Open Access

Five‑long non‑coding RNA risk score system for the effective prediction of gastric cancer patient survival

  • Authors:
    • Zunqi Hu
    • Dejun Yang
    • Yuan Tang
    • Xin Zhang
    • Ziran Wei
    • Hongbing Fu
    • Jiapeng Xu
    • Zhenxin Zhu
    • Qingping Cai
  • View Affiliations

  • Published online on: March 8, 2019     https://doi.org/10.3892/ol.2019.10124
  • Pages: 4474-4486
  • Copyright: © Hu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The prognosis for patients with gastric cancer (GC) is usually poor, as the majority of patients have reached the advanced stages of disease at the point of diagnosis. Therefore, revealing the mechanisms of GC is necessary for the identification of key biomarkers and the development of effective targeted therapies. The present study aimed to identify long non‑coding RNAs (lncRNAs) prominently expressed in patients with GC. The GC dataset (including 384 GC samples) was downloaded from The Cancer Genome Atlas database as the training set. A number of other GC datasets were obtained from the Gene Expression Omnibus database as validation sets. Following data processing, lncRNAs were annotated, followed by co‑expression module analysis to identify stable modules, using the weighted gene co‑expression network analysis (WGCNA) package. Prognosis‑associated lncRNAs were screened using the ‘survival’ package. Following the selection of the optimal lncRNA combinations using the ‘penalized’ package, risk score systems were constructed and assessed. Consensus differentially‑expressed RNAs (DE‑RNAs) were screened using the MetaDE package, and an lncRNA‑mRNA network was constructed. Additionally, pathway enrichment analysis was conducted for the network nodes using gene set enrichment analysis (GSEA). A total of seven modules (blue, brown, green, grey, red, turquoise and yellow) were obtained following WGCNA analysis, among which the green and turquoise modules were stable and associated with the histological grade of GC. A total of 12 prognosis‑associated lncRNAs were identified in the two modules. Combined with the optimal lncRNA combinations, risk score systems were constructed. The risk score system based on the green module [including ITPK1 antisense RNA 1 (ITPK1‑AS1), KCNQ1 downstream neighbor (KCNQ1DN), long intergenic non‑protein coding RNA 167 (LINC00167), LINC00173 and LINC00307] was the more efficient at predicting risk compared with those based on the turquoise, or the green + turquoise modules. A total of 1,105 consensus DE‑RNAs were identified; GSEA revealed that LINC00167, LINC00173 and LINC00307 had the same association directions with 4 pathways and the 32 genes involved in those pathways. In conclusion, a risk score system based on the green module may be applied to predict the survival of patients with GC. Furthermore, ITPK1‑AS1, KCNQ1DN, LINC00167, LINC00173 and LINC00307 may serve as biomarkers for GC pathogenesis.

Introduction

Gastric cancer (GC) originates from the lining of the stomach, and may metastasize to other tissues and organs, including the lungs, liver, lymph nodes and bones (1). It is estimated that 22,220 new cases of GC were diagnosed and 10,990 patients succumbed to GC in 2014 (2). GC is more common in males, and has a high incidence in East Asia and Eastern Europe (3). The most common inducer of GC is Helicobacter pylori infection, although other risk factors include pickled foods, smoking and obesity (4,5). Patients with GC usually have an unfavorable prognosis, as the majority reach the advanced stages of disease prior to diagnosis (6). Therefore, determining the mechanisms of GC is required for the identification of key biomarkers and the development of effective targeted therapies.

The human genome project indicates that only 1.2% of the mammalian genome encodes proteins (7), and that the majority of the genome is transcribed to tens of thousands of long non-coding RNAs (lncRNAs), which are >200 nt in length (8). lncRNAs function in various biological processes, including cellular development and differentiation (9). It has increasingly been suggested that the principal role of lncRNAs is the guidance of site specificity for chromatin-modifying complexes in order to effect epigenetic alterations (10). lncRNAs act through a number of mechanisms in the control of cancer. For instance, specific lncRNAs are key regulators of the protein signaling pathways underlying carcinogenesis (11). Additionally, other lncRNAs function as decoys, sequestering biomolecules and preventing cancerous cells from fulfilling their cellular roles (12,13). Numerous studies have reported the important role of lncRNAs in GC; lncRNA-H19 is upregulated in GC tissues and affects the progression and metastasis of GC by promoting isthmin 1 expression and inhibiting calneuron 1 expression (14). The downregulated expression of lncRNA maternally expressed gene 3 promotes cell proliferation and apoptosis, and predicts a poor prognosis in GC (15,16). Overexpression of the lncRNA colon cancer associated transcript 2 associates with the progression of GC and may serve as a promising prognostic marker for the disease (17). Antisense ncRNA in the INK4 locus acts as a growth regulator in GC by silencing microRNA (miR)-99a and miR-449a, and may indicate a potential prognostic biomarker and therapeutic target in GC (18,19). BRAF-activated non-coding RNA overexpression associates positively with tumor depth, clinical stage and tumor metastasis, and predicts a poor prognosis in patients with GC (20). However, the functions of numerous lncRNAs remain unclear; therefore, it is necessary to conduct a comprehensive assessment of the functions of lncRNAs in GC.

Bioinformatics analysis of gene expression profiles has been widely applied to investigate the pathogenesis of various diseases (21). In the current study, multiple GC datasets were searched and downloaded from open access databases. Using comprehensive bioinformatics analyses, certain prognosis-associated lncRNAs were identified. An optimal risk score system based on these lncRNAs was constructed to evaluate the risk of developing GC, the efficiency of which was determined using various independent datasets.

Subjects and methods

Data sources

The mRNA-sequencing data for GC, sequenced on the Illumina HiSeq 2000 RNA Sequencing platform (Illumina, Inc., San Diego, CA, USA), were downloaded from The Cancer Genome Atlas (TCGA; http://cancergenome.nih.gov/) database, which included 384 GC samples. Among the 384 samples, there were 122 samples from deceased patients due to GC, 238 samples from surviving patients (mean survival time, mean ± standard deviation, 16.17±16.96 months) and 24 samples without survival information.

From the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database, three kinds of datasets (dataset I–III) were searched and identified using ‘gastric cancer’ as the key words. Dataset I was searched according to the following criteria: i) The dataset was a gene expression profile; ii) the samples were tumor tissues from patients with GC; iii) the dataset was a human expression profile; and iv) the total number of samples was ≥100. Thus, GSE15459 (2226) (including 300 GC samples) and GSE54129 (including 111 GC samples) sequenced on the Affymetrix-GPL570 platform (Affymetrix; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were selected. The criteria for searching GC dataset II were as follows: i) The dataset was a gene expression profile; ii) the samples were tumor tissues from patients with GC; iii) the dataset was a human expression profile; iv) the samples contained survival information; and v) the total number of samples was ≥100. Only GSE62254 (27) (including 300 GC samples; Affymetrix-GPL570 platform; Affymetrix; Thermo Fisher Scientific, Inc.,) was selected, involving 135 samples from deceased patients due to GC, and 148 samples from surviving patients (mean survival time, mean ± standard deviation, 50.59±31.42 months), and 17 samples without survival information. Dataset III was selected according to the following criteria: i) The dataset was a gene expression profile; ii) the samples were tumor tissues from patients with GC; iii) there were control tissues; iv) the dataset was a human expression profile; and v) the total number of samples was ≥50. Ultimately, the GSE65801 (28) (including 32 GC samples and 32 control samples; Agilent GPL14550 platform; Agilent Technologies, Inc., Santa Clara, CA, USA), GSE29998 (29) (including 50 GC samples and 49 control samples; GPL6947 Illumina HumanHT-12 V3.0 platform; Illumina, Inc.), GSE33335 (3032) (including 25 GC samples and 25 control samples; GPL5175 [HuEx-1_0-st] platform) and GSE27342 (33,34) (including 80 GC samples and 80 control samples; GPL5175 [HuEx-1_0-st] platform) datasets were selected.

Data preprocessing

The data from the aforementioned databases were divided into three types based on the testing platforms. For the dataset from TCGA, the quantile standardization method in the R package preprocessCore (version 1.40.0; http://bioconductor.org/packages/release/bioc/html/preprocessCore.html) (35) was used for data normalization. For the CIMFast Event Language data sequenced on the Affymetrix platform, the R package oligo (version 1.41.1; http://www.bioconductor.org/packages/release/bioc/html/oligo.html) (36) was utilized for format conversion, missing data filling, background correction and data normalization. For the TXT data sequenced on the Agilent platform, the R package Limma (version 3.34.0; http://bioconductor.org/packages/release/bioc/html/limma.html) (37) was applied for log2 logarithmics and data normalization.

Subsequently, lncRNAs were annotated based on the Ref_seq and Transcript_ID provided by the annotation platform, and aligned with human genome sequences (version, GRCh38) on a platform using Clustal 2.1 software (http://www.clustal.org/clustal2/) (38). Subsequently, multiple annotation results were merged to identify the lncRNAs and their corresponding expression information (3941).

Weighted gene co-expression network analysis (WGCNA) to identify disease-associated modules

As a bioinformatics algorithm for building co-expression networks, WGCNA is used to identify disease-associated modules and thus screen for pathogenic processes and potential therapeutic targets (42). In the present study, based on the use of TCGA dataset as the training dataset, and GSE15459 and GSE54129 as the validation datasets, stable modules associated with GC were identified using the R package WGCNA (version 1.61; http://cran.r-project.org/web/packages/WGCNA/index.html) (43). Expression correlation between every two of the three datasets was calculated, and the adjacent function was defined as follows: WGCNA analysis was required to satisfy the precondition of scale-free network distribution, and thus the value of the adjacency matrix weighting parameter ‘power’ was investigated. Based on the RNA data, the squares of the correlation coefficients between log (k) and log [p (k)] were calculated for different ‘power’ values. A higher square value indicated that the network was closer to a scale-free network distribution. Following the definition of the adjacent function, module partition was conducted (the thresholds for module partition were that the module contained ≥150 RNAs and a cutHeight of 0.99). Combined with the clinical information from TCGA dataset, the correlation between each module and the clinical information was analyzed. Functional annotation was conducted for each stable module using the userListEnrichment function in the WGCNA package (43). Additionally, differential expression analysis of lncRNAs between tumor and control groups was performed for each module, with a P-value and false discovery rate (FDR) of <0.05.

Selection of prognosis-associated lncRNAs

Based on the lncRNAs obtained in the clinical factors-associated stable modules, univariate Cox regression analysis was performed using the R package survival (version 2.4; http://cran.r-project.org/web/packages/survival/index.html) (44) for the GC samples with survival information in the TCGA dataset, to identify prognosis-associated lncRNAs. A log-rank P-value of <0.05 was considered to indicate a statistically significant difference.

Construction and assessment of a risk score system

The lncRNAs in the stable modules that correlated significantly with notable clinical factors were analyzed separately. Using the Cox-Proportional Hazards (Cox-PH) model in the R package ‘penalized’ (http://bioconductor.org/packages/penalized/) (45), the optimal lncRNA combinations were selected. The parameter ‘lambda’ was obtained with 1,000 circulation calculations using a cross-validation likelihood algorithm (46). Subsequently, the risk score system was constructed, combined with the regression coefficient (β) and expression level (exprlncRNA) of each lncRNA in the optimal lncRNA combination. The risk score of each sample was calculated using the following formula:

Risk score = β lncRNA1 × exprlncRNA1 + β lncRNA2 × exprlncRNA2 + ··· + βlncRNAn × exprlncRNAn.

The samples in the TCGA dataset were divided into high-risk and low-risk groups according to the median of their risk scores. Kaplan-Meier survival curves were used to evaluate the correlation between the overall survival of the samples and the two groups. Using GSE15459 and GSE54129 as the validation datasets, the robustness of the risk score system in predicting sample risk and prognosis was assessed. Moreover, the predictive results of the risk score system in the training and validation datasets were compared to identify the optimal model for subsequent analyses.

Differential expression analysis of notable lncRNAs in multiple datasets

Using the MetaDE.ES algorithm in the R package MetaDE (version 1.0.5; http://cran.r-project.org/web/packages/MetaDE/) (47,48), consensus differentially expressed RNAs (DE-RNAs; between GC and control samples) were screened from the GSE65801, GSE29998, GSE33335 and GSE27342 datasets. τ2=0, Qpval>0.05, P<0.05 and FDR<0.05 were set as the cut-off criteria. The focus was the differential expression of the notable lncRNAs that were screened as disease or prognosis related-lncRNAs.

Analysis of lncRNA-associated pathways

Based on the correlation coefficients between notable lncRNAs and mRNAs that were located in the same WGCNA module, the lncRNA-mRNA network was constructed. Subsequently, pathway enrichment analysis was conducted for the network nodes using Gene Set Enrichment Analysis (GSEA; http://software.broadinstitute.org/gsea/index.jsp) (49). A nominal P-value of <0.05 was considered to indicate a statistically significant difference.

Results

Identification of GC-associated stable modules based on WGCNA

Following data preprocessing, a total of 988 lncRNAs and 15,127 mRNAs shared by TCGA dataset, GSE15459 and GSE54129 were identified. TCGA dataset was taken as the training dataset, whilst GSE15459 and GSE54129 were used as the validation datasets to screen for GC-associated RNA modules.

To ensure that the RNA expression levels in each dataset were comparable, expression consistency analysis was performed for the expression values of shared RNAs. As outlined in Fig. 1A, the correlation in expression between every two of the three datasets was >0.85, and P-values were <1×10−200, indicating significant positive correlations between every two datasets and suggesting that these data sets are comparable and suitable for further analysis. Additionally, the correlations of connectivity between nodes were >0.5, and the P-values were <1×10−200, suggesting that connection correlations between the RNAs of every two datasets were positive (Fig. 1B).

Following definition of the adjacent function, the power value of 6, for which the square value of the correlation coefficient reached 0.9 for the first time, was selected (Fig. 2A). Under a power value of 6, the mean connectivity degree of the RNAs was 2, which conformed to the small-world property in a scale-free network (Fig. 2B).

Following construction of the co-expression network (based on TCGA dataset), the stable modules associated with disease were screened. A total of seven modules (blue, brown, green, grey, red, turquoise and yellow) were obtained (Fig. 3A). The differentially expressed lncRNAs between the tumor and control groups in the seven modules are listed in Table I. Combined with the seven modules and the RNAs involved in each module, corresponding module partition was conducted in GSE15459 (Fig. 3B) and GSE54129 (Fig. 3C).

Table I.

Differentially expressed lncRNAs between the tumor and control groups in 7 modules.

Table I.

Differentially expressed lncRNAs between the tumor and control groups in 7 modules.

GroupModuleP-valueFDRlogFC
HOTAIRBlue 1.01×10−55 1.46×10−535.2805
MCF2L-AS1Blue 2.58×10−15 3.74×10−140.9188
GAS5Brown 8.85×10−7 3.38×10−60.1977
CASC2Green 5.41×10−11 3.92×10−100.7532
CECR3Green 2.06×10−11 1.66×10−102.8280
CPS1-IT1Green 1.58×10−2 2.48×10−21.2022
HCG27Green 2.35×10−3 4.49×10−30.3698
IGF2-ASGreen 3.12×10−6 1.08×10−52.2240
INHBA-AS1Green 1.85×10−2 2.86×10−20.9693
JAZF1-AS1Green 6.36×10−3 1.12×10−2−0.5507
KCNQ1DNGreen 1.59×10−2 2.48×10−21.8214
LINC00032Green 3.31×10−3 6.15×10−30.8562
LINC00112Green 1.50×10−2 2.42×10−22.1061
LINC00115Green 1.66×10−10 1.09×10−90.7638
LINC00163Green 2.81×10−2 4.16×10−2−1.2973
LINC00167Green 7.42×10−4 1.58×10−30.5133
LINC00242Green 1.84×10−6 6.67×10−60.6465
LINC00299Green 2.20×10−16 3.99×10−151.8836
LINC00326Green 1.72×10−3 3.47×10−34.7055
LINC00330Green 1.28×10−2 2.12×10−2−1.9390
LINC00410Green 4.51×10−7 1.92×10−64.2579
LINC00485Green 2.56×10−7 1.12×10−62.6578
LINC00486Green 3.22×10−4 8.06×10−41.6764
LINC00523Green 1.59×10−3 3.29×10−33.1421
LINC00607Green 5.97×10−7 2.40×10−61.4066
WDFY3-AS2Green 6.90×10−4 1.54×10−3−0.4393
ADARB2-AS1Grey 1.52×10−4 4.09×10−42.0305
TP53TG1Grey 5.11×10−6 1.65×10−5−0.2026
TTTY14Grey 4.86×10−4 1.12×10−3−1.0856
AGPAT4-IT1Red 5.75×10−6 1.77×10−50.8612
BPESC1Red 1.12×10−3 2.35×10−31.7243
BVES-AS1Red 1.82×10−4 4.71×10−4−1.6299
DGCR5Red 6.81×10−15 8.98×10−141.7023
DSCR9Red 8.94×10−14 1.08×10−121.1941
EPB41L4A-AS1Red 3.87×10−2 5.51×10−2−0.0787
HCG18Red 1.24×10−12 1.20×10−110.4696
LINC00029Red 2.01×10−3 3.94×10−31.7851
LINC00467Red 1.06×10−10 7.32×10−100.4418
LINC00470Red 5.90×10−6 1.78×10−51.6068
LINC00487Red 2.29×10−2 3.49×10−20.9837
LINC00574Red 8.80×10−5 2.45×10−40.7952
MAGI2-AS3Red 3.00×10−2 4.35×10−2−0.4123
MIR17HGRed 2.02×10−27 4.18×10−261.5489
MORC2-AS1Red 3.08×10−16 4.96×10−150.5989
SHANK2-AS3Red 1.49×10−8 8.00×10−81.6254
TTTY13Red 7.02×10−4 1.54×10−32.8019
ASMTL-AS1Turquoise 2.97×10−12 2.69×10−110.6011
C20orf166-AS1Turquoise 3.52×10−4 8.64×10−4−1.8050
CCDC26Turquoise 6.66×10−3 1.15×10−20.9937
CIRBP-AS1Turquoise 2.95×10−9 1.71×10−80.6040
CRNDETurquoise 2.43×10−3 4.58×10−30.4485
CSNK1G2-AS1Turquoise 3.76×10−9 2.10×10−81.2461
CYP1B1-AS1Turquoise 2.36×10−3 4.49×10−3−0.7444
DSCR10Turquoise 5.40×10−4 1.22×10−33.0388
ENO1-AS1Turquoise 1.73×10−4 4.56×10−40.4499
FBXL19-AS1Turquoise 3.49×10−32 1.01×10−300.9572
JPXTurquoise 3.27×10−5 9.30×10−50.2466
LGALS8-AS1Turquoise 1.04×10−6 3.87×10−60.4645
LINC00052Turquoise 2.46×10−6 8.70×10−63.7146
LINC00161Turquoise 4.63×10−4 1.08×10−30.9597
LINC00189Turquoise 2.11×10−8 1.09×10−71.2475
LINC00290Turquoise 2.47×10−2 3.73×10−22.6024
LINC00308Turquoise 4.33×10−3 7.85×10−32.1851
LINC00309Turquoise 2.65×10−4 6.75×10−42.2938
LINC00311Turquoise 1.32×10−2 2.14×10−20.6300
LINC00323Turquoise 6.57×10−3 1.15×10−20.5396
LINC00347Turquoise 1.63×10−3 3.32×10−32.4716
LINC00471Turquoise 3.59×10−6 1.18×10−51.0278
LINC00477Turquoise 4.83×10−3 8.64×10−31.4857
LINC00479Turquoise 4.59×10−4 1.08×10−31.1469
LINC00482Turquoise 1.02×10−4 2.80×10−40.8407
LINC00518Turquoise 6.17×10−8 2.98×10−72.5336
LINC00582Turquoise 4.52×10−4 1.08×10−3−1.5306
NBR2Turquoise 1.03×10−9 6.49×10−90.3945
NEAT1Turquoise 1.19×10−5 3.45×10−50.2762
NPSR1-AS1Turquoise 9.94×10−31 2.40×10−295.6701
PCBP1-AS1Turquoise 3.37×10−8 1.69×10−7−0.4557
RUSC1-AS1Turquoise 7.90×10−7 3.10×10−60.1934
ST7-AS2Turquoise 5.65×10−6 1.77×10−50.2453
ZNF295-AS1Turquoise 1.89×10−3 3.74×10−31.0881
ZNF503-AS2Turquoise 2.98×10−2 4.35×10−2−0.1638
C20orf203Yellow 3.31×10−2 4.75×10−20.7754
DLEU2Yellow 1.01×10−35 4.88×10−341.0471
FAM201AYellow 1.42×10−9 8.58×10−90.8587
FAM66CYellow 1.54×10−2 2.45×10−2−0.5395
HCG4BYellow 2.42×10−7 1.10×10−61.1311
HCG9Yellow 7.28×10−3 1.24×10−20.5412
HCP5Yellow 3.42×10−6 1.15×10−50.4477
INE1Yellow 4.51×10−12 3.85×10−110.8886
KIF25-AS1Yellow 1.51×10−7 7.06×10−71.7721
LINC00174Yellow 2.83×10−13 2.93×10−120.8178
LINC00265Yellow 4.80×10−7 1.99×10−60.5363
LINC00599Yellow 9.87×10−3 1.66×10−21.5708
LINC00606Yellow 1.28×10−2 2.12×10−24.1423
LY86-AS1Yellow 7.20×10−4 1.56×10−31.2773
PART1Yellow 6.79×10−6 2.01×10−5−1.5909
RHPN1-AS1Yellow 6.31×10−33 2.29×10−311.5361
SND1-IT1Yellow 1.31×10−13 1.46×10−121.1209
SOX2-OTYellow 2.75×10−2 4.11×10−2−0.4175
TP73-AS1Yellow 4.11×10−3 7.55×10−3−0.3515
TUG1Yellow 3.08×10−11 2.35×10−100.2871
ZNF252P-AS1Yellow 4.75×10−37 3.44×10−351.4731

[i] Differentially expressed lncRNAs were screened using the MetaDE.ES algorithm in the R package MetaDE (version 1.0.5; http://cran.r-project.org/web/packages/MetaDE/), P<0.05 and FDR<0.05 were set as the cut-off criteria. FDR, false discovery rate; FC, fold-change.

For TCGA dataset, the module partition and module correlations are presented in Fig. 4. The results illustrate that the RNAs in the same module tended to cluster together, including the green or blue nodes, indicating that the RNAs have more similar expression levels (Fig. 4A). The green and blue modules have the characteristics of independent branches (Fig. 4B).

The stabilities of the seven modules were assessed, and the blue, green, red, turquoise and yellow modules were deemed stable (preservation Z score >5). The top three modules were turquoise, green and yellow, according to the preservation Z score, and these three may be associated with GC pathogenesis. Functional annotation for each stable module revealed that the lncRNAs in the turquoise (including 46 lncRNAs), green (including 30 lncRNAs) and yellow (including 32 lncRNAs) modules were predominantly enriched in cell adhesion, immune response and digestion, respectively (Table II).

Table II.

Stabilities and functional annotations of the 7 modules of TCGA dataset.

Table II.

Stabilities and functional annotations of the 7 modules of TCGA dataset.

ModuleColorModule size, nmRNAlncRNAPreservation Z-scoreModule annotation
1Blue33633429.7094Pattern specification process
2Brown33132831.2017Epithelium development
3Green3182883019.0215Immune response
4Grey2,8562,822344.2851Cell-cell signaling
5Red2502133713.2273Digestive system process
6Turquoise9569104627.4163Cell adhesion
7Yellow3262943215.7692Digestion

[i] Z<5 indicates that the module is unstable; Z>5 indicates that the module is stable, and Z>10 indicates that the module is highly stable. Module annotation indicates the functional terms enriched for the modules. TCGA, The Cancer Genome Atlas; lncRNA, long non-coding RNA.

In addition, based on the clinical information in TCGA dataset, the correlation between each module and the clinical factors was analyzed. Among the 5 stable modules, the green and turquoise modules correlated significantly with histological grade (Fig. 4C). Therefore, the green and turquoise modules were further analyzed.

Selection of prognosis-associated lncRNAs

Based on the 76 lncRNAs in the green and turquoise modules, 12 prognosis-associated lncRNAs were identified in TCGA dataset using univariate Cox regression analysis. Among the 12 prognosis-associated lncRNAs, 5 belonged to the green module and 7 were from the turquoise module.

Construction and assessment of risk score system

Based on the expression levels of 12 prognosis-associated lncRNAs in TCGA dataset, the optimal lncRNA combinations that correlated with prognosis were selected using the Cox-PH model. 5-lncRNA, 5-lncRNA and 8-lncRNA (Table III) optimal combinations were separately screened from the prognosis-associated lncRNAs in the green, turquoise and green + turquoise modules, respectively. The risk score systems based on each optimal lncRNA combination were as follows: Risk score (green module) = (−0.9059377) × ExpITPK1-AS1 + (3.3537827) ExpKCNQ1DN + (−2.1388024) × ExpLINC00167 + (−1.037547) × ExpLINC00173 + (1.9587271) × ExpLINC00307. Risk score (turquoise module)=(−0.268429) × ExpASMTL-AS1 + (−0.3410407) × ExpCIRBP-AS1 + (1.0926567) × ExpDSCR10 + (−0.3433227) × ExpJPX + (0.5437058) × ExpLINC00479. Risk score (green + turquoise module)=(1.9685961) × ExpKCNQ1DN + (−0.6567239) × ExpLINC00167 + (−0.4293328) × ExpLINC00173 + (−0.246053) × ExpASMTL-AS1 + (−0.25746771) × ExpCIRBP-AS1 + (0.7023183) × ExpDSCR10 + (−0.3204003) × ExpJPX + (0.5495452) × ExpLINC00479.

Table III.

Optimal lncRNAs screened from the prognosis-associated lncRNAs in green, turquoise and green + turquoise modules.

Table III.

Optimal lncRNAs screened from the prognosis-associated lncRNAs in green, turquoise and green + turquoise modules.

ModuleslncRNAβ-valueP-valuesHazard ratio (95% CI)
Green ITPK1-AS1−0.90590.04960.0777 (0.0039–1.5300)
KCNQ1DN3.35380.005013.7200 (2.1360–18.1400)
LINC00167−2.13880.02840.0500 (0.0035–0.7191)
LINC00173−1.03760.04800.4930 (0.2229–1.0900)
LINC003071.95870.03572.0260 (1.0430–3.9380)
Turquoise ASMTL-AS1−0.26840.02700.6392 (0.4302–0.9498)
CIRBP-AS1−0.34100.04580.6489 (0.4146–1.0160)
DSCR101.09270.00394.4030 (1.5400–12.5900)
JPX−0.34330.04700.6624 (0.4243~1.0340)
LINC004790.54370.02311.9880 (1.0900–3.6260)
Green + turquoiseKCNQ1DN1.96860.00293.3500 (1.3101–5.3910)
LINC00167−0.65670.04760.0790 (0.0048–1.3100)
LINC00173−0.42930.04820.4338 (0.1693–1.1110)
ASMTL-AS1−0.24610.02380.7758 (0.5088–1.1830)
CIRBP-AS1−0.25750.04890.8482 (0.5322–1.3520)
DSCR100.70230.02082.2280 (1.6399–7.7580)
JPX−0.32040.01310.6847 (0.4187–1.1200)
LINC004790.54950.03371.9057 (1.0510–3.4550)

[i] P-values were generated by univariate cox regression, with P<0.05 as the threshold. lncRNA, long non-coding RNA; CI, confidence interval.

Based on the three risk score systems, the risk scores of the samples in TCGA dataset were calculated. The samples in the TCGA dataset were divided into high-risk and low-risk groups according to the median of their risk scores. Kaplan-Meier survival curves were used to evaluate the correlation between the overall survival of the samples and the two groups. The results revealed that the risk score system based on the optimal lncRNA combination [including ITPK1 antisense RNA 1 (ITPK1-AS1), KCNQ1 downstream neighbor (KCNQ1DN), long intergenic non-protein coding RNA 167 (LINC00167), LINC00173 and LINC00307] of the green module had the most significant predictive effect; therefore, the risk score system of the green module was the optimal system (Fig. 5). In this risk score system, the low-risk group (mean overall survival time, 16.71±18.26 months) had a greater overall survival time compared with the high-risk group (mean overall survival time, 13.63±15.76 months) for the TCGA training dataset. In addition, the correlation between overall survival and the two groups was significant (P=0.0049). For the validation dataset GSE62254, the low-risk group (mean overall survival time, 57.13±30.88 months; mean progression-free survival time, 42.34±30.26 months) also had a greater overall survival time and progression-free survival time relative to the high-risk group (mean overall survival time, 46.98±29.97 months; mean progression-free survival time, 30.91±27.97 months). Similarly, the two groups were significantly correlated with overall survival time (P=0.0251) and progression-free survival time (P=0.0006). Additionally, the associations between the risk score and survival status/lncRNA expression are displayed in Fig. 6. The risk score altered from low to high on the vertical axis; in the middle panels, red represents mortality, and black represents survival, which represented the distribution of mortality and survival at high and low risk in addition to the distribution of survival time. Fig. 6 reveals the expression trend of 5 genes from low risk to high risk (for example, LINC00307 expression tends to be decreased, while KCNQ1DN expression tends to be increased).

Differential expression analysis

There were a total of 1,105 consensus DE-RNAs (all were mRNAs) in the GSE65801, GSE29998, GSE33335 and GSE27342 datasets, including 22 mRNAs (2 upregulated and 20 downregulated) in the green module. The clustering heat map demonstrates that the different degrees and dysregulation directions of the DE-RNAs were essentially the same in the 4 datasets (Fig. 7).

Analysis of lncRNA-associated pathways

Based on the correlation coefficients of the 5 optimal lncRNAs in the green module, and the 22 mRNAs obtained as aforementioned, the lncRNA-mRNA network (involving 106 nodes) was constructed (Fig. 8A). GSEA analysis illustrated that 4 pathways [‘cell adhesion molecules (CAMS)’, ‘cytokine-cytokine receptor interaction’, the ‘chemokine signaling pathway’ and ‘leukocyte transendothelial migration’] had significant positive associations with 3 lncRNAs (LINC00167, LINC00173 and LINC00307) (Table IV). Moreover, the 4 pathways involved a total of 32 genes [including chemokine (C-C motif) ligand 22 (CCL22), chemokine (C-C motif) receptor 7 (CCR7), cluster of differentiation (CD) 274 molecule (CD274), CD40 ligand (CD40LG), chemokine (C-X-C motif) ligand 13, CXCL13; chemokine (C-X-C motif) receptor 5 (CXCR5), intercellular adhesion molecule 1 (ICAM1), matrix metalloproteinase 9 (MMP9) and vascular cell adhesion molecule 1 (VCAM1)], and these genes associated positively with the 4 pathways (Fig. 8B). Therefore, it was speculated that LINC00167, LINC00173 and LINC00307 may possess the same association directions with the 4 pathways and the 32 genes, and are involved in GC progression via these pathways.

Table IV.

Pathways that positively correlate with LINC00167, LINC00173 and LINC00307.

Table IV.

Pathways that positively correlate with LINC00167, LINC00173 and LINC00307.

LINC00167 LINC00173 LINC00307



PathwayESNESP-valueESNESP-valueESNESP-value
Cell adhesion molecules0.15981.17900.00960.16541.38000.00110.14650.86460.0058
Cytokine-cytokine receptor interaction0.22500.99470.04510.20521.04470.03570.11360.70580.0484
Chemokine signaling pathway0.23051.00600.04290.10520.53470.0469−0.1560−1.04900.0335
Leukocyte transendothelial migration−0.1690−0.71410.04610.23310.15630.0137−0.1860−0.77500.0480

[i] P-values were generated by GSEA analysis, with P<0.05 as the threshold. ES, enrichment score; NES, normalized enrichment score.

Discussion

In the present study, 5 stable modules (blue, green, red, turquoise and yellow) were identified using WGCNA. In particular, the green and turquoise modules associated significantly with histological grade. Subsequently, 12 prognosis-associated lncRNAs (5 lncRNAs in the green module and seven lncRNAs in the turquoise module) were identified. Moreover, 5-lncRNA, 5-lncRNA and 8-lncRNA optimal combinations were screened separately from the prognosis-associated lncRNAs in the green, turquoise and green + turquoise modules, respectively, which were used to construct risk score systems. Notably, the risk score system based on the optimal lncRNA combination (including ITPK1-AS1, KCNQ1DN, LINC00167, LINC00173 and LINC00307) of the green module had the most significant predictive effect and was thus identified as the optimal system. Differential expression analysis indicated that there were 1,105 consensus DE-RNAs in the GSE65801, GSE29998, GSE33335 and GSE27342 datasets. Following the construction of the lncRNA-mRNA network, 4 pathways had significantly positive associations with LINC00167, LINC00173 and LINC00307. Moreover, the 32 genes involved in the 4 pathways associated positively with the pathways.

Potassium voltage-gated channel subfamily E regulatory subunit 2 (KCNE2) is the β subunit of potassium voltage-gated channel subfamily Q member 1 (KCNQ1) in gastric parietal cells, and KCNQ1/KCNE2 is activated (accompanied with acid secretion) by certain pathways (50,51). Through mediating the expression of KCNQ1, atrial natriuretic peptide serves a role in the proliferation of the GC AGS cell line (52). KCNQ1 and insulin-like growth factor 2 mRNA-binding protein 2 polymorphisms may serve as independent predictive factors for chemotherapeutic response, and glucokinase (hexokinase 4) regulator polymorphisms may independently predict the survival of patients with metastatic GC (53). The KCNQ1 protein level was decreased in colorectal cancer samples, and was associated significantly with the unfavorable overall survival of patients with colorectal cancer (54). These observations demonstrated that KCNQ1DN may be involved in the prognosis of GC.

CCL22 functions in the development of GC by increasing the number of regulatory T cells, and CCL22 levels in sera predict the metastasis and recurrence of GC (55). CCR7 causes epithelial-mesenchymal transition by promoting Snail expression, which results in the migration and invasion of GC cells (56,57). A somatic mutation in CD274 induces its overexpression by disturbing miR-570 binding, and subsequently promotes immune evasion in GC by suppressing the activation and proliferation of T cells (58). The expression level of CXCL13 is a promising prognostic marker for patients with GC following surgical resection, and may be used to predict the response of these patients to postoperative adjuvant chemotherapy (59). CD40 contributes to CXCR5 expression, and the migration and accumulation of myeloid-derived suppressor cells in GC, indicating that CD40 may promote tumor growth by influencing immune evasion (60,61). ICAM1 overexpression is induced by leptin via the Rho/Rho-associated protein kinase pathway, which contributes to tumor cell migration in patients with GC (62). MMP9 in the blood has been identified as a novel tumor marker; in particular, the plasma level of MMP9 is a more effective predictor of GC development and progression compared with its serum level (63,64). VCAM1 functions in the perineural invasion (PNI) of GC by mediating the interaction between tumor cells and neural cells; therefore, VCAM1 inhibition suggests a promising approach for the treatment of PNI in patients with GC (65). LINC00167, LINC00173 and LINC00307 had the same association directions with the 4 pathways and 32 genes (including CCL22, CCR7, CD274, CD40LG, CXCL13, CXCR5, ICAM1, MMP9 and VCAM1), suggesting that LINC00167, LINC00173 and LINC00307 may associate positively with GC through their participation in the 4 pathways, and by mediating the expression of these genes.

Certain limitations of the present study should be considered. Bioinformatics analyses were used to obtain these results, and no experimental research was performed. Platform differences and data heterogeneities of the datasets may have influenced the accuracy of the risk score system. Therefore, further experiments are required to confirm the results.

In conclusion, 12 prognosis-associated lncRNAs were identified from the green and turquoise modules. In addition, the optimal risk score system may be used to predict the prognosis of patients with GC. lncRNAs ITPK1-AS1, KCNQ1DN, LINC00167, LINC00173 and LINC00307 may serve important roles in the pathogenesis of GC.

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (grant no. 81772955), the Natural Science Foundation of Shanghai (grant no. 17ZR1439300) and the Scientific Research Program of Shanghai Municipal Commission of Health and Family Planning (grant no. 201640269).

Availability of data and materials

The datasets used during the current study are available from the corresponding author on reasonable request.

Authors' contributions

ZH and DY performed data analyses and wrote the manuscript. YT, XZ, ZW, HF and JX contributed significantly in data analyses and manuscript revision. ZZ and QC conceived and designed the study. All authors read and approved the final manuscript.

Ethics approval and consent to participate

In the original article of the datasets, the trials were approved by the local institutional review boards of all participating centers, and informed consent was obtained from all patients.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Hu Z, Yang D, Tang Y, Zhang X, Wei Z, Fu H, Xu J, Zhu Z and Cai Q: Five‑long non‑coding RNA risk score system for the effective prediction of gastric cancer patient survival. Oncol Lett 17: 4474-4486, 2019
APA
Hu, Z., Yang, D., Tang, Y., Zhang, X., Wei, Z., Fu, H. ... Cai, Q. (2019). Five‑long non‑coding RNA risk score system for the effective prediction of gastric cancer patient survival. Oncology Letters, 17, 4474-4486. https://doi.org/10.3892/ol.2019.10124
MLA
Hu, Z., Yang, D., Tang, Y., Zhang, X., Wei, Z., Fu, H., Xu, J., Zhu, Z., Cai, Q."Five‑long non‑coding RNA risk score system for the effective prediction of gastric cancer patient survival". Oncology Letters 17.5 (2019): 4474-4486.
Chicago
Hu, Z., Yang, D., Tang, Y., Zhang, X., Wei, Z., Fu, H., Xu, J., Zhu, Z., Cai, Q."Five‑long non‑coding RNA risk score system for the effective prediction of gastric cancer patient survival". Oncology Letters 17, no. 5 (2019): 4474-4486. https://doi.org/10.3892/ol.2019.10124