LncRNA IUR Is Downregulated in Gastric Carcinoma and Associated with Poor Survival

GC patients

This study selected 60 GC patients (gender: 34 males and 26 females; age range: 29–68 years; mean age: 49.3 ± 6.4 years) from the 122 cases of GC patients admitted to Huanggang Central Hospital between March 2010 and January 2014. Inclusion criteria were: (1) all GC patients were newly diagnosed; (2) no initiated therapies were observed; and (3) patients willing to complete follow-up. Exclusion criteria were: (1) patients suffered from multiple clinical disorders; (2) patients with recurrent GC; and (3) patients with initiated therapies. Clinical staging was performed according to the standard established by AJCC. There were 12 (stage II), 22 (stage III), and 26 (stage IV) cases, respectively. Before the admission of patients, the aforementioned hospital's Ethics Committee approved this study. All these 60 GC patients signed informed consent.

GC tissues and cells

Biopsy was performed on all 60 GC patients before the initiation of therapies to collect nontumor gastric tissues and GC tissues. Histopathological examinations were performed to test all tissue samples, and all tissues samples were correct: (1) nontumor tissues contained no or less than 1% cancer cells; (2) cancer tissues contained more than 90% cancer cells.

Two human GC cell lines, SNU-16 and N87 (ATCC), were used. A mixture of 10% FBS and 90% RPMI-1640 medium was used for cell culture. Cell culture conditions were 5% CO₂, 37°C, and 95% humidity.

Follow-up

All 60 GC patients were monitored for 5 years. All patients were visited on a monthly manner through telephone. Patients who died of other diseases and the ones who failed to complete the follow-up were excluded.

Transient transfections

The vectors expressing IUR and ROCK1 were constructed by RIBOBIO (Guangzhou, China) using pcDNA3.1 vector. Transient transfections were performed using Lipofectamine 2000 reagent (Invitrogen) to transfect 10 nM vectors (empty vector as negative control, NC) into 10⁶ cells. Control (C) cells were untransfected. The following experiments were performed at 24 h post-transfections.

Quantitative polymerase chain reaction

Total RNAs in tissues and SNU-16 and N87 cells were isolated using TRIzol (Invitrogen). All RNA samples were quantified using NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific). Following DNase I digestion, RNA samples were used as template to synthesize cDNAs through reverse transcription using the TruScript Reverse Transcriptase Kit (NORGEN). To detect the expression of IUR and ROCK1 mRNA, SYBR Green Master Mix (Bio-Rad) was used to perform quantitative polymerase chain reaction (qPCR) with GAPDH as endogenous control. Three replicate qPCRs were set for each experiment. All qPCR data were normalized using 2^−ΔΔCT method.

Methylation-specific PCR

SNU-16 and N87 cells were subjected to DNA extraction using the Genomic DNA Extraction Kit (ab156900) from Abcam. DNA samples were converted using the DNA Methylation-GoldTM Kit (ZYMO RESEARCH). The Taq DNA Polymerase Kit (NEB) was then used to perform methylation-specific PCR (MSP) to analyze the methylation of ROCK1 gene.

Transwell assay

SNU-16 and N87 cells were harvested at 24 h post-transfections. After cell counting, RPMI-1640 medium was used to resuspend cells with a ratio of 1 mL per 4 × 10⁴ cells. A 96-well Transwell plate was used to cultivate cells (0.1 mL cell suspension per well in the upper chamber) under conditions of 5% CO₂, 37°C, and 95% humidity. The lower chamber contained RPMI-1640 medium (20% FBS). Membranes were coated with Matrigel (356234; Millipore) for 6 h at 37°C before invasion assay. Cell culture was performed for 12 h, following membrane staining for 15 min at room temperature using 1% Crystal Violet (Sigma-Aldrich). Cells were observed and counted under a light microscope.

Western-blot

RIPA solution (Sangon) was used to isolate total proteins. After protein denaturation in boiling water for 5 min, protein samples were separated through electrophoresis using SDS-PAGE gel (10%). The proteins were transferred onto PVDF membranes and blocked in FBS containing 5% nonfat milk for 1 h. PVDF membranes were first blotted with ROCK1 (ab156284, 1:1100; Abcam) and GAPDH (ab9485, 1: 1100; Abcam) primary rabbit antibodies at 4°C overnight, followed by blotting with goat anti-Rabbit IgG-HRP secondary antibody(1:1100, MBS435036; MyBioSource). Signals were developed by dropping ECL (Sigma-Aldrich) onto PVDF membranes. Gray values were normalized using ImageJ v.146 software.

Statistical analysis

Paired t test was used to compare GC and nontumor tissues. One-way ANOVA and Tukey's test were used to compare multiple groups. Linear regression was used to analyze correlations. Patients were divided into high and low IUR level groups according to the median value of the expression levels of IUR in GC tissues. Survival curves were plotted and compared by log-rank test. p < 0.05 was statistically significant.

IUR was downregulated in GC and low expression of IUR predicted poor survival

Expression levels of IUR in both GC and nontumor tissues of the 60 GC patients were measured by performing qPCR. It was observed that levels of IUR expression were significantly lower in GC tissues compared with nontumor tissues (Fig. 1A, p < 0.05). Survival curves were plotted for both high and low IUR level groups and compared. It can be observed that the 5-year overall survival rate of patients in low IUR group was significantly lower compared with patients in the high IUR group (Fig. 1B).

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FIG. 1.

Downregulation of IUR in GC predicted poor survival. Expression levels of IUR in both GC and nontumor tissues of the 60 GC patients were measured by performing qPCR and data were compared by paired t test (A). Patients were divided into high and low IUR level groups (n = 30) according to the median value of the expression levels of IUR in GC tissues. Survival curves were plotted and compared by log-rank test (B). Experiments were repeated three times and mean values were presented, *p < 0.05. GC, gastric carcinoma; qPCR, quantitative polymerase chain reaction. Color images are available online.

ROCK1 was inversely correlated with IUR in GC

Expression levels of ROCK1 mRNA in two types of tissues were also measured by performing qPCR. Levels of ROCK1 mRNA expression were significantly higher in GC tissues in comparison to nontumor tissues (Fig. 2A, p < 0.05). Correlation analysis showed that ROCK1 mRNA and IUR were significantly, inversely correlated across GC tissues (Fig. 2B). However, the correlation between ROCK1 mRNA and IUR was not significant in nontumor tissues (Fig. 2C).

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FIG. 2.

ROCK1 was inversely correlated with IUR in GC. Levels of ROCK1 mRNA expression were also measured by performing qPCR and compared by paired t test between GC and nontumor tissues (A). Correlations between ROCK1 mRNA and IUR in GC tissues (B) and nontumor tissues (C) were analyzed by linear regression. Color images are available online.

IUR overexpression downregulated ROCK1 in GC cells

SNU-16 and N87 cells were transfected with pcDNA3.1 vectors expressing IUR and ROCK1. Overexpression of IUR and ROCK1 was confirmed at 24 h post-transfection (Fig. 3A, p < 0.05). Moreover, compared with two controls, IUR overexpression was followed by ROCK1 downregulation (Fig. 3B, p < 0.05), while no alterations in IUR expression were observed in cells with ROCK1 overexpression (Fig. 3C).

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FIG. 3.

IUR overexpression was accompanied by ROCK1 downregulation in GC cells, SNU-16 and N87 cells were transfected with pcDNA3.1 vectors expressing IUR and ROCK1. Overexpression of IUR and ROCK1 was confirmed at 24 h post-transfection (A). The effects of IUR overexpression on ROCK1 were analyzed by qPCR and Western blot (B), and the effects of ROCK1 overexpression on IUR expression were analyzed by qPCR (C). Experiments were repeated three times and mean values were presented, *p < 0.05. Color images are available online.

IUR promoted the invasion and migration of GC cells

Transwell assay analysis showed that ROCK1 overexpression resulted in increased GC cell invasion (Fig. 4A, p < 0.05) and migration (Fig. 4B, p < 0.05) rates, while IUR overexpression resulted in decreased rates of GC cell invasion (Fig. 4A, p < 0.05) and migration (Fig. 4B, p < 0.05). Moreover, ROCK1 overexpression inhibited the role of IUR overexpression (p < 0.05).

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FIG. 4.

IUR promoted the invasion and migration of GC cells. The effects of IUR and ROCK1 overexpression on cell invasion and migration abilities were analyzed by performing Transwell invasion and migration assay. Experiments were repeated three times and mean values were presented, *p < 0.05. Color images are available online.

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FIG. 5.

IUR overexpression increased the methylation of ROCK1 in GC cells. MSP was performed to analyze the effects of IUR overexpression on the methylation of ROCK1 gene. Experiments were repeated three times and representative images were presented. U, unmethylation; M, methylation.

IUR overexpression increased the methylation of ROCK1 in GC cells

MSP was performed to analyze the effects of IUR overexpression on the methylation of ROCK1 gene. Compared with cells transfected with empty pcDNA3.1 vector, cells transfected with IUR expression vector showed increased methylation of ROCK1.