The lncRNA NEAT1/miRNA-766-5p/E2F3 Regulatory Axis Promotes Prostate Cancer Progression

Background Prostate cancer (PCa) is one of the most common malignancies in men. Increasing evidence has demonstrated that dysregulation of long noncoding RNAs (lncRNAs) is closely related to carcinogenesis and cancer progression. lncRNA NEAT1 has recently been identified as a carcinogenic regulator of multiple cancers; however, the role of NEAT1 on PCa is still poorly understood. Methods Kaplan–Meier was conducted to determine the overall survival rate in PCa patients with aberrant NEAT1 levels. qRT-PCR analysis was performed to detect expressions of NEAT1 and miR-766-5p in tissues and cells. In addition, CCK-8, colony formation, flow cytometry analysis, wound healing, and transwell assay were conducted to determine cell proliferation, cell arrest, apoptosis, migration, and invasion. The western blot assay was utilized to determine E2F3 and cell growth-related proteins. The relationship between NEAT1 and miR-766-5p or miR-766-5p and E2F3 was verified by correlation analysis and dual-luciferase reporter assay. Results Here, we find that NEAT1 is overexpressed in PCa tissues and cell lines. Besides, silencing of NEAT1 inhibits cell proliferation, invasion, and migration and promotes cell apoptosis and cell cycle arrest. Further mechanistic studies find that NEAT1 sponges miR-766-5p, and miRNA-766-5p is negatively correlated with the expression of NEAT1. In addition, the functional experiment shows that upregulation of miRNA-766-5p inhibits PCa proliferation, migration, and invasion. Furthermore, E2F transcription factor 3 (E2F3) is testified to be the downstream target gene of miRNA-766-5p. Finally, the rescue experiment revealed that miRNA-766-5p inhibition largely restores NEAT1 downregulation-mediated function on PCa progression, while E2F3 knockdown partly removes the effects of miRNA-766-5p inhibitor. Conclusions In conclusion, NEAT1 facilitates PCa progression by targeting the miRNA-766-5p/E2F3 axis.


Introduction
As one of the most common malignant tumors in the male urinary system, prostate cancer (PCa) is a malignant tumor occurring in the prostatic epithelium [1]. According to the latest world epidemiological statistics in 2019, the morbidity of PCa has surpassed that of lung cancer, ranking the first (20%, 174,560 cases), and the mortality rate is the second (10%, 31,620 cases) [2]. Although the incidence of PCa in China is lower than that in Europe and the United States, the incidence of PCa has been on the rise in recent years [3]. Although much progress has been made in diagnosing and treating PCa, the long-term prognosis is still poor [4,5]. So, new molecular mechanisms of PCa progression need to be explored urgently to design more effective treatment strategies.
lncRNA is a kind of RNA molecule with a length of more than 200 nucleotides that have no coding ability or encodes only a short polypeptide [6,7]. lncRNAs were previously considered a transcription by-product of cellular processes. However, with the development of epigenetics, people gradually realize its important biological role, finding that lncRNAs are involved in various cellular processes, including apoptosis, differentiation, and proliferation [8][9][10]. Accumulating research studies reveal that lncRNAs play an important role in accelerating or inhibiting tumor progression. According to the previous studies, lncRNAs regulate miRNAs expression in competitive endogenous RNAs (ceRNAs) form [11]. For instance, lncRNA SNHG14 promotes the tumorigenesis of PCa via targeting miR-5590-3p to regulate YY1 expression [12]. For another example, knockdown of lncRNA LOXL1-AS1 inhibits the occurrence and development of PCa via the miR-541-3p/CCND1 axis [13]. e oncogenic role of lncRNA NEAT1 had been reported in multiple cancers. However, the understanding of the regulatory role of NEAT1 in PC was very limited, so it was urgent to study and clarify the mechanism of NEAT1. Our study mainly elucidated the possible usefulness of NEAT1 on mediating PCa progression, providing a novel promising goal for PCa treatment.

Clinical Samples.
is study obtained a total of 50 paired PCa tumor tissues and adjacent nontumor tissues from Jiangsu College of Nursing. e experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Ethics Committee of Jiangsu College of Nursing. All participants had signed informed consent forms.

Expression of NEAT1 in GEO Database.
e expression of NEAT1 in prostate cancer tumor and adjacent nontumor tissues was obtained from Gene Expression Omnibus (GEO). e original data of GSE29079 were downloaded and analyzed by GEO 2R.

CCK-8 Assay.
Briefly, cells were placed in the 96-well plate with a density of 3 × 10 4 cells/well and then added CCK-8 (10 μL) reagent into each well for 2 h at 37°C. e optical density (OD) value was measured at 450 nm by using an ultraviolet spectrophotometer (Shimadzu, Kyoto, Japan). e membranes were then incubated with secondary antibody (1 : 1000; ab7090; Abcam, Shanghai, China) on the next day at 37°C for 2 h. An enhanced chemiluminescence kit and the ImageJ software were applied to protein quantification.

Colony Formation Test.
Logarithmic growth cells were inoculated into a 6-well plate. When visible clones appeared in the Petri dish, the culture was terminated. After discarding the supernatant, 4% paraformaldehyde was fixed, and GIMSA staining solution was dyed for 10-30 min. Finally, cell clone numbers were counted under a microscope (Optical-SH, Shanghai, China).

Wound
Healing Assay. Firstly, cells were seeded into the 6-well plate until they generated a confluent monolayer. en, using a sterile pipette tip, a scratch was made in the confluent cells. After 48 h, the scratch-induced wound was observed under a microscope and analyzed by ImageJ.

Transwell Analysis.
According to the previous study, the migration and invasion of PCa cells were detected by the transwell assay [14]. 100-150 μl of cell suspension was added to the upper chamber of the transwell with or without precoated with Matrigel (BD Bioscience, Beijing, China) for the invasion and migration assay, respectively. Meanwhile, 600-800 μl of medium containing 10% serum was added to the lower chamber, and the medium was incubated at room temperature for 48 h. en, the medium was transferred to a well prefilled with approximately 800 μl of methanol and fixed for 30 min at room temperature. e chamber was removed, the upper chamber fixative was aspirated, transferred to wells prefixed with approximately 800 μl Giemsa staining solution, and stained for 15-30 min at room temperature.
e cells were carefully wiped on the bottom membrane surface of the upper chamber with a wet cotton 2 Journal of Oncology swab. Random fields of view were taken under the microscope for counting, and the results were tallied.

Flow Cytometry.
For apoptosis, cells were collected and suspended with 100 μL of binding buffer. Next, FITC annexin V and propidium iodide (PI) were used to stain cells in the dark. After 10 min, 400 μL of binding buffer was added to each tube, and the apoptosis was assessed by flow cytometry (BD Bioscience, CA, USA). For the cell cycle, to put it simply, the cells were fixed with 70% ethanol, digested with RNase for 30 min, stained with propidium iodide, and incubated for 15 min. Finally, flow cytometry was performed to measure the cell cycle.

RNA-Binding Protein Immunoprecipitation (RIP) Assay.
e RIP experiment was carried out with the Magna RIP kit (Magna, ON, CAN). Briefly, the lysed cells were incubated in the RIP buffer solution, and the magnetic beads were labeled with anti-Ago2 or IgG. e abundance of NEAT1 and miRNA-766-5p was verified by qRT-PCR.

Statistical
Analysis. Data were presented as the mean ± standard deviation (SD). Comparisons were determined using the unpaired Student's t-test and one-way ANOVA followed by Bonferroni's post hoc test. P < 0.05 indicated statistically significant.

NEAT1 Was Highly Expressed in PCa Tissue and Cell Lines.
We firstly measured the levels of NEAT1 by qRT-PCR analysis. As shown in Figure 1(a), NEAT1 was overexpressed in PCa tissues compared to adjacent normal tissues. More interestingly, we found that the level of NEAT1 in PCa patients with tumor stage III + IV was higher than that of stage I + II (Figure 1(b)).
is result is consistent with the gene expression profiles obtained from the Gene Expression Omnibus (GEO) database (GSE29079) ( Figure S1). Meanwhile, the Kaplan-Meier curve showed that the survival rate of the low-level NEAT1 group was superior to that of the high-level group (Figure 1(c)). In addition, compared with RWPE-1, NEAT1 was highly expressed in PCa cell lines, especially in LNCaP and PC3, which were chosen for the following experiments. Altogether, these results indicated the carcinogenic role of NEAT1 in PCa.

Knockdown of NEAT1 Inhibited Cell Growth in Prostate
Cancer. Next, sh-NEAT1 and its control sh-NC were used to treat LNCaP and PC3 cells to explore the functions of NEAT1 in PCa. qRT-PCR analysis showed that sh-NEAT1 transfection markedly inhibited the expression of NEAT1 in LNCaP and PC3 cells (Figure 2(a)). e CCK-8 assay revealed that the cell viability was significantly inhibited by NEAT1 knockdown in LNCaP and PC3 cells (Figure 2(b)). e colony formation assay showed that silencing of NEAT1 decreased the colony number of LNCaP and PC3 cells (Figure 2(c)). Furthermore, flow cytometry analysis detected whether NEAT1 was involved in cell cycle progression. e results indicated that NEAT1 silencing significantly increased the percentage of G0/G1 phase cells and decreased S and G2/M phase cells (Figure 2(d)). Meanwhile, we also found that NEAT1 knockdown decreased cyclin D1 expression and increased p21 expression in LNCaP and PC3 cells ( Figure 2(e)). Moreover, flow cytometry analysis and western blot analysis illustrated that NEAT1 knockdown positively affected apoptosis, including increasing apoptosis rate, upregulating Bax, cleaved-caspase-9, and cleaved-caspase-3 expression and decreasing the expression of Bcl-2 (Figures 2(f) and 2(g)).

Downregulation of NEAT1 Repressed Cell Migration and Invasion in PCa.
On the other hand, the scratch test and transwell analysis were used to examine the functions of NEAT1 on cell migration and invasion. e results illustrated that NEAT1 silencing dramatically reduced the migration and invasion in LNCaP and PC3 cells (Figures 3(a) and 3(b)). Consistently, the expressions of migration-and invasion-associated proteins (Cox-2, MMP-2, and MMP-9) were lower in the sh-NEAT1 group compared with the sh-NC group (Figure 3(c)). All these data suggested the inhibition of NEAT1 repressed PCa progression.

NEAT1 Exerts Its Function by Sponging miRNA-766-5p.
We detected the cellular position of NEAT1 to investigate the specific mechanism that NEAT1 modulated in PCa. In LNCaP and PC3, NEAT1 was mainly located in the cytoplasm (Figure 4(a)). Since lncRNAs exerted their function by      Journal of Oncology sponging miRNAs, we used the StarBase database to predict the target of NEAT1 and identify miRNA-766-5p and found that there were promising binding sites between NEAT1 and miR-766-5p (Figure 4(b)). e luciferase reporter vectors NEAT1-WT or NEAT1-Mut were constructed to transfect into LNCaP and PC3 cells to verify their connection. From the results, miR-766-5p mimic greatly reduced the luciferase activities of NEAT1-WT but had no effect on NEAT1-Mut (Figure 4(c)). Moreover, miRNA-766-5p was low expressed in PCa tissues and cell lines, especially in LNCaP and PC3 (Figures 4(d) and 4(e)). Additionally, RIP experiments demonstrated that NEAT1 and miRNA-766-5p were largely abundant in Ago2-precipitated RNA-induced silence complexes (Figure 4(f )). en, we found that sh-NEAT1 highly expressed miRNA-766-5p in LNCaP and PC3, and there was a negative relationship between NEAT1 and miRNA-766-5p in clinical samples (Figures 4(g) and 4(h)).

E2F3 Was a Target Gene of miRNA-766-5p.
To study the downstream functional genes of miRNA-766-5p, the Star-Base software was used to predict that E2F3 might be a target gene of miRNA-766-5p (Figure 6(a)) since it has been reported to exert major roles in multiple cancers. A luciferase reporter assay was performed to verify this prediction. We found that in the E2F3-WT group, miRNA-766-5p mimic obviously reduced the luciferase activity in LNCaP and PC3 cells, whereas it did not affect the activity in the E2F3-Mut group (Figure 6(b)). Moreover, E2F3 was highly expressed in PCa tissues and cell lines, especially in LNCaP and PC3 (Figures 6(c) and 6(d)). To further evaluate the relationship between miRNA-766-5p and E2F3, we used miRNA-766-5p mimic to transfect LNCaP and PC3 cells. As shown in Figures 6(e) and 6(f ), overexpression of miRNA-766-5p inhibited the protein and mRNA expression of E2F3. Moreover, we found that E2F3 expression was negatively correlated with miRNA-766-5p expression (Figure 6(g)).

Discussion
Growing evidence has indicated that lncRNAs are involved in various pathologic processes of cancer by regulating gene expressions at epigenetic, transcriptional, and post-transcriptional levels [15]. For PCa oncogenesis, abundant lncRNAs with carcinogenic or anticancer roles have been proved to be involved in this process [16,17]. In our study, we conducted a series of validation and functional experiments to assess the effect of NEAT1 in the development and occurrence of PCa, finding that knockdown of NEAT1 inhibited PCa tumorigenesis. Simply put, NEAT1 was an oncogenic gene in PCa. lncRNA NEAT1 (nuclear-enriched abundant transcript 1) was firstly reported in 2007. It is enriched in the nucleus and is an important component of near-nuclear paraspeckles, regulating gene expression mainly through stabilizing mRNA in the nucleus. NEAT1 is located on chromosome 11q13, with two variants: NEAT1_1 (3.7 kb) and NEAT1_2 (23 kb) [18]. Recently, accumulating evidence suggests that NEAT1 acts as tumor-promoting in the occurrence and development of most tumors, such as breast cancer [19], multiple myeloma [20], and pancreatic cancer [21]. Li et al. found that in hepatocellular carcinoma, NEAT 1, as a sponge for miR-204, suppresses its expression to increase ATG3 expression to promote autophagy and enhances the resistance of the cells to sorafenib [22]. Qi et al. revealed that NEAT1 competes against let-7a to promote cell proliferation and metastasis in non-small cell lung cancer (NSCLC) [23]. Additionally, NEAT1 expression is positively correlated with poor prognosis. Chen et al. reported that NEAT1 expression in esophageal squamous cell carcinoma (ESCC) is significantly upregulated and is closely related to tumor size and clinical staging, suggesting that the high expression of NEAT1 is one of the independent indicators of poor prognosis in patients with ESCC [24].
Although NEAT1 is reported to be the transcriptional target of p53, it plays an important role in the tumor suppressive function of p53 in osteosarcoma, breast cancer,     [27]. NEAT1 drives PCa growth by changing the epigenetic landscape of the target gene promoter to facilitate transcription [28]. Besides, Xiong et al. found that NEAT1 promotes PCa cells growth via modulating the SRC3/IGF1R/AKT axis [29]. In our study, NEAT1 was overexpressed in PCa tissues and cells, which is consistent with the previous result [30]. NEAT1 silencing suppressed the migration, proliferation, and invasion of PCa. Hence, NEAT1 was a cancer-promoting gene in PCa.
Increasing reports have exhibited that miRNAs participate in numerous biological behaviors of tumor progression, including invasion and apoptosis [31]. Lots of research showed that lncRNA took effects on tumorigenesis via regulation of specific miRNA. In our research, we first discovered that NEAT1 was mainly distributed in the cytoplasm of PCa cells through subcellular separation and qRT-PCR analysis, revealing that NEAT1 might regulate the expression of its downstream genes after transcription. Subsequently, bioinformatics prediction and luciferase reporter gene analysis determined that NEAT1 served as a ceRNA for miRNA-766-5p and negatively regulated miRNA-766-5p expression. miRNA-766-5p has been reported to exert major roles in multiple cancers, such as lung adenocarcinoma [32], colorectal cancer [33], and glioma [34]. e present study further verified that miRNA-766-5p expression was greatly downregulated in PCa tissues and cells, and miRNA-766-5p overexpression restrained the migration, proliferation, and invasion.
Next, we further explored the molecular mechanisms of miRNA-766-5p in PCa. Using StarBase websites, we predicted the target gene of miRNA-766-5p and identified E2F3. E2F3, located on chromosome 6p22, has been shown to be significantly upregulated in cancers and is associated with malignancy progression, including proliferation and cell cycle process [35,36]. In this study, E2F3 was positively correlated with NEAT1 whereas was negatively correlated with miRNA-766-5p. Also, rescue experiments demonstrated that E2F3 overexpression reversed that miRNA-766-5p inhibitor-induced positive function on the proliferation, migration, and invasion in the sh-NEAT1 stable cell line. However, the present study also has some limitations. e main limitation is the lack of in vivo experiments. erefore, a complete in vivo experiment should be employed to verify our results.
In conclusion, this study demonstrated the main roles of the NEAT1/miRNA-766-5p/E2F3 axis in the proliferation, migration, and invasion of PCa cells. NEAT1 promoted PCa cell proliferation, migration, and invasion via sponging miR-776-5p and consequently increased E2F3 expression, suggesting that NEAT1 might be a potential therapeutic target for PCa.

Data Availability
Data archiving will be made available on reasonable request. Runyuan Ji is responsible for the data.
Ethical Approval e experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Animal Ethics Committee of Jiangsu College of Nursing.