- Top of page
- MATERIALS AND METHODS
- FUNDING SUPPORT
- CONFLICT OF INTEREST DISCLOSURES
- 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%. 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.
Long noncoding RNAs (lncRNAs) are functional noncoding RNA molecules greater than 200 nucleotides in length. 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). CCAT1, which is activated by c-Myc, promotes cell migration and proliferation. Zhao et al reported that the overexpression of HULC promoted proliferation and invasion and inhibited GC cell apoptosis. 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). 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.
- Top of page
- MATERIALS AND METHODS
- FUNDING SUPPORT
- CONFLICT OF INTEREST DISCLOSURES
- Supporting Information
The mainstream tumorigenesis processes involved in GC are characterized by phenotypic, multistep progression cascades. GC is a highly heterogeneous disease. 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. 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. In addition, patients with low levels of MEG3 had a relatively poor prognosis. Arita et al confirmed the existence of circulating lncRNA H19 in plasma. 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. 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. 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. 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, 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. 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. 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. 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.
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.