OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2016.5297 OL-0-0-5297 Articles Plasma lncRNA-GACAT2 is a valuable marker for the screening of gastric cancer TanLin 1 YangYunben 2 ShaoYongfu 1 ZhangHaiqiang 1 GuoJunming 2 Department of General Surgery, The Affiliated Hospital of Ningbo University School of Medicine, Ningbo, Zhejiang 315010, P.R. China Department of Biochemistry and Molecular Biology, Zhejiang Key Laboratory of Pathophysiology, Ningbo University School of Medicine, Ningbo, Zhejiang 315211, P.R. China Correspondence to: Dr Yongfu Shao, Department of General Surgery, The Affiliated Hospital of Ningbo University School of Medicine, 247 Renming Road, Ningbo, Zhejiang 315010, P.R. China, E-mail: shaoyongfu1173@163.com Dr Junming Guo, Department of Biochemistry and Molecular Biology, Zhejiang Key Laboratory of Pathophysiology, Ningbo University School of Medicine, 818 Fenghua Road, Ningbo, Zhejiang 315211, P.R. China, E-mail: guojunming@nbu.edu.cn 12 2016 19 10 2016 12 6 4845 4849 26072015 16092016 Copyright © 2016, Spandidos Publications 2016

Long non-coding RNAs (lncRNAs) are crucial in contributing to gastric tumorigenesis and development. However, the diagnostic value of the majority of lncRNAs in gastric cancer (GC) are not clear. The present study investigated the diagnostic value of gastric cancer associated transcript 2 (GACAT2), a lncRNA that is aberrantly expressed in GC tissues. A total of 343 plasma samples from 80 healthy individuals, 29 patients with gastric dysplasia (GD) and 117 paired preoperative and postoperative patients with GC were collected. Plasma GACAT2 levels were subsequently measured by reverse transcription-quantitative polymerase chain reaction. Finally, the associations between plasma GACAT2 levels and various clinicopathological features of patients with GC were assessed. The results demonstrated that plasma GACAT2 levels in preoperative patients with GC were significantly higher than those in the postoperative group (P=0.031). Compared with healthy individuals, plasma GACAT2 levels were significantly increased in patients with GD (P<0.001) and preoperative patients with GC (P=0.040). Moreover, the individual relative changes of plasma GACAT2 expression following surgery were significantly associated with lymphatic metastasis (P=0.034), distal metastasis (P=0.035) and perineural invasion (P=0.039). Therefore, the results of the current study suggest that plasma-based GACAT2 may be developed as a tumor marker to screen and predict the prognosis of GC patients.

 long non-coding RNA GACAT2 gastric cancer tumor marker Introduction

Gastric cancer (GC) is one of the most prevalent malignant tumors (1). A number of cancer prevention studies have demonstrated that the most effective way to reduce GC-associated mortality is through early diagnosis and appropriate treatment (2–4). However, the diagnosis rate of early GC remains extremely low (<10%) (5). Patients with GC are usually diagnosed following identification of tumors on B-mode ultrasound, computerized tomography, or magnetic resonance imaging scans. However, early GC tumors are often not large enough to be detected by conventional imaging techniques. Furthermore, the sensitivity and specificity of traditional tumor markers are not satisfactory (6). Thus, investigations into new, more efficient tumor markers are important to improve early diagnosis of this disease.

Long non-coding RNAs (lncRNAs) are a class of regulatory non-coding RNAs (7). Previous studies have investigated the roles of lncRNAs in tumorigenesis and cancer development. For example, Zang et al (8) reported that lncRNA-PEG10 expression was upregulated in esophageal cancer tissues, and that it regulated the proliferation and invasion of cancer cells. Furthermore, Shao et al (9) demonstrated that lncRNA-AA174084 expression was downregulated in gastric cancer tissues. A previous study determined that gastric cancer associated transcript 2 (GACAT2) was not only significantly downregulated in gastric cancer tissue, but was also aberrantly expressed in gastric precancerous lesions and gastric cell lines (10).

Blood is one of the most commonly used samples in cancer screening, therefore in the present study, plasma GACAT2 expression was compared among groups of healthy individuals, patients with GD, and patients with preoperative or postoperative GC. The aim of the current study was to investigate whether plasma GACAT2 levels may be used as a novel tumor marker to screen and predict the prognosis of patients with GC.

 Materials and methods <sec> <title>Plasma and clinical data collection

A total of 343 plasma samples were collected from 80 healthy individuals, 29 patients with GD, 117 preoperative and 117 postoperative patients with GC at the Department of Gastroenterology, Affiliated Hospital of Ningbo University School of Medicine (Ningbo, China) between March 2013 and July 2014. Postoperative plasma samples were collected 14 days following surgery. No patients underwent chemotherapy or radiotherapy ahead of sample collection. Tumors were classified using the tumor-node-metastasis staging system (11). Histological grade was evaluated by the National Comprehensive Cancer Network clinical practice guidelines (V.1.2011) (12). Written informed consent was provided by all patients, and all aspects of the study were approved by the Human Research Ethics Committee of Ningbo University (IRB No. 20120303).

 RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from plasma using the TRIzol® LS reagent (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol as previously reported (13). Complementary DNA was subsequently generated using the GoScript™ Reverse Transcription system (Promega Corporation, Madison, WI, USA), following the manufacturer's protocol, as previously reported (14). In order to detect plasma GACAT2 levels, RT-qPCR was performed with GoTaq qPCR Master mix (Promega Corporation) on a Mx3005P qPCR system (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) following the manufacturer's protocol. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, which maintains a stable level in human plasma (9), was used as a control for plasma GACAT2 detection. Primers for GAPDH and GACAT2 were synthesized by Sangon Biotech, Co., Ltd. (Shanghai, China). The primer sequences were as follows: GACAT2 forward, 5′-TGGATGCTTACAAAGGACTGG-3′ and reverse, 5′-CTGCAATTACGGAAAGAGCTG-3′; GAPDH forward, 5′-ACCCACTCCTCCACCTTTGAC-3′ and reverse, 5′-TGTTGCTGTAGCCAAATTCGTT-3′. The conditions of thermal cycling were as follows: 95°C at 5 min for a hot-start, followed by 45 cycles at 94°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec. The level of GACAT2 was calculated using the 2−ΔΔCq method (9). A lower quantification cycle (Cq) indicates a higher level of plasma GACAT2. The ΔΔCq method (ΔCqpost-op-ΔCqpre-op) was used to evaluate individual relative changes of plasma GACAT2 following surgery, and all results were expressed as the mean ± standard deviation of three independent experiments.

 Detection of serum carcinoembryonic antigen (CEA) and serum carbohydrate antigen (CA)19-9

To measure levels of CA19-9 and CEA, an Elecsys 2010 machine (Roche Diagnostics, Basel, Switzerland) was used. The cut-off values of CA19-9 and CEA were set at 35 U/ml and 5 ng/ml, respectively (15).

 Statistical analysis

Data were analyzed using SPSS software v19.0 (IBM SPSS, Armonk, NY, USA). The difference in plasma GACAT2 levels among healthy controls, patients with GD, and preoperative and postoperative patients with GC were analyzed by one-way analysis of variance (ANOVA). Associations between plasma GACAT2 levels and clinicopathological factors were analyzed by ANOVA and Student's t-test. Graphs were plotted using SigmaPlot v12.1.0 (Systat Software Inc., San Jose, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Plasma GACAT2 levels are significantly decreased following surgery

GACAT2 levels in preoperative and postoperative patients with GC were detected by RT-qPCR. As presented in Fig. 1, the level of plasma GACAT2 in the postoperative gastric cancer patients was significantly lower than that in the preoperative group (P=0.031), indicating that GACAT2 expression significantly decreased following surgery.

 Plasma GACAT2 levels are significantly increased in patients with GD and GC

Plasma GACAT2 levels were measured at two stages of gastric carcinogenesis to gain further information regarding their effect on gastric mucosal damage. The data indicated that plasma GACAT2 levels in patients with GD (P<0.001) and patients with preoperative GC (P=0.040) were significantly higher than those in the healthy controls (Fig. 2). However, there was no significant difference in GACAT2 expression between the two patient groups.

To investigate whether preoperative plasma GACAT2 may be used as a tumor biomarker for GC screening, a receiver operating characteristic (ROC) curve was constructed to evaluate its clinical value. The area under the curve (AUC) was 0.622 (95% confidence interval, 0.551–0.694; P<0.05; Fig. 3), and the optimal cut-off value was 6.625, with which the sensitivity and specificity were 87.2 and 28.2%, respectively (Fig. 3). This implies that plasma GACAT2 levels may be used as a reliable biomarker in the screening of GC.

 Identification of RT-qPCR products of plasma GACAT2

Blood is the primary material used in the diagnosis of diseases; therefore, to confirm the results and prove the existence of GACAT2 in plasma, the RT-qPCR products of plasma GACAT2 were sequenced. Fig. 4 demonstrates that the sequence of plasma GACAT2 was completely consistent with that from the genome database (http://www.ncbi.nlm.nih.gov/nuccore/NR_120598.1).

 Association between plasma GACAT2 levels and clinicopathological factors of patients with GC

Based on the aforementioned results, associations between plasma GACAT2 levels and the clinicopathological features of patients with GC were investigated. The individual relative changes of plasma GACAT2 following surgery were significantly associated with lymphatic metastasis (P=0.034), distal metastasis (P=0.035) and perineural invasion (P=0.039) (Table I), indicating a correlation between GACAT2 and invasive GC.

 Discussion

Previous studies have identified the differential expression of lncRNAs in various diseases and suggested their possible associations with tumorigenesis (16). Advanced cancer genomic techniques have demonstrated that lncRNAs are important in determining GC occurrence and development (17).

Blood plasma is the most commonly used sample in clinical diagnosis (18). A number of changes in tumor cells may be detected by measuring the expression of different proteins in plasma. RT-qPCR may provide a method to detect changes in the expression of DNA or RNA associated with tumorigenesis. The present study investigated whether plasma-based lncRNAs could be used as biomarkers for the screening of GC and prediction of prognosis in patients with the disease. Plasma GACAT2 levels between preoperative and postoperative patients with GC were compared. The results demonstrated that plasma GACAT2 levels in postoperative patients with GC were significantly lower than those in the preoperative group (Fig. 1). To obtain further information regarding the change in GACAT2 expression during gastric tumorigenesis, plasma GACAT2 levels in two stages of gastric carcinogenesis were measured. Compared with healthy controls, plasma GACAT2 levels were not only increased in the gastric cancer group as a whole (P=0.040), they were also significantly higher in patients with GD (P<0.001; Fig. 2). The AUC was 0.622 (Fig. 3). When the optimal cut-off value was 6.625, the sensitivity and specificity were 87.2 and 28.2%, respectively. Taken together, the results of the current study indicated that plasma GACAT2 has the potential value for the early screening of GC.

A previous study demonstrated that GACAT2 expression was significantly downregulated in gastric tissue and gastric cancer cell lines (10). The reason plasma GACAT2 expression increased in patients with GC and GD in the current study remains unknown. A recent study compared lncRNA-LINC00152 levels between plasma and exosomes extracted from the blood and observed that no difference between the two (19). One hypothesis suggests that lncRNAs that are stable in human plasma are protected by exosomes (19). It has been demonstrated that exosomes serve an important role in the protection and secretion of non-coding RNAs, including miRNAs and lncRNAs (20,21), and cells affected by stimuli, such as oxidative stress, may increase the secretion of exosomes (22,23). In addition, GC cells are able to release related RNAs into the extracellular environment via exosomes during tumorgenesis (24). Therefore, GACAT2 may be released in a similar manner, leading to elevated GACAT2 levels in the blood plasma of patients with malignant GC.

Lymphatic metastasis, distal metastasis and perineural invasion are important factors affecting GC prognosis (25,26). The present study demonstrated that individual relative changes of plasma GACAT2 following surgery were significantly associated with lymphatic metastasis (P=0.034), distal metastasis (P=0.035) and perineural invasion (P=0.039). Higher GACAT2 levels in plasma therefore are correlated with a worse pathological situation; therefore plasma-based GACAT2 may be used as a tumor marker to predict the prognosis of patients with GC.

In conclusion, our study demonstrated that plasma GACAT2 levels were significantly increased in patients with GD and preoperative patients with GC. In addition, the individual relative changes of plasma GACAT2 expression following surgery were significantly associated with lymphatic metastasis, distal metastasis and perineural invasion. Our findings suggested that plasma-based GACAT2 as a tumor marker has a potential diagnostic value for the screening of GC and prediction of prognosis.

 Acknowledgements

The present study was supported by the Zhejiang Provincial Natural Science Foundation of China (grant no. LY14C060003), the Applied Research Project on Nonprofit Technology of Zhejiang Province (grant no. 2014C33222), the National Natural Science Foundation of China (grant no. 81171660), the Medical Scientific Research Project of The Affiliated Hospital of Ningbo University School of Medicine (grant no. XYY14009) and the K.C. Wong Magna Fund in Ningbo University (grant no. 2016001).

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Reverse transcription-quantitative polymerase chain reaction was used to detect GACAT2 levels in pre-op and post-op patients with gastric cancer. Plasma GACAT2 expression decreased following surgery (*P<0.05). A higher ΔCq indicates a higher level of plasma GACAT2. GACAT2, gastric cancer associated transcript 2; Cq, quantification cycle; pre-op, preoperative; post-op, postoperative.

Plasma GACAT2 levels were increased in GD and pre-op patients with gastric cancer as compared with healthy controls (*P<0.05 and ***P<0.001). A lower ΔCq indicates a higher level of plasma GACAT2. Healthy volunteers, n=80; GD, n=29; pre-op patients, n=117; GACAT2, gastric cancer associated transcript 2; GD, gastric dysplasia; pre-op, preoperative; Cq, quantification cycle.

ROC curve for differentiating gastric cancer plasma from healthy controls. ROC, receiver operating characteristic; AUC, area under the curve.

Sequencing result of the product of plasma GACAT2 by RT-qPCR, demonstrating that the sequence was the same as the sequence of GACAT2 in the genomic database. GACAT2, gastric cancer associated transcript 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Association of plasma GACAT2 level changes (ΔΔCq) between postoperative and preoperative patients with gastric cancer and clinicopathological factors.

Characteristics N Mean ± SD P-value
Age, years 0.889
  ≥60 81 1.544±4.253
  <60 36 1.340±3.996
Gender 0.449
  Male 81 1.143±4.443
  Female 36 2.243±3.344
Tumor location 0.969
  Sinuses ventriculi 57 1.547±4.149
  Cardia 21 1.027±4.205
  Corpora ventriculi 21 1.206±3.103
  Others 18 2.123±5.797
Diameter, cm 0.252
  ≥5 60 0.735±4.393
  <5 57 2.266±3.777
Differentiation 0.057
  Well   6 5.070±1.725
  Moderate 57 2.608±4.290
  Poor 54 −0.108±3.571
TNM stage 0.326
  Early 24 2.775±3.990
  Advanced 93 1.147±4.155
Borrmann type 0.224
  I and II 33 2.386±3.091
  III and IV 60 0.466±4.567
Pathological diagnosis 0.342
  Signet ring cell cancer 18 −0.117±5.671
  Adenocarcinoma 99 1.752±3.831
Invasion 0.223
  T1 and T2 36 2.701±3.917
  T3 and T4 81 0.939±4.168
Lymphatic metastasis 0.034a
  N0 45 3.232±3.805
  N1–3 72 0.387±4.005
Distal metastasis 0.035a
  M0 96 2.070±3.720
  M1 21 −1.210±5.093
Venous invasion 0.246
  Negative 60 2.236±3.916
  Positive 57 0.687±4.292
PNI 0.039a
  Negative 63 2.731±3.326
  Positive 54 0.022±4.563
Blood CEA 0.767
  Positive 51 1.722±5.144
  Negative 66 1.295±3.246
Blood CA19-9 0.793
  Positive 39 1.232±5.011
  Negative 78 1.606±3.708

P<0.05. ΔΔCq=ΔCqpost-op-ΔCqpre-op. TNM, tumor-node-metastasis; PNI, perineural invasion; CEA, carcinoembryonic antigen; CA, carbohydrate antigen; SD, standard deviation; GACAT2, gastric cancer associated transcript 2; Cq, quantification cycle.