Enhanced expression of long non-coding RNA NEAT1 is associated with the progression of gastric adenocarcinomas

GAC samples were obtained from 131 surgical patients of the Department of Gastroenterology, Zhonghan Hospital of Hubei Province. All 131 tumors were adenocarcinomas and other types of tumors (squamous cell, carcinoid, and stromal tumors) were excluded. A summary of cohort characteristics was shown in Table 1. Case-matched adjacent normal mucosa, located at least 3 cm far from the macroscopically unaffected margins of the tumor, were defined as normal controls. All the samples were stored in liquid nitrogen immediately after the operations. Gastric carcinomas were staged according to the TN classification system and graded into five groups: stage 0 (TisN0M0, n = 0), stage 1 (T1N0–1M0, n = 4), stage 2 (T1N2M0, T2N0–1M0, n = 30), stage 3 (T2N2M0, T3N1–2M0, T4N0M0, n = 66), stage 4 (T4N1–2M0,T1–3N3M0, TanyNanyM1, n = 31). Matched samples of GACs and adjacent normal tissues (ANTs) were immediately stored in liquid nitrogen after operation. For each sample, a portion of tissue was disposed to make frozen sections and then micro-dissected by the Eppendorf Microdissection System (Siskiyou MX160L micromanipulator and PixeLink PL-A662 color CCD firewire camera). Then, the dissected tissues were subjected to real-time PCR analysis. All patients were informed about the aims of specimen collection and gave signed written consent in accordance with the ethical guidelines of Zhongshan University. The investigations were conducted according to the Declaration of Helsinki principles and the study was approved by the ethical committee of Zhongshan Hospital of Hubei Province (EC201402348).

Total RNA was isolated from tissues by using AxyPrepTM Blood Total RNA MiniPrep Kit (Axygen) according to the manufacturer’s instruction. First-strand complementary DNA (cDNA) was synthesized with RevertAid™ First Stand cDNA Synthesis Kit (Fermentas) using a random hexamar primer. Quantitative PCR was performed through BioRad Chromo4 real-time PCR system. The primer sets for amplifying NEAT1 and reference gen (ACTB) are the following: NEAT1-F: TGG ACT AGCT CAG GGA CTT CAG; NEAT1-R: TCT CCT TGA CCA AGA CTT CCT TC; ACTB-F: GAC CTG ACT GAC TAC CTC ATG AAG AT; ACTB-R: GTC ACA CTT CAT GAT GGA GTT GAA GG. At the end point of PCR cycles, melt curves were made to check product purity. The level of NEAT1 was expressed as a ratio relative to the β-actin messenger RNA (mRNA) in each sample. Exploratory data analysis using scatter plot was applied to visually identify the expression level of target mRNA. Statistical analysis was performed using paired t test or non-parametric test. P values less than 0.05 were considered statistically significant.

Human gastric cancer cell lines MKN-45, BGC823, MGC803, SGC7901, AGS, and MKN28 were obtained from the Cobioer Biosciences Co., Ltd. (Shanghai, China) where they were characterized by mycoplasma detection, DNA fingerprinting, isozyme detection, and cell vitality detection. These cell lines were purchased in August 2014 and immediately expanded and frozen such that they could be restarted every 3 to 4 months from a frozen vial of the same batch of cells. MKN28 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10 % fetal bovine serum (PAA) and 1 % penicillin/streptomycin (Life Technologies, Inc.).

Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) was used. The pcDNA3.1-NEAT1 and empty vector (used as a negative control) was purchased from Invitrogen, Shanghai, China. Cells were seeded in 96 plates 24 h before the experiment. MGC803 cells were transfected with pcDNA3.1-NEAT1 or negative control. The MGC803 was transfected.

NEAT1-set small interfering RNA (siRNA)/small hairpin RNA (shRNA)/RNAi Lentivector as well as control shRNA vector were purchased from Nanjing EnoGene Biotechnology Limited Corporation (catalog no. ES000103). To generate lentiviruses expressing MALAT-1 shRNA and control shRNAs, HEK293T cells grown on a 10-cm dish were transfected with 6 μg of NEAT1-set shRNA lentivector or control vector, 6 μg of pREV, 6 μg of pGag/Pol, and 2 μg of pVSVg. Twelve hours after transfection, cells were cultured with DMEM medium containing 20 % FBS for an additional 36 h. The culture medium containing lentivirus particles was centrifuged at 10,000 ×g for 2 min and then used for infection. Twenty-four hours after infection, cells were cultured with fresh medium for another 24 h, followed with further experiment. The knockdown efficiency was evaluated by real-time PCR analysis.

Cell proliferation was measured by WST-1 assay. Cells were plated in 96-well culture plates (1 × 10³ per well); WST-1 (Roche) assay measuring the activity of mitochondrial dehydrogenases was performed following the manufacturer’s instruction at 0-, 1-, 2-, 3-, 4-, and 5-day time points.

Migration assays were performed using 24-well transwell units with 8-mm pore size polycarbonate inserts (BD Biosciences). Transwells were coated overnight with 10 mg/mL of fibronectin in phosphate-buffered saline at 48.8 °C, followed by incubation with 1 % bovine serum albumin for 1 h at 37 °C. The MKN45 and AGS cells transfected with NEAT1 shRNA or nonsense strand were detached with trypsin/ethylene diamine tetraacetic acid, washed once with DMEM containing 10 % fetal bovine serum (FBS), and re-suspended in DMEM containing 1 % FBS at 2 × 10⁵ cells/mL. Aliquots (100 mL) of cell suspensions were directly added to the upper side of each chamber. Following incubation for 12 h, the cells on the upper side of the membrane were removed, whereas the cells that migrated to the underside were fixed with 3 % formaldehyde and stained with 0.3 % crystal violet for 10 min. The number of cells on the underside of the membrane was counted in five different fields with a light microscope at 20 °C, and the mean and standard deviation were calculated from three independent experiments.

GraphPad Prism software (Version 5.0) was used to analyze the obtained data. Results of the NEAT1 lncRNA expression for paired GACs and ANTs or paired GACs and local lymph node metastases were compared using paired t test. Results of the NEAT1 lncRNA expression in different GAC groups were compared using non-parametric Mann–Whitney test. P values less than 0.05 were considered statistically significant.

The expression levels of NEAT1 lncRNA in the collected GAC samples were examined using real-time PCR assay. As shown in Fig. 1a, the expression levels of NEAT1 lncRNA were markedly enhanced in GACs compared to ANTs (P < 0.001). There was a statistical difference between the group of early-stage GACs and advanced GACs (P < 0.01). This result was in line with the reported findings in NSCLCs and HCCs [16, 17].

Next, we compared the NEAT1 lncRNA expression between tumor groups with (n = 85) or without lymph node metastasis (n = 46). As shown in Fig. 2a, there was a marked difference of NEAT1 expression between GAC with or without lymph node metastasis (LNM) (P = 0.004). In the GACs with LNM (n = 85), we also compared the expression of NEAT1 lncRNA in primary tumors and metastases. As shown in Fig. 2b, the analysis using paired t test showed a minor but significant increase of NEAT1 lncRNA expression in the metastases compared to the corresponding primary tumors (P = 0.0392). The association between NEAT1 expression and GAC metastasis might need further investigation.

It was reported that increased NEAT1 lncRNA expression could drive oncogenic growth by altering the epigenetic landscape of target gene promoters to favor transcription [18]. To further investigate the role of NEAT1 lncRNA in GAC progression, in vitro studies using GAC cell lines were performed. We first compared the expression of NEAT1 lncRNA among six cell lines including MKN45, BGC823, MGC803, SGC7901, AGS, and MKN28. As shown in Fig. 3a, MKN-45 and AGS showed relatively higher expression of NEAT1, which were utilized for the following knockdown experiments.

The efficiencies of overexpression and knockdown of NEAT1 in GAC cells were shown in Fig. 3b, c. Then, WST-1 assay was carried out to evaluate the influence of altered expression of NEAT1 on GAC cell proliferation. As shown in Fig. 3d, down-regulation of NEAT1 lncRNA significantly impaired the proliferation of MKN45 and AGS cells, while overexpression of NEAT1 lncRNA resulted in enhanced proliferation in MGC803 cells.

The migration ability of MKN45 and AGS cells transfected with NEAT1 shRNA or scramble RNA was evaluated by transwell assay. We found that the MKN45 as well as AGS cells with decreased expression of NEAT1 showed slightly impaired migration compared with controls (Fig. 3e). Thus, altered expression NEAT1 lncRNA might have minor effect on GAC cells’ migration.