sina.com.

Guoxi Zhang, Email: zgx8778@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material 1 (DOCX 1049 kb) (1MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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