Serum LINC01133 combined with CEA and CA19-9 contributes to the diagnosis and survival prognosis of gastric cancer.

1. Introduction
Gastric cancer (GC) is one of the major malignancies of the digestive system caused by various factors.[1] Every year, about 990,000 people were diagnosed with GC worldwide, of whom approximately 738,000 died.[1,2] Gastritis caused by inflammation of gastric mucosa is also a common disease and multimorbidity in gastroenterology department.[3,4] Among them, frequently occurring chronic superficial gastritis (CSG) and chronic atrophic gastritis (CAG) are more at risk of progression to GC.[4–6] What’s worse, early GC was difficult to be detected in a timely manner because of lacking some specific symptoms and signs.[7,8] Thus, the early diagnosis of gastritis and GC is essential for the timely treatment of GC patients. At present, the main diagnostic biomarkers of GC were carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9), which were commonly used for early screening and disease monitoring of clinical GC.[9–11] However, due to the limited sensitivity and specificity of conventional biomarkers, the combined detection of several markers was inherent.[12] Therefore, the exploration of novel biomarkers is still significant for the early diagnosis of GC.
Studies revealed that noncoding RNAs were able to participate in the regulation of various disease processes, especially in formation and progression of malignant tumors.[13,14] Notably, some aberrantly expressed long noncoding RNAs (lncRNAs) in circulatory system could be detected easily, and reflect the process of cancer initiation and progression.[15,16] Currently, some functional lncRNAs have been revealed in GC, which could participate in the progression of GC and exert certain biological functions.[17] For instance, a recent study has revealed that the expression of lncRNA MNX1-AS1 was significantly increased in GC tissues and associated with the poor prognosis of GC patients.[18] Additionally, Xian et al found that lncRNA HULC and ZNFX1-AS1 could distinguish GC patients from healthy controls, and were proposed as novel biomarkers for GC diagnosis.[19] In addition, a previous study demonstrated that the aberrant expression of long intergenic nonprotein coding RNA 1133 (LINC01133) specifically downregulated in GC tissues.[20] Moreover, LINC01133 could inhibit the progression of GC by regulating miR-576-5p, miR-106a-3p.[21,22] These studies all demonstrated that LINC01133 had important biological functions in GC, but the clinical significance of its aberrant expression is still unknown in GC.
The present study collected data from 156 GC, 96 CSG, 77 CAG patients and 89 healthy controls, and aimed to evaluated the clinical significance of LINC01133 combined with the available biomarkers, including CEA and CA19-9, in patients with GC.

2. Materials and methods

2.1. Patients and serum sample collection
In this study, 5 mL venous blood samples were collected from 156 GC patients who underwent radical resection at Affiliated Hospital of Weifang Medical University from 2013 to 2016. Serum samples were isolated after centrifugation at 2000 rpm for 10 minutes and stored at −80°C. All GC patients in this study conformed to the following inclusion criteria: (I) the tumor tissues were histopathologically diagnosed as GC; (II) all patients did not receive any antitumor treatment before surgery; and (III) had complete demographic and clinical data. Specific exclusion criteria were as follows: (I) patients with a history of other types of cancer other than GC; (II) age < 18 years; (III) pregnant or lactating women; and (IV) cases with incomplete clinical data or no follow-up available. In addition, venous blood samples from 96 CSG patients, 77 CAG patients and 89 healthy controls were collected, serum samples were isolated and preserved. All gastritis patients were confirmed by histopathological examination. All GC patients underwent a 5-year follow-up with telephone or face-to-face for survival and recorded their survival information. Cases who died of other unrelated events were excluded from this study. A signed informed consent was obtained from participants before sampling. Above protocols for blood collection and analysis were all in accordance with the guideline of the Ethics Committee and has approved by the Ethics Committee of Affiliated Hospital of Weifang Medical University.

2.2. Detection of CEA and CA19-9 in serum samples
The serum levels of CEA and CA19-9 were detected by electrochemiluminescence immunoassay using detection reagents in an Elecsys 2010 analyzer (Roche, Basel, Switzerland) following the manufacturer’s protocols. Each sample was detected for 3 times.

2.3. RNA extraction
Total RNA was isolated from the 300 μL serum using a Serum Extract Kit (Biotake Corporation, Beijing, China). NanoDrop 2000 was used to analyze the OD ratio (260/280 nm) with the OD ratio of 1.8 to 2.0 available for reverse transcription. Then, the synthesis of cDNA from the RNA was performed using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, MA). All operations were performed according to the manufacturer’s instructions.

2.4. Quantitative real-time PCR
The expression levels of LINC01133 were determined by quantitative real-time PCR (qRT-PCR) with the SYBR Green PCR Kit (Bio-Rad, Shanghai, China) on the Applied Biosystems 7900 Real-Time PCR system (CA). β-actin was used as an endogenous control for LINC01133. Primer sequences were as follows: LINC01133: 5’-CCTGTGGTGGAGAGAATGGA-3’, 5’-CCCCACCTTTCCAGATCCAAA-3’; β-actin: 5’-ATGTGGCCGAGGACTTTGATT-3’, 5’-AGTGGGGTGGCTTTTAGGATG-3’. The final expression level was calculated using the 2−ΔΔCt method.

2.5. Statistical analysis
In this study, all data were presented as mean ± standard deviation and analyzed using SPSS 21.0 (SPSS, Inc, Chicago, Illinois) and GraphPad 7.0 (GraphPad Software, Inc, San Diego, CA). Student t test was used to compare the differences between 2 groups, and one-way ANOVA followed by Tukey’s test was used for differences between multiple groups. Student t test and Chi-square test were used to assess the relationship between LINC01133 expression and the clinicopathological characteristics of GC patients. Receiver operating characteristics (ROC) curve was used to evaluate the diagnostic value of LINC01133, CEA, and CA19-9 individually for GC patients. Regarding the diagnostic value of the joint detection of LINC01133, CEA, and CA19-9, the probability value after the joint detection of all 3 needed to be calculated using logistic regression analysis firstly, and then were assessed using ROC curve analysis. Kaplan–Meier method and log-rank test were used to conduct survival comparison analysis of GC patients. Cox regression analysis was used to screen the independent prognostic factors for GC. P < .05 was considered to indicate a statistically significant difference.

3. Results

3.1. Basic characteristics of study subjects
The data analyzed in this study were obtained from 156 GC patients, 96 CSG patients, 77 CAG patients and 89 healthy controls. All baseline characteristics of the study subjects were included in Table 1. The results showed that GC patients had significant differences in the expression levels of CEA and CA19-9 compared with CSG patients, CAG patients and healthy controls (all P < .01). However, no significant differences were found in age, gender, smoking and drinking between GC patients and CSG patients, CAG patients, healthy controls (all P > .05).

3.2. Differentially expressed LINC01133 between GC, CSG, CAG, and healthy controls
The expression levels of LINC01133 in GC patients, CSG patients, CAG patients and healthy controls were measured by qRT-PCR. The results revealed that the expression levels of LINC01133 were all decreased in GC patients, CSG patients, and CAG patients compared with healthy controls (Fig. 1, all P < .05). The expression levels of LINC01133 were also decreased by degrees and had statistical difference among GC patients, CSG patients, and CAG patients (Fig. 1, all P < .05).

3.3. Association of LINC01133 with the clinicopathological characteristics of GC patients
According to the median expression, 156 GC patients were divided into low LINC01133 expression group (n = 80) and high LINC01133expression group (n = 76). Then, Student t test was used to analyze differences in CEA and CA19-9 between the and high LINC01133 expression groups, and Chi-square test was used to analyze the associations between other clinicopathological characteristics and LINC01133 expression. The results of the analysis are shown in Table 2, the expression levels of LINC01133 in GC patients were associated with CEA (P < .001), CA19-9 (P < .001), tumor size (P = .025), differentiation (P = .003), lymph node metastasis (P = .007) and tumor node metastasis (TNM) stage (P = .016), but there was no correlation with age, gender, smoking and drinking (all P > .05)

3.4. Diagnostic value of LINC01133, CEA, CA19-9 to distinguish GC patients from healthy individuals
ROC analysis was used to evaluate the diagnostic value of LINC01133, CEA, and CA19-9 for GC patients. First, we evaluated respectively the diagnostic value of LINC01133, CEA, and CA19-9 in discriminating GC patients from healthy controls. As shown in Figure 2A and Table 3, LINC01133 had relatively high diagnostic performance with an area under the curve (AUC) of 0.914, and the sensitivity and specificity were 85.9% and 84.27%, respectively, at a cutoff value of 1.625. Figure 2B and C showed the ROC curves of CEA (AUC = 0.755) and CA19-9 (AUC = 0.889), which also had diagnostic value in GC as expected. Given the correlation found between LINC01133, CEA, and CA19-9, this study further evaluate the diagnostic performance of the joint detection of the 3 molecules. The results showed that the joint detection of LINC01133, CEA, and CA19-9 had the highest accuracy in distinguishing GC patients from healthy controls (Figure 2D, AUC = 0.979) with the diagnosis sensitivity and specificity were 91.03% and 95.51%, respectively.

3.5. Discrimination abilities of LINC01133, CEA, and CA19-9 between GC and gastritis patients
In this study, we also analyzed the discrimination abilities of LINC01133, CEA, and CA19-9 in GC and gastritis (CSG and CAG) patients. First, we analyzed the expression level differences of LINC01133, CEA, and CA19-9 in CSG, CAG, and GC patients. The results were shown in Figure 3, the expression levels of LINC01133 were decreased in GC patients compared with CSG and CAG patients, and the expression levels of CEA and CA19-9 were increased in GC patients compared with CSG and CAG patients (Fig. 3A–C, all P < .05). Meanwhile, the expression levels of LINC01133 and CA19-9 also had statistical difference in CAG compared with CSG patients (Fig. 3A and C, all P < .05). Although the expression level of CEA had a slightly increased in CAG patients compared with CSG patients, there was no statistical difference (Fig. 3B, all P < .05).
In addition, the diagnostic values of LINC01133, CEA, and CA19-9 between GC and gastritis patients were evaluated using ROC curve. Results as shown in Figure 3D and Table 3, LINC01133 (AUC = 0.866), CEA (AUC = 0.705), and CA19-9 (AUC = 0.808) all had the ability to diagnose GC patients from gastritis patients. Moreover, the joint detection of LINC01133, CEA, and CA19-9 showed the highest diagnostic accuracy (AUC = 0.937).

3.6. Low LINC01133 is associated with poor overall survival in GC patients
From the 5-year follow-up records, it was known that 74 GC patients died, accounting for 47.44%, and 19 GC patients lost to follow-up, accounting for 12.18%. Statistical results showed that the expression level of LINC01133 in non-survivor was lower than in survivor (Fig. 4A, P < .05). According to the median expression, all GC patients were divided into low LINC01133 expression group (n = 80) and high LINC01133 expression group (n = 76). Kaplan–Meier results indicated that GC patients with low LINC01133 expression had poor overall survival compared to GC patients with high LINC01133 expression (Fig. 4B, log-rank P = .0001).
In addition, the relationship between LINC01133 and GC overall survival in the Cancer Genome Atlas database was also analyzed in this study. Kaplan–Meier results as shown in Figure 4C, GC patients with low LINC01133 were associated with poor overall survival compared with high LINC01133 expression, which was consistent with the results of this study analysis (log-rank P = .0043).

3.7. Independent prognostic factors predicted the overall survival of GC patients
Multivariate Cox regression analysis was used to screen the independent prognostic factors for GC in present study. Results as shown in Table 4 revealed that lymph node metastasis, TNM stage and LINC01133 were all independently associated with GC survival and all could serve as independent prognostic factors for GC. While age, gender, smoking, drinking, CEA, CA19-9, tumor size, and differentiation did not had independent association with GC survival.

4. Discussion
Multiple studies have reported that LINC01133 was involved in disease progression through aberrant expression in a variety of diseases.[22–24] First, in the cancer field most relevant to the present study, LINC01133 was found to be downregulated in GC tissues and cell lines and was positively correlated with GC progression and metastasis.[22] And, Zhang et al found that LINC01133 was significantly downregulated in colorectal cancer tissues.[23] Besides, Song et al revealed that the overexpression of LINC01133 inhibited invasion and metastasis both in vitro and in vivo in breast cancer.[25] In addition, Yotova et al also found that aberrant expressed LINC01133 was upregulated in ectopic endometriotic lesions.[24] In the present study, the expression levels of LINC01133 were found to be decreased in GC patients and gastritis patients compared with healthy controls, and was associated with some clinicopathological characteristics of GC patients. Therefore, we suggested that the downregulated LINC01133 may be involved in the regulation of GC progression.
CEA and CA19-9 were currently commonly major biomarkers for GC early diagnosis screening and disease monitoring.[26,27] For instance, Feng et al found that the elevation of CEA level was an independent risk factor for the poor prognosis of early GC.[26] For another study, anti-helicobacter pylori antibody combined with serum CA724, CA19-9, and CEA also had diagnostic and prognostic value for young patients with early GC.[27] In this study, it is found that GC patients had significantly different expression levels of CEA and CA19-9 compared with healthy controls, which validated the diagnostic value of CEA and CA19-9 as diagnostic biomarkers for GC. In addition, this study found that the expression levels of LINC01133 in GC patients were associated with CEA and CA19-9. LINC01133 has been uncovered as a potential diagnostic biomarker for cervical squamous carcinoma.[28] ROC curve and logistic regression analysis results showed that although all 3 alone could diagnose GC patients, the joint detection of LINC01133, CEA, and CA19-9 had the highest diagnostic accuracy in distinguishing GC patients from healthy controls. Therefore, we speculated that the joint detection of LINC01133, CEA, and CA19-9 may be suitable for more accurate diagnosis of GC.
As mentioned above, the deregulated expression of LINC01133 may be involved in the progression of GC, but its significance in prognosis was not yet clear. Studies have shown that multiple lncRNAs have been considered as potential prognostic biomarkers for GC.[29,30] For example, Zhu et al revealed that LINC001006 levels in cancer tissues were significantly lower than control, and LINC001006 might be a novel prognostic biomarker for GC.[29] Besides, high expressed PCAT-1 was correlated with poor overall survival and proposed as a prognostic biomarker of GC.[30] Additionally, Yao et al also suggested that high expression of UCA1 was associated with shorter overall survival time, suggesting that UCA1 might be a prognostic biomarker in GC.[31] In this study, the relationship between LINC01133 and GC overall survival was also analyzed. We found that the expression level of LINC01133 in non-survivor was lower than that in survivor, and GC patients with low LINC01133 were associated with poor overall survival. More critically, LINC01133 was independently associated with GC survival and could serve as independent prognostic factor. Therefore, we speculated that LINC01133 might be a potential prognostic biomarker to predict the overall survival of GC patients.
Gastritis carried a risk of progression to gastric cancer,[32] it was essential to early diagnose gastritis and GC and perform timely treatment. In distinguishing GC patients from gastritis patients, multiple factors were revealed as potential diagnostic biomarkers, such as Circ_0004771,[33] LncRNA HCP5[34] and the gastric microbiome altered in early GC.[35] Data from gastritis patients were also included in this study and corresponding statistical analysis was performed. We found that LINC01133, CEA, and CA19-9 were all abnormally expressed in GC patients compared with gastritis patients. Additionally, LINC01133, CEA, and CA19-9 had diagnostic value in distinguishing GC patients from gastritis patients, the joint detection of LINC01133, CEA, and CA19-9 showed the highest diagnostic accuracy. Therefore, we speculate that the joint detection of LINC01133, CEA, and CA19-9 might improve the early diagnosis of GC.
The limitation of this study was the failure to collect longer period of prognostic data, especially follow-up data of gastritis patients. More data of gastritis patients with developing into GC in gastritis population would be collected if the follow-up time can be lengthened, which will further confirm the risk factors of gastritis progressing into GC. Next, we will determine whether LINC01133 in gastritis patients can predict the occurrence of GC, thereby the role of LINC01133 involved in GC would be further clarified.
In conclusion, the expression levels of LINC01133 were decreased in GC patients compared with both healthy controls and gastritis patients. In GC patients, LINC01133 was related with tumor size, differentiation, lymph node metastasis, TNM stage, CEA, and CA19-9. Low levels of LINC01133 were associated with GC patients’ overall survival, and might serve as an independent prognostic biomarker. In addition, the joint detection of LINC01133, CEA, and CA19-9 showed significantly better diagnostic performance in distinguishing GC patients from healthy controls and gastritis patients, which may provide novel insight for early diagnosis of GC with high accuracy. In one word, serum LINC01133 may help to develop new methods to guide the diagnosis and prognosis of GC.

Author contributions
Investigation: Xiaomei Sui.
Formal analysis: Qifu Zhang.
Methodology: Yanfang Chen.
Project administration: Qifu Zhang.
Resources: Meili Hao.
Software: Yanfang Chen.
Supervision: Xiaomei Sui.
Writing – original draft: Xiaomei Sui.
Writing – review & editing: Xiaomei Sui.