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Keywords:

  • gastric cancer;
  • long noncoding RNA;
  • lncRNA;
  • biomarker;
  • gastric juice;
  • molecular diagnosis;
  • AA174084

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

BACKGROUND

Long noncoding RNAs (lncRNAs) play a crucial role in tumorigenesis. However, the value of lncRNAs in the diagnosis of gastric cancer remains unknown. To identify whether lncRNA-AA174084 is a potential marker for the early diagnosis of gastric cancer (GC), the authors investigated its levels in tissues, blood, and gastric juices from patients with various stage of gastric tumorigenesis.

METHODS

Total RNA in 860 specimens from patients and healthy controls was extracted. Levels of AA174084 in 134 paired GC tissues, 127 gastric mucosal tissues, 335 plasma samples, and 130 gastric juice samples at each stage of gastric tumorigenesis were measured using real-time reverse transcriptase-polymerase chain reaction analysis. The potential association between AA174084 levels and patients' clinicopathologic features were analyzed. A receiver operating characteristic (ROC) curve was constructed for differentiating GC patients from controls.

RESULTS

Expression levels of AA174084 were down-regulated significantly in 95 of 134 GC tissues (71%) compared with the levels in paired, adjacent, normal tissues (P < .001). AA174084 levels had significant, negative correlations with age (P = .031), Borrmann type (P = .016), and perineural invasion (P = .032). Plasma AA174084 levels in patients with GC dropped markedly on day 15 after surgery compared with preoperative levels (P < .001) and were associated with invasion (P = .049) and lymphatic metastasis (P = .042). AA174084 levels in gastric juice from patients with GC were significantly higher than the levels in normal mucosa or in patients with minimal gastritis, gastric ulcers, and atrophic gastritis (P < .001). The area under ROC was up to 0.848 (P < .001).

CONCLUSIONS

AA174084 may have potential as marker for the early diagnosis of GC. Cancer 2014;120:3320–3328. © 2014 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

Gastric cancer (GC) is 1 of the most prevalent types of cancer and is the second leading cause of global cancer deaths, especially in Eastern Asia, Eastern Europe, and South America.[1, 2] Despite efforts to understand the pathophysiologic mechanisms of cancer and to develop clinical treatments, there has been little progress in improving the prognosis of patients with GC, and the 5-year overall survival rate remains at <25%.[3] Advances in increasing the disease-free survival of patients with GC have been severely limited because of the lack of desirable tumor markers for early diagnosis, individualized treatments, prognosis evaluation, and postoperative prediction of the risk of recurrence.[2, 3] Therefore, the challenge ahead lies in the reliable identification of GC progression-specific targets to enable the use of new molecular markers for detection in clinical practice.[4]

Long noncoding RNAs (lncRNAs) are functional noncoding RNA molecules greater than 200 nucleotides in length.[5] In recent years, lncRNAs have demonstrated multiple biologic functions that are widely involved in the regulation of gene expression networks at the epigenetic, transcriptional, and post-transcriptional levels.[6, 7] Many investigators have reported that the deregulated expression of lncRNAs is associated with a variety of multigenetic diseases.[5, 8, 9] In addition, recent research has demonstrated that some lncRNAs exhibit distinct gene expression patterns in human cancers,[6, 10] some of which are associated with tumor development, invasion, metastasis, and patient prognosis.[9, 11] It has been suggested that some lncRNAs, such as H19, hepatocellular carcinoma up-regulated lncRNA (HULC), and colon cancer-associated transcript 1 (CCAT1), play a functional role in gastric cancer.[12-14] Zhuang et al demonstrated that H19-produced microRNA-675 (miR-675) regulates gastric cancer cell proliferation by targeting the tumor suppressor runt-related transcription factor 1 (RUNX1).[12] CCAT1, which is activated by c-Myc, promotes cell migration and proliferation.[13] Zhao et al reported that the overexpression of HULC promoted proliferation and invasion and inhibited GC cell apoptosis.[14] These findings strongly suggest a decisive role of lncRNAs in the molecular etiology of stomach cancer.

In the current study, we focused on AA174084. This is 1 of the lncRNAs we previously identified that aberrantly expressed in GC tissues in our lncRNA array screening (Gene Expression Omnibus no. GSE47850; available at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47850; accessed June 1, 2014).[10] We tested the potential correlations between tissue, plasma, and gastric juice AA174084 levels and clinicopathological factors in patients with GC. Our data demonstrate that AA174084 may be a potential biomarker for screening early GC and predicting prognosis of GC.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

Patients, Specimens, and Clinical Data Collection

Clinical samples were collected from 3 Chinese centers for gastroenterology (Yinzhou People's Hospital, The First Hospital of Ningbo, and The Affiliated Hospital of Ningbo University School of Medicine) from February 2011 to November 2013. The 134 GC tissues and the paired, adjacent, nontumorous tissues located 5 cm away from the edge of tumor were obtained from surgical excision. The 37 healthy gastric mucosa (HGM) samples, 16 gastric ulcer (GU) tissues, 18 erosive gastritis (EG) tissues, and 28 paired gastric dysplasia (GD) tissues were obtained from biopsy specimens. All tissues were preserved in RNA fixer (Bioteke, Beijing, China) at−80°C until use.

Peripheral blood samples were collected from 120 healthy volunteers, 29 patients with dysplasia, 83 preoperative GC patients, and 103 postoperative (2 weeks) GC patients. Blood samples were collected in ethylene diamine tetraacetic acid (EDTA) anticoagulation tubes (Kangjian, Taizhou, China) and centrifuged at ×3000g for 10 minutes within 20 minutes after collection. The plasma was separated into a 2-mL RNase-free centrifuge tube (Axygen, Union, Calif) and stored at −80°C until RNA isolation.

Endoscopy was performed using an Olympus device (GIF H260; Olympus Corporation, Tokyo, Japan) after a 12-hour overnight fast. Then, a matching washing pipe (PW-2L-1; Olympus Corporation) was used to collect gastric juices from 45 healthy volunteers, 30 patients with GU, 16 patients with chronic atrophic gastritis (AG), and 39 patients with GC. Gastric juice samples were collected in 4-mL centrifuge tubes (Axygen) and centrifuged at ×2000g for 30 minutes within 20 minutes after collection. Then, the supernatant was separated into another 2-mL RNase-free centrifuge tube (Axygen) and stored at −80°C until RNA isolation.

The diagnosis of each case was confirmed histopathologically. Tumors were staged according to the tumor-lymph node-metastasis (TNM) staging system of the International Union Against Cancer (5th edition). Histologic grade was assessed following the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines for Oncology (V.1.2011). No patient received any treatment before the upper gastrointestinal endoscopy examination or surgery. All clinical samples and data were collected in a double-blind manner. This study was approved by the Human Research Ethics Committee of Ningbo University. Written informed consent was obtained from all participants.

Total RNA Preparation, Reverse Transcription, and Quantitative Reverse Transcriptase-Polymerase Chain Reaction Detection

Total RNA was extracted from tissues using Trizol reagent (Ambion, Carlsbad, Calif), whereas plasma and gastric juices were processed using Trizol LS reagent (Ambion) according to the manufacturer's instructions. The quality of total RNA was detected at an A260/A280 ratio using 1% agarose gel electrophoresis (Supporting Fig. 1; see online supporting information). The GoScript Reverse Transcription System (Promega, Madison, Wis) was used to generate combinational DNA. To evaluate AA174084 levels, real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was achieved using the GoTaq qPCR Master Mix (Promega) on an Mx3005P Real-Time PCR System (Stratagene, La Jolla, Calif). The sequences of the PCR primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and AA174084 were as follows: 5′-ACCCACTCCTCCACCTTTGAC-3′ (sense) and 5′-TGTTGCTGTAGCCAAATTCGTT-3′ (antisense) for GAPDH; and 5′-ctggttcttcatccctgctatg-3′ (sense) and 5′-cctgctcctctttgtgttctct-3′ (antisense) for AA174084. The conditions of thermal cycling were as follows: 10 minutes at 95°C for a hot start; then 45 cycles at 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The cycle threshold (Ct) values were recorded for both AA174084 and GAPDH. The expression levels of AA174084 were calculated using the ΔCt method with GAPDH as the control to normalize the data. Lower ΔCt values indicate higher expression of AA174084. Relative expression was calculated using the 2−ΔΔCt method.[10] All results were expressed as the mean±standard deviation of 3 independent experiments.

Cloning and Sequencing of qRT-PCR Products

The qRT-PCR products of GAPDH and AA174084 were purified using the UNIQ-10 PCR Product Purification Kit (Sangon Biotech, Shanghai, China) and then were cloned into the pUCm-T vector (Sangon Biotech). Finally, PCR product sequencing was performed by the same company.[10]

Detection of Gastric Juice Carcinoembryonic Antigen Levels

Gastric juice carcinoembryonic antigen (CEA) levels were measured using an enzyme-linked immunosorbent assay CEA kit (KBH Diagnosis, Shanghai, China) with a SpectraMax M5 Microplate Reader (Molecular Devices Inc., Sunnyvale, Calif) as previously reported.[15] The cutoff value was 10 ng/mL.

Detection of CEA and CA 19-9 Expression in Tissues

The paraffin tissue sections were incubated in primary anti-CEA or anticarbohydrate antigen 19-9 (anti-CA 19-9) (DAKO, Glostrup, Denmark) for 1 hour at room temperature. After incubation with broad-spectrum second antibody K5007 (DAKO), the tissues were incubated in diaminobenzidine (DAKO) for color development. The results were interpreted according to the 2010 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines.

Statistical Analysis

All statistical analyses were performed using Statistical Program for Social Sciences (SPSS) 20.0 software (SPSS, Chicago, Ill), GraphPad Prism 5.0 (GraphPad Software, La Jolla, Calif), or SigmaPlot 12.3 (Systat Software, San Jose, Calif). One-way analyses of variance, rank-sum tests, Student t tests, and chi-square tests were used according to the actual conditions. A receiver operating characteristic (ROC) curve was constructed to evaluate the diagnostic value of AA174084 levels. P values <.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

AA174084 Expression Is Changed During Gastric Carcinogenesis

To determine whether AA174084 was aberrantly expressed in GC tissues, we used qRT-PCR to detect its levels in 134 GC tissues compared with adjacent normal tissues. The results demonstrated that AA174084 was down-regulated in 95 of 134 GC tissues (71%) compared with the paired, adjacent, normal tissues (P < .001) (Fig. 1). The average expression level in adjacent normal tissues was 3.18 times the average level in GC tissues.

image

Figure 1. This chart compares AA174084 expression levels in gastric cancer tissues with the levels in adjacent normal tissues (n=134). AA174084 expression levels were calculated using the Δ cycle threshold (ΔCt) method, and data are expressed as the mean±standard error of the mean from 3 independent experiments. Triple asterisks indicate P < .001.

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On the basis of these findings, we further explored AA174084 expression levels in tissues from various stages of gastric carcinogenesis. Compared with HGM, AA174084 expression was significantly decreased in GD and GC tissues, as indicated in Figure 2A. In addition, AA174084 levels differed between GD tissues and GC tissues.

image

Figure 2. Differences in AA174084 expression levels are illustrated among various types of gastric mucosa. (A) AA174084 levels were decreased significantly in gastric dysplasia (GD) and gastric cancer (GC) tissues compared with the levels in healthy gastric mucosa (HGM). AA174084 expression levels in HGM (n=37), GD (n=28), and GC (n=134) were determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). (B) AA174084 expression had a good correlation with gastric mucosal inflammatory lesion depth. AA174084 expression levels in 37 HGM tissues, 18 erosive gastritis (EG) tissues, and 16 gastric ulcer (GU) tissues were determined by qRT-PCR. AA174084 expression levels were calculated using the Δ cycle threshold (ΔCt) method, and the data shown are the means±standard deviations from 3 independent experiments. Asterisks indicate statistically significant differences (single asterisk, P < .05; double asterisks, P < .01; triple asterisks, P < .001).

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Finally, we investigated AA174084 levels in HGM, EG tissues, and GU tissues to obtain more information about AA174084 expression and gastric mucosal damage. The levels in GU tissues were much lower than those in chronic AG and HGM tissues, as expected, indicating a good correlation with gastric mucosal inflammatory lesion depth (Fig. 2B).

Correlation Between AA174084 Levels and Clinicopathologic Factors in Patients With GC

AA174084 levels were associated with several clinicopathologic factors, including age (P = .031), Borrmann type (P = .016), and perineural invasion (P = .032), as indicated in Supporting Table 1 (see online supporting information). An ROC curve was constructed for differentiating GC tissues from other benign gastric lesions. The results indicated that the area under the ROC curve (AUC) was up to 0.676 (95% confidence interval [CI], 0.612-0.740; P < .001) (Supporting Fig. 2; see online supporting information). The optimal cutoff value was 11.62, which had 57% sensitivity and 73% specificity.

The Presence of AA174084 in Human Plasma and Gastric Juice

Because body fluid is the main material used in the diagnosis of diseases, we explored whether AA174084 could be detected by qRT-PCR. To make sure that AA174084 and GAPDH were present in human plasma and gastric juices, we sequenced their qRT-PCR products. We observed that, as expected, their sequences (Supporting Fig. 3; see online supporting information) were completely consistent with those from the database (available at: http://www.ncbi.nlm.nih.gov/nucest/AA174084 and http://www.ncbi.nlm.nih.gov/gene/2597, respectively; accessed June 1, 2014).

GAPDH Is a Satisfactory Reference for the Measurement of lncRNAs in Human Plasma and Gastric Juices

To date, no ideal reference has been identified for the detection of lncRNAs in body fluids, and especially gastric juices, using qRT-PCR. Because GAPDH, a housekeeping gene that is stably expressed in most human tissues, is regarded as an ideal reference for the detection of lncRNAs in tissues by qRT-PCR, we detected GAPDH levels in 232 plasma samples and 130 gastric juice samples. The results indicated that GAPDH levels in human plasma and gastric juices were not affected by common factors like age, sex, and pathology (Supporting Tables 2 and 3; see online supporting information) and that GAPDH levels are stable in human plasma and gastric juices and can be used as a reference for the detection of plasma or gastric juice lncRNAs by qRT-PCR.

AA174084 Levels in Plasma Decrease Significantly After Surgery and May Be a Potential Biomarker for Evaluating Prognosis

On the basis of the findings described above, we investigated whether AA174084 could be used as a biomarker for the diagnosis of GC. The results from 120 healthy individuals, 29 patients with dysplasia, 84 preoperative GC patients, and 103 postoperative GC patients unexpectedly indicated that there was no difference in AA174084 levels among healthy individuals, patients with dysplasia, and preoperative GC patients; however, the levels sharply declined in 63 of 83 GC patients (76%) on day 15 after surgery (P < .001) (Fig. 3).

image

Figure 3. AA174084 levels were decreased only in postoperative plasma from patients with gastric cancer (GC). AA174084 levels in 120 healthy individuals, 29 patients with dysplasia, 84 preoperative (Pre-op) patients with GC, and 103 postoperative (Post-op) patients with GC were determined by quantitative reverse transcriptase-polymerase chain reaction. There was no difference between healthy individuals, patients with dysplasia, and preoperative GC patients; however, a sharp decrease was observed in postoperative patients with GC at day 15 after surgery. AA174084 expression levels were calculated using the Δ cycle threshold (ΔCt) method, and data are expressed as the means±standard deviations from 3 independent experiments. Asterisks indicate statistically significant differences (double asterisks, P < .01; triple asterisks, P < .001).

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This symbolic reduction in plasma AA174084 levels after surgery aroused our great interest in exploring the potential relation between postoperative AA174084 plasma levels and clinicopathologic features in patients with GC. The individual relative changes in AA174084 levels after surgery were calculated using the ΔΔCt method (ΔCtpreoperation−ΔCtpostoperation). Changes in the AA174084 level had a significant, positive association with invasion (P = .049) and lymphatic metastasis (P = .042), as indicated in Supporting Table 4 (see online supporting information). These data indicated that, the higher the postoperative AA174084 level in plasma, the worse pathologic result.

Gastric Juice AA174084 Is a Potential Screening Biomarker for Early GC

For the purpose of screening patients with early GC, the measurement of plasma-based AA174084 has serious limitations, because the level does not differ in human plasma between patients with GC and controls. Because of the high specificity of gastric juice for gastric organs, we turned our attention to detection of the AA174084 level in human gastric juice. We compared the gastric juice AA174084 level between individuals with normal mucosa or minimal gastritis (NMMG), patients with GU, patients with chronic AG, and patients with GC. The results indicated that the AA174084 level in gastric juice from patients with GC was significantly higher than the levels in the other groups (Fig. 4A). An ROC curve also was constructed for differentiating the GC group from the other groups. The results indicated that the AUC was up to 0.848 (95% CI, 0.776-0.921; P<.001) (Fig. 4B), which was higher than the AUC for using the tissue level of AA174084 as the marker (Supporting Fig. 2; see online supporting information). The optimal cutoff value was 0.88, sensitivity was 0.46, and specificity was 0.93.

image

Figure 4. Gastric juice AA174084 may be a potential marker of gastric cancer (GC). (A) AA174084 levels in gastric juice from patients with GC were significantly higher than the levels in other groups. AA174084 levels in gastric juice from 45 patients with normal mucosa or minimal gastritis (NMMG), 30 patients with gastric ulcers (GU), 16 patients with atrophic gastritis (AG), and 39 patients with GC were detected by quantitative reverse transcriptase-polymerase chain reaction. There was no difference between the patients with NMMG, GU, and AG. AA174084 expression levels were calculated using the Δ cycle threshold (ΔCt) method, and data are expressed as the means±standard deviations from 3 independent experiments. Asterisks indicate statistically significant differences (single asterisk, P < .05; double asterisks, P < .01). (B) The area under the receiver operating characteristic curve (AUC) is illustrated for AA174084 (P < .001).

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Next, we investigated whether the gastric juice AA174084 level could be used as a biomarker for the early diagnosis of GC. Our data indicated that there was an obvious increase in the AA174084 level in the early GC group (Fig. 5A). On the basis of cutoff values from the ROC curves (Fig. 4B), when the gastric juice AA174084 level was used as a screening marker, the positive detection rate for early GC was up to 57.1% (Supporting Table 5; see online supporting information). The combined use of gastric juice AA174084, CEA, or serum CEA levels may improve the detection rate (Supporting Table 5; see online supporting information). These data demonstrated that the gastric juice AA174084 level has great potential as a screening biomarker of early GC.

image

Figure 5. Gastric juice AA174084 levels changed in the early stage of gastric cancer (GC). (A) AA174084 levels in gastric juice from 75 patients with normal mucosa or benign lesions (NMBL), 16 patients with chronic atrophic gastritis (AG), and 7 patients with early gastric cancer (EGC) were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Gastric juice AA174084 levels were increased significantly in patients with EGC. (B) AA174084 levels in gastric juice from 23 patients with benign giant ulcers and 20 patients with malignant ulcers were detected by qRT-PCR, and the levels were significantly higher in the group with malignant ulcers. AA174084 expression levels were calculated using the Δ cycle threshold (ΔCt) method, and data are expressed as the means±standard deviations from 3 independent experiments. Asterisks indicate statistically significant differences (single asterisks, P<.05; triple asterisks, P<.001).

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Identifying the benign or malignant ulcer is 1 of the difficult problems in upper gastroendoscopy. Therefore, we tested the diagnostic value of gastric juice AA174084 for distinguishing between benign giant GUs (≥2 cm) and malignant ulcers. We observed that the gastric juice AA174084 level in patients with malignant ulcers was significantly higher than that in patients who had benign giant ulcers (Fig. 5B).

Correlation Between Gastric Juice AA174084 Levels and Clinicopathologic Factors in Patients With GC

An additional analysis was performed to assess the correlation between gastric juice AA174084 levels and the clinicopathologic features of patients with GC. Gastric juice AA174084 levels were associated with tumor size (P=.026), tumor stage (P=.034), Lauren type (P=.021), and gastric juice CEA levels (P=.039), as indicated in Table 1. In addition, the patients with benign giant ulcers had relatively high levels of AA174084 gastric juices (Supporting Table 6; see online supporting information).

Table 1. Relation Between AA174084 Levels (Δ Cycle Threshold) in Gastric Juice and Clinicopathologic Factors of Patients With Gastric Cancer
CharacteristicNo. of Patients (%)AA174084 Level: Mean±SDaP
  1. Abbreviations: CA 19-9, cancer antigen 19-9; CEA, carcinoembryonic antigen; SD, standard deviation.

  2. a

    AA174084 expression levels were calculated using the Δ cycle threshold (ΔCt) method.

Age, y   
≥6025 (64)5.611±5.432.585
<6014 (36)6.643±5.938 
Sex   
Men29 (74)5.380±5.963.255
Women10 (26)7.727±3.941 
Smoking   
Yes26 (67)5.292±5.712.281
No13 (33)7.360±5.192 
Drinking alcohol   
Yes28 (72)5.997±5.903.978
No11 (28)5.941±4.851 
Helicobacter pylori infection   
Positive21 (54)6.241±5.372.758
Negative18 (46)5.679±5.921 
Tumor location   
Sinuses ventriculi16 (41)4.985±5.439.308
Corpora ventriculi14 (36)5.286±5.054 
Gastric angle4 (10)7.271±7.209 
Cardia5 (13)10.085±5.723 
Greatest tumor dimension, cm   
>610 (26)2.651±5.412.026
≤629 (74)7.130±5.224 
Differentiation   
Moderate7 (18)4.239±4.871.367
Poor32 (82)6.363±5.702 
Tumor stage   
Early7 (18)1.990±2.276.034
Advanced32 (82)6.855±5.706 
Borrmann type   
I6 (19)7.482±4.777.705
II8 (25)8.041±6.179 
III12 (37)6.953±5.786 
IV6 (19)4.450±6.498 
Lauren type   
Intestinal29 (74)7.178±5.581.021
Diffuse and mixed10 (26)2.512±3.976 
CEA   
Gastric juice   
Positive27 (69)4.767±5.601.039
Negative12 (31)8.713±4.587 
Blood   
Positive25 (64)6.761±5.781.248
Negative14 (36)4.590±6.143 
CA 19-9: Blood   
Positive9 (23)4.770±4.405.464
Negative30 (77)6.345±5.883 

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

The mainstream tumorigenesis processes involved in GC are characterized by phenotypic, multistep progression cascades.[4] GC is a highly heterogeneous disease.[16] The reliable identification of GC progression-specific targets has huge implications for the prevention and treatment of GC. However, identification of the molecular mechanisms underlying tumorigenesis still remains a challenge.

Recently, it was demonstrated that lncRNAs play oncogenic and tumor-suppressor roles in tumorigenesis.[12-14] Several associations between altered lncRNAs in cancers and clinical significance were observed, and some have attempted to develop these as therapeutic targets.[12-14] In addition to H19, HULC, and CCAT1, mounting evidence indicates that altered lncRNAs, including gastric cancer-associated transcript 1 (GACAT1), long intergenic noncoding protein RNA 152 (LINC00152), maternally expressed gene 3 (MEG3), SUMO1 pseudogene 3 (SUMO1P3), and the long intergenic noncoding RNA BM742401 (a sequence tag), are related to GC pathophysiology. We previously observed that expression levels of GACAT1, LINC00152, and SUMO1P3 were significantly correlated with tumor size, differentiation, metastasis, and invasion.[11, 17, 18] Park et al demonstrated that BM742401 was down-regulated in GC tissues, and its down-regulation was associated with poor survival.[19] Sun et al observed that MEG3 levels were markedly decreased in GC tissues, and MEG3 down-regulation could promote cell proliferation and inhibit cell apoptosis.[20] In addition, patients with low levels of MEG3 had a relatively poor prognosis.[20] Arita et al confirmed the existence of circulating lncRNA H19 in plasma.[21] In the current study, we considered whether plasma or gastric juice lncRNAs could be used as biomarkers for predicting clinical prognosis and early diagnosis of GC.

First, we discovered that the AA174084 expression level was significantly down-regulated in GC tissues (Fig. 1). Then, we further explored the AA174084 expression level in each stage of gastric carcinogenesis. The results indicated that, compared with human HGM, AA174084 levels were significantly decreased in GD and GC tissues (Fig. 2A). An ROC curve was constructed for differentiating GC tissues from other benign gastric lesions, and the results demonstrated that the AUC was up to 0.676 (95% CI, 0.612-0.740; P<.001) (Supporting Fig. 2; see online supporting information). These results suggest that AA174084 may play crucial roles during cancer occurrence and progression.

Some studies have demonstrated that full-blown cancer is the final outcome of long-standing biologic processes, including a progressive accumulation of genotypic and phenotypic changes in which a subset of cases are triggered by persistent inflammatory conditions.[4, 22, 23] Inflammatory responses exert tumor-promoting roles in cancer development, including tumor initiation, progression, metastasis, and prognosis.[24] It is established that EG and GUs are the inflammatory lesions of gastric mucosa. The main difference between them is that there is more extensive damage from ulcers than from erosion. In our study, we observed that the AA174084 level was negatively correlated with the degree of inflammatory lesions (Fig. 2B). This suggests that AA174084 may be associated with inflammation in cancer development.

Borrmann type and perineural invasion (PNI) are independent prognostic factors in patients with advanced GC. Borrmann type is a valuable predictor for lymph node metastasis and survival, whereas the presence of PNI has been identified as an independent prognostic factor for survival.[25, 26] Our results demonstrate that the AA174084 level is associated with both Borrmann type (P=.016) and PNI (P=.032) (Supporting Table 1; see online supporting information). Our data also indicate that AA174084 may be used as a biomarker for predicting the clinical prognosis of patients with GC.

The measurement of tumor biomarkers is not only an effective means for monitoring tumor recurrence but also an important way to evaluate prognosis.[27, 28] qRT-PCR, a reliable method for studying lncRNA expression, requires reference genes as appropriate normalization for accurate and reliable results.[29] Although several genes (such as GAPDH, β-actin, and 18S rRNA) have been used as reference genes in the detection of tissue lncRNA levels, there is still no reference being used for the detection of plasma and gastric juice lncRNAs. Thus, we tested whether GAPDH could be a satisfactory reference for plasma and gastric juice lncRNA measurement. Our data indicated that GAPDH levels in human plasma and gastric juice were not affected by age, sex, or pathology (Supporting Tables 2 and 3; see online supporting information). To our knowledge, this is the first demonstration that GAPDH is a satisfactory reference for the detection of plasma and gastric juice lncRNAs.

On the basis of the findings described above, we also explored the existence of AA174084 in human plasma and gastric juice for the first time. By sequencing the qRT-PCR products of AA174084 (Supporting Fig. 3; see online supporting information), we confirmed their existence in plasma and gastric juice. Then, we observed that plasma AA174084 levels dropped markedly in 63 of 84 patients with GC (75%) on day 15 after surgery (P<.001) (Fig. 3).

Comparing tumor marker levels between a patient's own blood from samples before and after surgery is an effective method for evaluating their prognosis. Hotta et al demonstrated that the postoperative/preoperative serum CEA ratio was a predictor of prognosis after surgery for patients with stage III rectal cancer.[30] In our study, we observed that the individual relative change in plasma AA174084 level after surgery was associated with invasion (P=.049) and lymphatic metastasis (P=.042). Moreover, our data indicated that patients with higher AA174084 postoperative plasma levels had the worst pathologic results (Supporting Table 4; see online supporting information). Our investigation indicates that plasma lncRNA-AA174084 may be a candidate biomarker for evaluating the prognosis of patients with GC.

For the diagnosis of early GC, the measurement of plasma-based AA174084 has obvious limitations, because AA174084 levels in plasma do not differ between healthy individuals and patients with GC (Fig. 3). Gastric juice, with a single source and tissue specificity, is an ideal sample that can easily be obtained by using a string test or an endogastric capsule, which is both acceptable by patients and economic.[31, 32] In our study, we explored the gastric juice AA174084 level as a biomarker for early GC screening. The experimental data indicated that gastric juice AA174084 levels were increased only in patients with GC (Fig. 4A). There were no significant differences between individuals with NMMG, GU, and AG. These results suggest that benign gastric diseases like GU and AG, which have always been considered to affect the secretion of gastric juice,[33] do not affect the AA174084 level in gastric juice. The AUC was up to 0.848 (Fig. 4B), which was higher than the AUC when the tissue AA174084 level was used as a marker (Supporting Fig. 2; see online supporting information). More valuable is the possibility that the gastric juice AA174084 level also may be detectable at an early stage (Fig. 5A). The AA174084 level in gastric juice was significantly increased in patients who had GC (especially those who had early GC) compared with the level in normal individuals (Figs. 4A, 5A). This does not mean that the AA174084 level in gastric juice will decrease along with the increasing severity of GC. This is because the small number of patients with early stage GC (n=7). However, as indicated in Table 1, we can conclude that there may be a positive correlation between the AA174084 level in gastric juices and tumor progression. Our data indicate that gastric juice AA174084 has great potential as screening biomarker of early GC.

A giant GU is defined as an ulcer that measures >2 cm in greatest dimension. Poor response to drug treatment, longer healing time, and chronic penetration or perforation are its clinical characteristics. Moreover, it is very important to differentiate a giant GU from a malignant ulcer. However, the identification of a benign or malignant giant ulcer is 1 of the challenges in endoscopic examination. In the current study, first, we observed that the level of gastric juice AA174084 was closely related to an ulcer's greatest dimension (Supporting Table 6; see online supporting information). Patients who had giant GUs had relatively high levels of gastric juice AA174084. Then, we tested the diagnostic value of gastric juice AA174084 in distinguishing a benign giant GU from a malignant ulcer. We observed that gastric juice AA174084 levels from patients with malignant ulcers were significantly higher than the levels from patients with benign giant GUs (Fig. 5B). This result suggests that gastric juice AA174084 has potential value in the differential diagnosis of GC.

The greatest tumor dimension, cancer stage, and Lauren type are independent prognostic factors in patients with GC.[34] In this study, we assessed the correlation between gastric juice AA174084 levels and the clinicopathologic features of patients with GC. The result indicated that the gastric juice AA174084 level was associated with greatest tumor dimension (P=.026), tumor stage (P=.034), Lauren type (P=.021), and gastric juice CEA level (P=.039) (Table 1). Our data also revealed that a higher AA174084 level in gastric juice suggested a worse pathologic result for patients with GC. These correlations indicate that gastric juice AA174084 also may be a candidate biomarker for predicting the clinical prognosis of patients with GC.

It is noteworthy that we observed significantly lower levels of AA174084 in gastric cancer tissues compared with the levels in adjacent normal tissues and HGM. However, AA174084 levels were elevated in gastric juice from patients with GC. This unexpected result is interesting. Previous studies have demonstrated that the levels of some noncoding RNAs, such as miR-200a, miRNA-21, and miR-106a, in body fluids are inconsistent with the their levels in tissues.[35, 15] Exosomes, which are automatically secreted, homogenous membrane vesicles, may transfer noncoding RNA to the body fluids.[36, 37] Recent research has provided evidence that some lncRNAs can be enriched in exosomes and selectively released from normal cells and malignant cells.[38] The high level of AA174084 in gastric juice may be caused in part by secretion through exosomes or other pathway. In addition, the tumor microenvironment plays a major role in tumorigenesis. Tumor cells can influence their homeostatic activities and support the neoplastic nature of the tumor.[36] Exosomes are increasingly recognized as important mediators of cell-to-cell communication.[36, 37] They can transfer receptors, proteins, and RNA to target cells through interaction with specific receptors.[37]

In conclusion, lncRNA-AA174084 potentially may play a role during GC development. The plasma AA174084 level also has potential as a biomarker for prognosis evaluation, and the level of AA174084 in gastric juice has potential use in the early diagnosis and differential diagnosis of GC.

FUNDING SUPPORT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

This work was supported by the National Natural Science Foundation of China (no. 81171660), the Zhejiang Provincial Natural Science Foundation of China (no. LY14C060003), the Applied Research Project on Nonprofit Technology of Zhejiang Province (no. 2014C33222), the Scientific Innovation Team Project of Ningbo (no.2011B82014), the Natural Science Foundation of Ningbo (no. 2012A610207), the Project of Key Disciplines in Ningbo (no. XKL11D2127 and no. XKL11D2128), the Postgraduate Scientific Innovation Foundation of Ningbo University (no. G14069), and the K. C. Wong Magna Fund in Ningbo University.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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cncr28901-sup-0001-suppinfo01.doc11663K

Supplementary Information

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