Sage Journals: Discover world-class research

Abstract

Colorectal cancer is a major public health problem and fourth guiding cause of cancer-induced mortality worldwide. The five-year survival rate for patients with colorectal cancer remains poor, and almost half of colorectal cancer patients present recurrence and die within five years. The increasing studies showed that long non-coding RNA (lncRNA) was involved in colorectal cancer. Therefore, this study was used to explore molecular mechanisms of nuclear paraspeckle assembly transcript 1 (NEAT1) in colorectal cancer. The real-time quantitative polymerase chain reaction (RT-qPCR) was employed to estimate the expression levels of NEAT1, Nuclear receptor 4 A1 (NR4A1), and miR-486-5p in colorectal cancer tissues and cells. Kaplan-Meier curve was conducted to analyze relationship between survival time of colorectal cancer patients and level of NEAT1. The protein levels of NR4A1, $\beta$ -catenin, c-Myc, and cyclinD1 were assessed with western blot assay. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) and flow cytometry assays were performed to evaluate proliferation and apoptosis of colorectal cancer cells, respectively. The migration and invasion abilities of cells were examined by transwell assay. The relationship between miR-486-5p and NEAT1 or NR4A1 was confirmed by dual-luciferase reporter assay. We found NEAT1 and NR4A1 were highly expressed in colorectal cancer tissues and cell lines compared with controls. Loss-functional experiments revealed that knockdown of NEAT1 or NR4A1 repressed proliferation and motility, while inducing apoptosis of colorectal cancer cells. The gain of NR4A1 could abolish NEAT1 silencing-induced effects in colorectal cancer cells. In addition, NEAT1 contributed to colorectal cancer progression through mediating NR4A1/Wnt/ $\beta$ -catenin signaling pathway. In conclusion, NEAT1 stimulated colorectal cancer progression via acting as competing endogenous RNA to sponge miR-486-5p and regulate NR4A1/Wnt/ $\beta$ -catenin signaling pathway.

Keywords

LncRNA NEAT1 miR-486-5p NR4A1/Wnt/β-catenin colorectal cancer

1. Introduction

Colorectal cancer is commonly diagnosed cancers worldwide, and its incidence and mortality rates are increasing per year, especial in developed regions and countries [1, 2]. In the past few decades, chemotherapy was a mainstream treatment method to suppress invasion and metastatic of tumor cells for colorectal cancer patients at advanced stages, which resulted in huge burden of economy and body for colorectal cancer patients. Besides, formation of drug resistance is a major obstacle for increasing the overall survival time of colorectal cancer patients [3]. Therefore, enormous efforts are needed to clarify the pathogenesis for colorectal cancer.

Recently, long non-coding RNAs (lncRNAs), more than 200 nucleotides (nt) in length and without coding-protein ability [4], have been widely acknowledged as pivot roles in multiple biological activities, including cell differentiation [5], maintains active chromatin [6], and carcinomas development [7]. Besides, previous studies have shown that nuclear paraspeckle assembly transcript 1 (NEAT1) was dysregulation in various cancers, such as gastric cancer [8], ovarian cancer [9], and breast cancer [10]. Importantly, recent studies have highlighted that NEAT1 was highly expressed and acted as a biomarker for survival prognosis in colorectal cancer [11]. Therefore, functional effects of NEAT1 was investigated in colorectal cancer.

Additionally, microRNAs (miRNAs) are a group of non-coding RNA molecules with 18–25 nucleotides [12]. Although miRNAs are no ability to encode protein, it is closely associated with the occurrence and development of human malignant tumors [13]. For instance, Liu et al. indicated that miR-19a was involved in occurrence and progression of colorectal cancer via mediating proliferation and migration of colorectal tumor cells [14]. As for miR-486-5p, a previous research revealed that miR-486-5p was decreased and functioned as an inhibitor in the development of colorectal cancer [15]. Zhang et al. also confirmed that transfection with miR-486-5p mimic into colorectal cancer cells could inhibit migratory and invasive ability of tumor cells [16].

Nuclear receptor 4 A1 (NR4A1), a transcription factor belong to the member of the steroid/thyroid hormone receptor family, is regulated by multiple factors, including serum, inflammatory factors, and stress [17, 18]. In addition, it has been suggested that frequent dysregulation of NR4A1 was closely associated with the development of most cancers by acting oncogene or tumor inhibitor [19, 20]. This adverse function of NR4A1 possible because of the different environments and regulatory patterns of tumors. In summary, NR4A1 could be served as biomarkers and targets for cancer diagnosis and treatment. Therefore, this study aimed to explore molecule mechanism of NEAT1, along with association relationship among NEAT1, miR-486-5p, and NR4A1 in colorectal cancer.

2. Materials and methods

2.1 Patient specimens

All colorectal cancer tissues ( $N=$ 40) and paired normal tissues were obtained from patients experienced surgery at The Second Hospital of Anhui Medical University, and 5-year overall survival investigation was conducted from 2013–2018. In addition, the colorectal cancer patients were divided into two groups using the median value of NEAT1 expression level as the cutoff (high expression of NEAT1 group $>$ median value, low expression of NEAT1 group $<$ median value). This study was performed with approval from the Ethics Committee of The Second Hospital of Anhui Medical University, and the written informed consent was provided from all volunteer. All tissues samples were frozen in liquid nitrogen immediately and then stored at $-$ 80 ${{}^{\circ}}$ C until next used.

2.2 Cells culture

Human NCM460 cells (INCELL, San Antonio, TX, USA) were maintained in Dulbecco’s Modified Eagle Medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; PAN, Aidenbach, Bavaria, Germany). SW620 and HT29 cells were purchased from Shanghai Cell Bank at the Chinese Academy of Sciences (Shanghai, China) and cultured in 1640 medium (GIBCO BRL) containing 10% FBS (PAN). The above cell lines were cultured at 37 ${{}^{\circ}}$ C in a humidified incubator with 5% CO ${}_{2}$ .

2.3 RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted with TRIzol reagent (Solarbio, Beijing, China) from colorectal cancer tissues or cells. Afterward, qualified RNA was reverse transcribed to complementary DNA by Prime Script RT Reagent kit (Takara, Dalian, China). The transcription levels of NEAT1, NR4A1, and miR-486-5p was estimated by SYBR-Green Reagent kit (Qiagen, Hilden, Germany) under ABI Prism 7900 (Applied Biosystems, Foster City, CA). The expression level of RNA was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endogenous small nuclear RNA U6 by using 2 ${}^{\rm-\Delta\Delta Ct}$ method.

The sequences of primers were listed:

NEAT1 (Forward, 5’-GUCUGUGUGGAAGGAGGAATT-3’; Reverse, 5’-UUCCUCCUUCCACACAGACTT-3’); NR4A1 (Forward, 5’-AGGGCTGCAAGGGCTTCT-3’; Reverse, 5’-GGCAGATGTACTTGGCGTTTTT-3’); miR-486-5p (Forward, 5’-CAGTCCTGTACTGAGCTGC-3’; Reverse, 5’-GTGCAGGGTCCGAGGT-3’); GAPDH (Forward, 5’-TCCCATCACCATCTTCCAGG-3’; Reverse, 5’-GATGACCCTTTTGGCTCCC-3’); U6 (Forward, 5’-AACGCTTCACGAATTTGCGT-3’; Reverse, 5’-CTCGCTTCGGCAGCACA-3’).

2.4 Western blot assay

Total protein was extracted from tissues or transfected cells by cell lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). The 60 $\mu$ g of protein was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore Corp, Billerica, MA, USA). The membranes were washed by Tris-buffered saline (TBST) which containing 0.1% Tween-20, and then incubated antibodies against NR4A1 (1:1500 dilution, cwbiotech, Beijing, China), $\beta$ -catenin (1:1500 dilution, Santa Cruz Bio-technology, Santa Cruz, CA, USA), c-Myc (1:1500 dilution, Santa Cruz Bio-technology), and cyclinD1 (1:1500 dilution, Santa Cruz Bio-technology), with GAPDH (1:3000 dilution, Santa Cruz Bio-technology) as the internal control. After washing with TBST buffer, the membranes were incubated with the secondary antibody (1:2000 dilution, cwbiotech) with horseradish peroxidase conjugated. Finally, the intensity of the bands was analyzed with enhanced chemiluminescence method.

2.5 RNA transfection and vector construction

The Specific small interfering RNA (siRNA) for NEAT1 (si-NEAT1), siRNA for NR4A1 (si-NR4A1) and its negative control si-NC, overexpressed plasmid of NR4A1 (NR4A1) and overexpressed plasmid of NEAT1 (NEAT1) and its negative control pcDNA, miR-486-5p mimic, and its negative control miR-NC were obtained from GenePharma (Shanghai, China). The oligonucleotides and vectors were transfected into SW620 and HT29 cells utilizing Lipofectamine 3000 (Thermo Fisher Scientific) referring to manufacturer’s instructions. Transfected cells were incubated in a humidified incubator at 37 ${{}^{\circ}}$ C with 5% CO ${}_{2}$ . After 48 h, SW620 and HT29 cells were harvested for subsequent experimentations.

2.6 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl- 2H-tetrazol-3-ium bromide (MTT) assay

The transfected SW620 and HT29 were seeded into 96-well culture plates at the density 4 $\times$ 10 ${}^{3}$ cells/well. After incubation at 37 ${{}^{\circ}}$ C for 24 h, 48 h, and 72 h, 20 $\mu$ L of MTT (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was added to each well for incubation another 4 h. Next, the dimethyl sulfoxide was used to dissolve formazan crystals. After incubation for another 10 min, the cell viability was examined by measuring optical density at wavelength of 490 nm per well on a microplate reader (Applied Biosystems).

2.7 Flow cytometry

Flow cytometry was conducted for cell apoptosis assay. In brief, SW620 and HT29 cells were harvested and washed with cold phosphate buffer saline. Apoptotic cells were stained with 5 $\mu$ L of Annexin V labeled with fluorescein isothiocyanate (FITC) and 5 $\mu$ L of propidium iodide (PI) (Thermo Fisher Scientific) in the dark at the room temperature for 15 min. The apoptotic cells were counted and analyzed using flow cytometer (Applied Biosystems) and FlowJo software (Tree Star Corp, Ashland, OR), respectively.

2.8 Transwell assay

The migration experiment was conducted with transwell plates that had 8- $\mu$ m pore size polycarbonate membranes. Approximately 2 $\times$ 10 ${}^{5}$ SW620 or HT29 cells was placed in the upper chamber with 200 $\mu$ L of serum-free medium, and then 600 $\mu$ L complete medium was added to the lower chamber (serum concentration: 10%) to allow cells migration. After 48 h of culturing, migrated cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 15 min, then photographed on light microscopy (Applied Biosystems). The invasion assay was performed using membranes coated with Matrigel matrix (BD Science, Sparks, MD, USA), while other steps were the same as migration assay.

Figure 1.

The expression levels of NEAT1 and NR4A1 in colorectal cancer tissues and cell lines. (A and B) The relative expression level of NEAT1 was determined by RT-qPCR assay in colorectal cancer tissues and adjacent non-tumor tissues, as well as in colorectal cancer cell lines (HT29 and SW620) and normal colonic epithelial cells (NCM460). (C) Kaplan-Meier curve for survival time of colorectal cancer patients was displayed with log-rank test. (D, E, F and G) RT-qPCR and western blot assays were performed to assess the expression level of NR4A1 in colorectal cancer tissues and cell lines (HT29 and SW620), along with matched controls. (H) The correlation between NEAT1 and NR4A1 expression was analyzed in colorectal cancer tissues. ${}^{*}P<$ 0.05.

2.9 Dual-luciferase reporter assay

The online bioinformatics prediction software Starbase (http://starbase.sysu.edu.cn/) was applied to predict binding site between miR-486-5p and NEAT1 or NR4A1. The fragments of NEAT1 or NR4A1 3’UTR containing miR-486-5p binding site as well as its mutant form were synthesized and inserted into vector to product the luciferase reporter, named as NEAT1 WT/MUT and NR4A1WT/MUT. Subsequently, SW620 and HT29 cells co-transfected of these luciferase reporters and miR-486-5p mimic or miR-NC with Lipofectamine 3000 (Thermo Fisher Scientific). After transfection for 48 h, SW620 or HT29 cells were harvested for luciferase activity assay by Dual-Luciferase Reporter Assay System (Thermo Fisher Scientific).

2.10 Statistical analysis

$P$ -value $<$ 0.05 meant significant difference by using Student’s $t$ -test or one-way analysis of variance in indicated time. Kaplan-Meier curve was performed to analyze survival time differences between two groups from different patients. All data were expressed as mean $\pm$ standard deviation, and statistical analysis was conducted with SPSS 21.0 software (IBM, Somers, NY, USA).

Figure 2.

The function effects of NEAT1 silencing on proliferation, apoptosis, migration, and invasion of colorectal cancer cells. SW620 and HT29 cells were transfected with si-NEAT1 or si-NC. (A) The relative expression level of NEAT1 was assessed by RT-qPCR assay in SW620 and HT29 cells. (B and C) MTT assay was carried out to evaluate cell viability in SW620 and HT29 cells post-transfection. (D) Apoptosis of transfected SW620 and HT29 cells was measured by flow cytometry. (E and F) Transwell assay was employed to measure cell abilities of migration and invasion of SW620 and HT29 cells. ${}^{*}P<$ 0.05.

3. Results

3.1 NEAT1 and NR4A1 were highly expressed in colorectal cancer tissues and cells

To begin with, we first examined NEAT1 level in colorectal cancer tissues and adjacent non-tumor tissues by RT-qPCR assay. As presented in Fig. 1A, when compared with the normal tissues, the expression level of NEAT1 was significantly upregulated in colorectal cancer tissues. The same conclusion was confirmed in Fig. 1B, RT-qPCR results indicated that NEAT1 was higher expression in SW620 and HT29 cells than that in normal colonic epithelial cells NCM460. In addition, Kaplan-Meier analysis results implied that patients with low NEAT1 expression had a longer overall survival time than those with high NEAT1 expression (Fig. 1C). We also discovered that NR4A1 also was overexpressed in colorectal cancer tissues when compared with normal tissues. No matter mRNA and protein (Fig. 1D–E). Moreover, our data also revealed that NR4A1 was upregulated in colorectal cancer cell lines in comparison with NCM460 cells (Fig. 1F–G). A positive correlation between NR4A1 and NEAT1 expression was found in colorectal cancer tissues (Fig. 1H). Above data suggested that overexpression of NEAT1 and NR4A1 was correlated with the development of colorectal cancer.

3.2 Knockdown of NEAT1 repressed proliferation, migration, and invasion while induced apoptosis of colorectal cancer cells

To identify potential effects of NEAT1 silencing on proliferation, apoptosis, migration, and invasion in colorectal cancer cells, SW620 and HT29 cells were transfected with si-NEAT1 or si-NC. Transfection efficiency was measured by RT-qPCR assay, and the results implied that knockdown of NEAT1 led to an obvious reduction of NEAT1 expression (Fig. 2A). Additionally, MTT analysis revealed that NEAT1 knockdown effectively impeded the proliferation of SW620 and HT29 cells (Fig. 2B–C). Conversely, SW620 and HT29 cells transfected si-NEAT1 showed higher apoptosis rate when compared with si-NC-transfected cells (Fig. 2D). Furthermore, the results of transwell assay indicated that the migration ability of SW620 and HT29 cells was greatly wakened following NEAT1 knockdown (Fig. 2E). Similar results were observed in invasion assay (Fig. 2F). These results demonstrated that NEAT1 knockdown obviously facilitated apoptosis and constrained proliferation and motility of colorectal cancer cells.

Figure 3.

Knockdown of NR4A1 regulated proliferation, apoptosis, migration, and invasion of colorectal cancer cells. SW620 and HT29 cells were transfected with si-NR4A1 or si-NC. (A and B) The mRNA and protein levels of NR4A1 were measured by RT-qPCR and western blot assays in SW620 and HT29 cells, respectively. (C and D) The effect of si-NR4A1 on cellular viability was assessed by MTT assay. (E) Flow cytometry assay was conducted to test apoptosis of transfected SW620 and HT29 cells. (F and G) Cell migration and invasion capabilities were evaluated by transwell assay in transfected SW620 and HT29 cells. ${}^{*}P<$ 0.05.

3.3 Inhibition of NR4A1 suppressed proliferation, migration, and invasion, and enhanced apoptosis of colorectal cancer cells

As NR4A1 was upregulated in colorectal cancer tissues and cells, we knocked down the expression of NR4A1 in SW620 and HT29 cells by performing loss-functional experiment. As shown in Fig. 3A, compared with si-NC-transfected SW620 and HT29 cells, the expression level of NR4A1 was decreased in SW620 and HT29 cells transfected with si-NR4A1. Moreover, knockdown of NR4A1 evidently repressed protein expression level of NR4A1 in SW620 and HT29 cells by western blot assay (Fig. 3B). Analogously, NR4A1 silencing impeded proliferation in in SW620 and HT29 cells when compared with the control group (Fig. 3C-D). We further probed the effect of NR4A1 knockdown on the apoptosis in SW620 and HT29 cells, and results implied that apoptosis of SW620 and HT29 cells was dramatically increased after inhibition of NR4A1 (Fig. 3E). Additionally, the migration and invasion results suggested that SW620 and HT29 cells transfected with si-NR4A1 remarkably inhibited the cell capabilities of migration and invasion in comparison with si-NC group (Fig. 3F-G). The above data indicated knockdown of NR4A1 might act as a tumor suppressor in colorectal cancer.

Figure 4.

Overexpression of NR4A1 abolished the effects of NEAT1 silencing on proliferation, motility, and apoptosis of colorectal cancer cells. (A and B) The expression level of NR4A1 was measured by RT-qPCR and western blot assays in SW620 and HT29 cells transfected with si-NEAT1 or si-NC. SW620 and HT29 cells were transfected with si-NC, si-NEAT1, si-NEAT1+pcDNA, or si-NEAT1+NR4A1. (C and D) Cell viability of SW620 and HT29 cells was assessed by MTT assay. (E) Flow cytometry showed apoptosis rate of SW620 and HT29 cells after transfection. (F and G) Transwell analysis was used to show the migration and invasion abilities of SW620 and HT29 cells. ${}^{*}P<$ 0.05.

3.4 Overexpression of NR4A1 mitigated NEAT1 silencing-mediated effects on proliferation, motility, and apoptosis of colorectal cancer cells

It reported that NEAT1 and NR4A1 were involved in proliferation, motility, and apoptosis of colorectal cancer cells in above results. As presented in Fig. 4A–B, RT-qPCR and western blot assays implied that the expression level of NR4A1 was inhibited in SW620 and HT29 cells after transfection with si-NEAT1 (Fig. 4A–B). In addition, overexpression of NR4A1 could significantly reduce cell viability loss in SW620 and HT29 cells caused by silencing NR4A1 (Fig. 4C–D). Flow cytometry results showed apoptosis rate was notably decreased in SW620 and HT29 cells transfected with si-NEAT1+NR4A1 when compared with cells only transfected with si-NEAT1 (Fig. 4E). Finally, transwell assay indicated that overexpression of NR4A1 effectively prevented knockdown of NEAT1-induced inhibitory effects on migration and invasion (Fig. 4F–G). Collectively, these data suggested that NEAT1 silencing repressed proliferation, motility, and facilitated apoptosis in colorectal cancer cells by regulating NR4A1 expression.

3.5 NEAT1 targeted miR-486-5p to regulate NR4A1 expression in colorectal cancer cells

The online bioinformatics tool was used to predict target miRNA of NEAT1. The possible binding sites between miR-486-5p and NEAT1, as well as its mutant sites were shown in Fig. 5A. Moreover, overexpression miR-486-5p conspicuously reduced the relative luciferase activity of WT-NEAT1 group, but not that of the MUT-NEAT1 (Fig. 5B–C). Furthermore, the increased expression of miR-486-5p was observed in SW620 and HT29 cells after silencing NEAT1, while overexpression of NEAT1 significantly repressed miR-486-5p expression in SW620 and HT29 cells (Fig. 5D–E). Besides, we found miR-486-5p could bind to the 3’UTR of NR4A1 by bioinformatics prediction (Fig. 5F). Subsequently, dual-luciferase reporter assay was applied to measure luciferase activity in SW620 and HT29 cells. As indicated in Fig. 5G–H, the luciferase activity in NR4A1 3’UTR-WT group was distinctly decreased by miR-486-5p mimic, while miR-486-5p mimic failed to affect luciferase intensity in NR4A1 3’UTR-WT group. In addition, western blot analysis displayed that transfection of miR-486-5p mimic prominently inhibited protein levels of NR4A1 in SW620 and HT29 cells, while transfection with NR4A1 evidently blocked this reduction (Fig. 5I–J). In summary, NEAT1 mediated NR4A1 expression in colorectal cancer cells by targeting miR-486-5p.

Figure 5.

Target assay of NEAT1 and miR-486-5p. (A) The putative binding sites between NEAT1 and miR-486-5p were shown. (B and C) Relative luciferase activity was determined in SW620 and HT29 cells co-transfected with NEAT1 WT/MUT luciferase reporter and miR-486-5p mimic/miR-NC. (D and E) The RT-qPCR assay was conducted to test miR-486-5p level in SW620 and HT29 cells transfected with si-NC, si-NEAT1, pcDNA, or NEAT1. (F) The pairing regions between miR-486-5p and NR4A1, as well as their mutant regions were presented. (G and H) Dual-luciferase reporter assay was employed to examine luciferase activity in SW620 and HT29 cells. (I and J) The protein expression level of NR4A1 was determined by western blot assay in SW620 and HT29 cells transfected with miR-NC, miR-486-5p, miR-486-5p+pcDNA, or miR-486-5p+NR4A1. ${}^{*}P<$ 0.05.

3.6 Downregulation of NR4A1 induced by miR-486-5p could be recovered by transfection with NEAT1 in colorectal cancer cells

To further analyze the interaction relationship among NEAT1, miR-486-5p, and NR4A1, gain-functional experiment was performed to assess both mRNA and protein levels of NR4A1 by RT-qPCR and western blot analysis. The results elucidated that overexpression of NEAT1 could mitigate suppressive effect on NR4A1 expression in colorectal cancer cells caused by upregulation of miR-486-5p (Fig. 6A–C). Thus NEAT1/miR-486-5p/NR4A1 axis might play key function in colorectal cancer.

Figure 6.

NEAT1 regulated NR4A1 expression in colorectal cancer cell lines via affecting miR-486-5p. (A, B and C) The mRNA and protein expression levels of NR4A1 were assessed by RT-qPCR and western blot assays in SW620 and HT29 cells transfected with miR-NC, miR-486-5p, miR-486-5p+pcDNA, or miR-486-5p+NEAT1. ${}^{*}P<$ 0.05.

Figure 7.

NEAT1 mediated Wnt/ $\beta$ -catenin signaling pathway via regulating NR4A1. (A) Western blot assay was used to measure the expression levels of $\beta$ -catenin, c-Myc, and cyclinD1 in SW620 cells transfected with pcDNA, pcDNA-NEAT1, NEAT1+si-NC, or NEAT1+si-NR4A1. (B) The expression levels of $\beta$ -catenin, c-Myc, and cyclinD1 were assessed with western blot assay in SW620 cells transfected with si-NC, si-NEAT1, si-NEAT1+pcDNA, si-NEAT1+NR4A1.

3.7 NEAT1 mediated Wnt/

\beta

-catenin signaling pathway via regulating NR4A1

It had reported that Wnt/ $\beta$ -catenin signaling pathway was closely related to cell proliferation and invasion. Therefore, we detected expression levels of $\beta$ -catenin, c-Myc, and cyclinD1 in SW620 cells. As presented in Fig. 7A, overexpression of NEAT1 markedly activated Wnt/ $\beta$ -catenin signaling pathway by increasing $\beta$ -catenin, c-Myc, and cyclinD1 expression, while the enhancement effects could be attenuated by silencing NR4A1 in SW620. Conversely, overexpression of NR4A1 restored knockdown of NEAT1-induced low expression of $\beta$ -catenin, c-Myc, and cyclinD1 in SW620 cells (Fig. 7B). These data supported that NEAT1 was critical regulator for Wnt/ $\beta$ -catenin signaling pathway in colorectal cancer cells by affecting NR4A1 expression at least.

4. Discussion

Currently, our results suggested that NEAT1 was dramatically increased in colorectal cancer tissues and cells compared with controls; at the meanwhile, knockdown of NEAT1 effectively repressed the abilities of proliferation and motility while enhanced apoptosis of colorectal cancer cells by targeting miR-486-5p/NR4A1 axis.

A series of studies highlighted the importance of lncRNA in colorectal cancer pathogenesis [21]. The regulation mechanism by which lncRNA mediated cellular behaviors was complex, which was involved multiple factors [22]. Typically, increasing studies have reported that lncRNAs functioned as miRNA sponges to regulate multiple cellular behaviors. Jiang et al. discovered that NEAT1 involved in the development of lung cancer by acting as a competing RNA to sponge hsa-mir-98-5p [23]. Similar results were reported by Luo et al. [24], NEAT1 acted as an oncogene in colorectal cancer by sponging miRNA-34a. At the present study, we demonstrated that NEAT1 was a biomarker in clinical outcome of colorectal cancer patients. Mechanistic investigation results suggested that NEAT1 could serve as competing RNA via sponging miR-486-5p to mediated NR4A1 expression in colorectal cancer.

Furthermore, Liu et al. reported that miR-486-5p was notably decreased in colorectal cancer tissues, especial in advanced stage cancer (stage III and IV), while neuropilin-2 was a target of miR-486-5p [15]. Surely, miR-486-5p could target several other pathways, like phosphatidylinositol 3-kinase regulatory subunit 1 (PIK3R1) [16]. In this study, our data revealed that miR-486-5p negatively targeted NR4A1 expression in colorectal cancer cells. Consistent with previous results [25], NR4A1 acted as carcinogenic gene in the development and progression of colorectal cancer. Furthermore, knockdown of NEAT1-induced effects on proliferation, apoptosis, migration, and invasion could be abolished by overexpression of NR4A1. Additionally, Wnt/ $\beta$ -catenin signaling pathway could mediate cellular behaviors, including proliferation and invasion [26]. Interestingly, it had been confirmed that NEAT1 could activate the Wnt/ $\beta$ -catenin signaling pathway in multiple myeloma and glioblastoma cells [27, 28]. Luo et al. also revealed that NEAT1 was involved in pathogenesis and development of colorectal cancer by mediating the Wnt/ $\beta$ -catenin signaling pathway [24]. Therefore, it was value to probe the association between NEAT1 dysregulation and activation of Wnt/ $\beta$ -catenin signaling pathway in colorectal cancer. Our result showed that upregulation of NEAT1 exhibited enhancement effects on $\beta$ -catenin, cyclinD1, and c-Myc expression in colorectal cancer cells. In converse, knockdown of NEAT1 obviously inhibited $\beta$ -catenin, c-Myc, and cyclinD1 expression, which indicated that NEAT1 was associated with the Wnt/ $\beta$ -catenin signaling pathway in colorectal cancer. Thus, it was concluded that NEAT1 contributed to colorectal cancer progression via sponging miR-486-5p and activating of NR4A1/Wnt/ $\beta$ -catenin signaling pathway at least.

In summary, this research reported the cancerogenic roles of NEAT1 in colorectal cancer progression through mediating proliferation, metastasis, and apoptosis of colorectal cancer cells through regulating NR4A1/Wnt/ $\beta$ -catenin signaling pathway by sponging miR-486-5p, which might offer a novel theoretical target for colorectal cancer diagnosis in the future.

Footnotes

Conflict of interest

The authors declare that they have no competing interests.

References

Arnold

Sierra

M.S.

Laversanne

Soerjomataram

Jemal

and Bray

, Global patterns and trends in colorectal cancer incidence and mortality, Gut 66(4) (2017), 683.

Parkin

D.M.

Bray

Ferlay

and Pisani

, Global cancer statistics, CA: A Cancer Journal for Clinicians 55(2) (2005), 74–108.

Longley

D.B.

Allen

W.L.

and Johnston

P.G.

, Drug resistance, predictive markers and pharmacogenomics in colorectal cancer, Biochimica et Biophysica Acta (BBA) – Reviews on Cancer 1766(2) (2006), 184–196.

Ponting

C.P.

Oliver

P.L.

and Reik

, Evolution and functions of long noncoding RNAs, Cell 136(4) (2009), 629–641.

Fatica

and Bozzoni

, Long non-coding RNAs: New players in cell differentiation and development, Nature Reviews Genetics 15 (2013), 7.

Wang

K.C.

Yang

Y.W.

Liu

Sanyal

Corces-Zimmerman

Chen

Lajoie

B.R.

Protacio

Flynn

R.A.

Gupta

R.A.

Wysocka

Lei

Dekker

Helms

J.A.

and Chang

H.Y.

, A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression, Nature 472 (2011), 120.

Gibb

E.A.

Brown

C.J.

and Lam

W.L.

, The functional role of long non-coding RNA in human carcinomas, Molecular Cancer 10(1) (2011), 38.

J.W.

Kong

and Sun

, Long noncoding RNA NEAT1 is an unfavorable prognostic factor and regulates migration and invasion in gastric cancer, Journal of Cancer Research and Clinical Oncology 142(7) (2016), 1571–1579.

Chai

Liu

Zhang

and Liu

, HuR-regulated lncRNA NEAT1 stability in tumorigenesis and progression of ovarian cancer, Cancer Medicine 5(7) (2016), 1588–1598.

10.

Choudhry

Albukhari

Morotti

Haider

Moralli

Smythies

Schödel

Green

C.M.

Camps

Buffa

Ratcliffe

Ragoussis

Harris

A.L.

and Mole

D.R.

, Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival, Oncogene 34 (2014), 4482.

11.

Chen

Tan

Zheng

Wang

Zhao

and Li

, NEAT expression is associated with tumor recurrence and unfavorable prognosis in colorectal cancer, Oncotarget 6(29) (2015), 27641–27650.

12.

Bartel

D.P.

, MicroRNAs: Genomics, biogenesis, mechanism, and function, Cell 116(2) (2004), 281–297.

13.

Lee

Y.S.

and Dutta

, MicroRNAs in cancer, Annu Rev Pathol 4 (2009), 199–227.

14.

Liu

Yang

Cheng

Chen

Cui

Sun

You

Liu

Sun

Wang

Zhang

and Chen

, miR-19a promotes colorectal cancer proliferation and migration by targeting TIA1, Mol Cancer 16(1) (2017), 53.

15.

Liu

Shi

Liu

and Lian

, miR-486-5p attenuates tumor growth and lymphangiogenesis by targeting neuropilin-2 in colorectal carcinoma, OncoTargets and Therapy 9 (2016), 2865–2871.

16.

Zhang

and Qin

, miR-486-5p regulates the migration and invasion of colorectal cancer cells through targeting PIK3R1, Oncology Letters (2018).

17.

Uemura

Mizokami

and Chang

, Identification of a new enhancer in the promoter region of human TR3 orphan receptor gene. A member of steroid receptor superfamily, J Biol Chem 270(10) (1995), 5427–5433.

18.

Winoto

and Littman

D.R.

, Nuclear hormone receptors in T lymphocytes, Cell 109(Suppl) (2002), S57–66.

19.

Zhou

Drabsch

Dekker

T.J.

de Vinuesa

A.G.

Hawinkels

L.J.

Sheppard

K.A.

Goumans

M.J.

Luwor

R.B.

de Vries

C.J.

Mesker

W.E.

Tollenaar

R.A.

Devilee

C.X.

Zhu

Zhang

and Dijke

P.T.

, Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-β signalling, Nat Commun 5 (2014), 3388.

20.

Mullican

S.E.

Zhang

Konopleva

Ruvolo

Andreeff

Milbrandt

and Conneely

O.M.

, Abrogation of nuclear receptors Nr4a3 and Nr4a1 leads to development of acute myeloid leukemia, Nat Med 13(6) (2007), 730–735.

21.

Xie

Tang

Xiao

Y.F.

Xie

B.S.

Dong

Zhou

J.Y.

and Yang

S.M.

, Long non-coding RNAs in colorectal cancer, Oncotarget 7(5) (2016), 5226–5239.

22.

Huarte

, The emerging role of lncRNAs in cancer, Nat Med 21(11) (2015), 1253–1261.

23.

Jiang

Wang

Huang

and Feng

, NEAT1 upregulates EGCG-induced CTR1 to enhance cisplatin sensitivity in lung cancer cells, Oncotarget 7(28) (2016), 43337–43351.

24.

Luo

Chen

J.-J.

Qin

Huang

Y.-Z.

M.-H.

and Zhong

, Long non-coding RNA NEAT1 promotes colorectal cancer progression by competitively binding miR-34a with SIRT1 and enhancing the Wnt/β-catenin signaling pathway, Cancer Letters 440–441 (2019), 11–22.

25.

Huang

Xie

Song

Chang

Liang

and Huang

, MiR-506 suppresses colorectal cancer development by inhibiting orphan nuclear receptor NR4A1 expression, Journal of Cancer 10(15) (2019), 3560–3570.

26.

Clevers

and Nusse

, Wnt/β-catenin signaling and disease, Cell 149(6) (2012), 1192–1205.

27.

Chen

Cai

Wang

Liu

Yang

Zhou

Kang

and Jiang

, Long noncoding RNA NEAT1, regulated by the EGFR pathway, contributes to glioblastoma progression through the WNT/beta-catenin pathway by scaffolding EZH2, Clin Cancer Res 24(3) (2018), 684–695.

28.

Geng

Guo

Zhang

Wang

Liu

and Xiong

, Resveratrol inhibits proliferation, migration and invasion of multiple myeloma cells via NEAT1-mediated Wnt/β-catenin signaling pathway, Biomedicine & Pharmacotherapy 107 (2018), 484–494.

Upregulation lnc-NEAT1 contributes to colorectal cancer progression through sponging miR-486-5p and activating NR4A1/Wnt/ β -catenin pathway

Abstract

Keywords

1. Introduction

2. Materials and methods

2.1 Patient specimens

2.2 Cells culture

2.3 RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR)

2.4 Western blot assay

2.5 RNA transfection and vector construction

2.6 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl- 2H-tetrazol-3-ium bromide (MTT) assay

2.7 Flow cytometry

2.8 Transwell assay

2.10 Statistical analysis

3.1 NEAT1 and NR4A1 were highly expressed in colorectal cancer tissues and cells

3.2 Knockdown of NEAT1 repressed proliferation, migration, and invasion while induced apoptosis of colorectal cancer cells

3.5 NEAT1 targeted miR-486-5p to regulate NR4A1 expression in colorectal cancer cells

4. Discussion

Footnotes

Conflict of interest

References