Abstract
Background: LncRNA ZEB1-AS1 has been identified as a tumor oncogene in hepatocellular carcinoma. However, the clinical significance in esophageal squamous cell carcinoma (ESCC) is still unknown. The aim of this study was to explore ZEB1-AS1 expression levels and evaluated its clinical significance in ESCC patients. Methods: LNCRNA ZEB1-AS1 expression was determined by quantitative real-time PCR (QRT-PCR) in 87 pairs of ESCC specimens and adjacent non-tumor tissues. Then, the association of ZEB1-AS1 expression with clinicopathological factors or survival of ESCC patients were determined. Results: LNCRNA ZEB1-AS1 was found up-regulated in ESCC tissues compared to adjacent non-tumor tissues. Increased lncRNA ZEB1-AS1 expression was significantly associated with tumor grade, depth of invasion, and lymph node metastasis. Kaplan-Meier analysis revealed that ESCC patients with high ZEB1-AS1 expression had a poorer overall survival and disease-free survival. Furthermore, multivariate analysis suggested that ZEB1-AS1 expression was identified as an independent prognostic factor in patients with ESCC. Conclusion: These results indicated that lncRNA ZEB1-AS1 was associated with tumor progression and could be an independent prognostic factor for ESCC patients.
Keywords: Esophageal cancer, lncRNA ZEB1-AS1, overall survival, disease-free survival
Introduction
Esophageal cancer is the eighth most common cancer worldwide and the sixth most common cause of death from cancer [1]. Approximately half of the esophageal cancer cases that are newly diagnosed each year occur in China [2]. There are two common types of esophageal cancer: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) [3]. Despite the recent advances in ESCC treatment, the prognosis is still unfavorable, and the overall 5-year survival rate is less than 10% [4]. Therefore, it is necessary to search novel biomarkers for ESCC, which can improve therapeutic strategies and predict clinical outcome.
In recent years, genome-wide transcriptional studies found that only approximately 1% of the human genome serves as blueprints for proteins, whereas a much larger proportion of the genome is transcribed into non-coding RNAs (NCRNAs) [5,6]. Among these ncRNAs are long non-coding RNAs (lncRNAs) which are more than 200 nucleotides in length with little protein-coding potential [7]. In recent years, several lncRNAs have been shown to be involved in carcinogenesis and cancer progression. For example, Zhang et al showed that lncRNA MALAT1 was up-regulated in clear cell renal cell carcinoma and correlated with advanced clinical features and shorter overall survival time [8]. Li et al found that increased expression of the lncRNA ANRIL promoted lung cancer cell metastasis and correlated with poor prognosis [9]. Zhou et al suggested that lncRNA LET was down-regulated in gastric cancer and associated with tumor progression, furthermore, they indicated that lncRNA LET might act as a novel prognostic indicator in gastric cancer [10].
Recently, Li et al found that lncRNA ZEB1 antisense1 (ZEB1-AS1) was up-regulated in hepatocellular carcinoma and correlated with poor prognosis of HCC patients, furthermore, they revealed that ZEB1-AS1 could induce epithelial to mesenchymal transition and cancer metastasis [11]. However, to our knowledge, expression of ZEB1-AS1 in ESCC and the relationship between ZEB1-AS1 expression and ESCC remains unclear.
In the present study, we determined the expression patterns of lncRNA ZEB1-AS1 in ESCC tissues and adjacent non-tumor tissues. Moreover, we explored the correlation between ZEB1-AS1 dysregulation and clinical characteristics and prognosis of ESCC patients.
Materials and methods
Tissue specimens
A total of 87 pairs of primary ESCC tissues and adjacent non-tumor tissues were obtained from patients who underwent surgery at the Department of Thoracic Surgery, The First Affiliated Hospital of Xinxiang Medical University between 2006 and 2008. All specimens were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. No patient received chemotherapy or radiotherapy prior to surgery. Written informed consent was obtained fromall patients prior to participation in the study. The medical ethics committee of First Affiliated Hospital of Xinxiang Medical University approved the study.
RNA isolation and quantitative real-time PCR (QRT-PCR)
The total RNA was extracted from tissues with Trizol reagent (Invitrogen) according to the manufacturer’s protocol. 1.5 μg total RNA was reverse transcribed in a final volume of 20 μl using random primers under standard conditions using the Prime Script RT Master Mix (Takara). After the RT reaction, the quantitative real-time PCR (QRT-PCR) was performed using the SYBR Select Master Mix (Applied Bio systems) with 0.5 μl complementary DNA (CDNA) on ABI 7900 system (Applied Biosystems) according to the manufacturer’s instructions. By using GAPDH as an internal control, ZEB1-AS1 expression level was determined by qRT-PCR using the following primer sequences: forward, 5’-ATTGTTAGGAAAGGTTATAAAATTT-3’; and reverse, 5’-ACCCAAACTATATAAAAAATTACAC-3’. GAPDH forward primer, 5’-CGCTCTCTGCTCCTCCTGTTC-3’, GAPDH reverse primer, 5’-ATCCGTTGACTCCGACCTTCAC-3’. All experiments were performed using the 2-ΔΔCt method. Each experiment was performed in triplicate.
Statistical analysis
Statistical analyses were performed using SPSS version 18.0. The chi-square test was used to assess ZEB1-AS1 expression with respect to clinicopathological factors. The survival curves of the patients were determined using the Kaplan-Meier method and the log-rank test was used for statistical evaluations. A Cox proportional hazards model was used for multivariate analysis. All data are presented as the mean ± SD from at least three independent experiments. P<0.05 was considered statistically significant.
Results
LncRNA ZEB1-AS1 was up-regulated in ESCC
To determine whether lncRNA ZEB1-AS1 was involved in the tumorigenesis of ESCC, QRT-PCR was performed to detect the differential expression of ZEB1-AS1 in 87 pairs of ESCC tissues and matched adjacent non-tumor tissues. As shown in Figure 1A, ZEB1-AS1 expression in ESCC tissues was significantly higher than that in adjacent non-tumor tissues, indicating that ZEB1-AS1 might play an oncogenic role in ESCC progression.
Figure 1.
LncRNA ZEB1-AS1 expression were up-regulated in ESCC. The relative ZEB1-AS1 expression levels were determined using qRT-PCR and demonstrated using the comparative ΔCt method. A. Higher relative ZEB1-AS1 levels were detected in ESCC tissues than in adjacent non-tumor tissues. B. ZEB1-AS1 expression was classified into a low ZEB1-AS1 expression group and a high ZEB1-AS1 expression group according to the median value of relative ZEB1-AS1 expression. *P<0.05.
Over expression of lncRNA ZEB1-AS1 was associated with advanced clinicopathologic factors of ESCC patients
The 87 ESCC patients were classified into two groups according to the median expression level of ZEB1-AS1: ESCC patients expressing ZEB1-AS1 less than the median expression level were assigned to the low expression group (n=43), and those samples with expression equal or above the median expression level were assigned to the high expression group (n=44) (Figure 1B). The association between clinicopathologic factors and ZEB1-AS1 expression were shown in Table 1. Over expression of ZEB1-AS1 was significantly associated with tumor grade, depth of invasion, and lymph node metastasis (P<0.05, Table 1). In contrast, there was no correlation was detected in the expression level of ZEB1-AS1 with other clinicopathological factors of ESCC patients, including age, gender, tumor size, and tumor location (P>0.05, Table 1). These data indicated that lncRNA ZEB1-AS1 might play an important role in ESCC progression.
Table 1.
Correlation between lncRNA ZEB1-AS1 expression and clinicopathologic factors of ESCC patients
| Parameters | Group | Total | ZEB1-AS1 expression | P value | |
|---|---|---|---|---|---|
|
| |||||
| Low | High | ||||
| Age (years) | <60 | 41 | 21 | 20 | 0.752 |
| ≥60 | 46 | 22 | 24 | ||
| Gender | Male | 54 | 29 | 25 | 0.307 |
| Female | 33 | 14 | 19 | ||
| Tumor size (cm) | <4 cm | 51 | 26 | 25 | 0.730 |
| ≥4 cm | 36 | 17 | 19 | ||
| Tumor location | Upper | 22 | 10 | 12 | 0.216 |
| Middle | 41 | 24 | 17 | ||
| Lower | 24 | 10 | 14 | ||
| Tumor grade | High + Middle | 38 | 26 | 12 | 0.002 |
| Low | 49 | 17 | 32 | ||
| Depth of invasion | T1-T2 | 36 | 30 | 6 | 0.000 |
| T3-T4 | 51 | 13 | 38 | ||
| Lymph nodes metastasis | Absence | 53 | 33 | 20 | 0.003 |
| Presence | 34 | 10 | 24 | ||
Correlation between lncRNA ZEB1-AS1 expression and ESCC patients’ survival
The correlation of lncRNA ZEB1-AS1 expression with prognosis in ESCC patients was further investigated by Kaplan-Meier analysis and log-rank test. Our results showed that 5-year overall survival (P>0.05, Figure 2A) and disease-free survival (P>0.05, Figure 2B) of ESCC patients with high ZEB1-AS1 expression were shorter compared to those patients with low ZEB1-AS1 expression. Furthermore, multivariate analysis using the Cox proportional hazard model revealed that tumor grade, depth of invasion, lymph node metastasis and ZEB1-AS1 expression were independent prognostic factors for overall survival (HR=2.371, 95% CI: 1.284-6.115; P<0.05), as well as disease-free survival (HR=2.695, 95% CI: 1.379-8.352; P<0.05) of ESCC patients after esophagectomy (Table 2).
Figure 2.
Kaplan-Meier survival analysis of association between lncRNA ZEB1-AS1 expression level and (A) overall survival and (B) disease-free survival of ESCC patients. Patents expressing high level of ZEB1-AS1 have a significantly shorter overall survival and disease-free survival compared with patients with low level of ZEB1-AS1. (P<0.05, log-rank test).
Table 2.
Multivariate Cox proportional hazard model analysis of overall survival and disease-free survival inESCC patients
| Overall survival | Disease-free survival | |||||
|---|---|---|---|---|---|---|
|
|
||||||
| HR | 95% CI | P | HR | 95% CI | P | |
| Age (years) | 0.886 | 0.528-1.837 | 0.203 | 0.966 | 0.674-2.073 | 0.261 |
| Gender | 1.225 | 0.413-2.938 | 0.447 | 1.336 | 0.692-3.431 | 0.387 |
| Tumor size (cm) | 1.861 | 0.557-4.286 | 0.159 | 1.525 | 0.492-4.273 | 0.118 |
| Tumor location | 1.513 | 0.769-5.115 | 0.214 | 1.703 | 0.783-6.827 | 0.139 |
| Tumor grade | 2.607 | 1.804-5.312 | 0.021 | 2.874 | 2.016-7.726 | 0.009 |
| Depth of invasion | 2.036 | 1.413-4.627 | 0.015 | 2.412 | 1.797-6.082 | 0.011 |
| Lymph nodes metastasis | 2.941 | 1.184-7.153 | 0.008 | 3.102 | 1.263-9.173 | 0.003 |
| ZEB1-AS1 expression | 2.371 | 1.284-6.115 | 0.013 | 2.695 | 1.379-8.352 | 0.007 |
HR: hazard ratio; 95% CI: 95% confidence interval.
Discussion
LncRNAs are commonly defined as transcribed RNA molecules which are longer than 200 nucleotides, possessing no potential protein-coding capacity [12]. Accumulating evidences suggested that lncRNAs play critical roles in various physiological and pathological processes [13,14]. Recently, lncRNAs were considered to be novel biomarkers for types of human cancer, including ESCC [15,16]. For example, Tong et al demonstrated that lncRNA POU3F3 could serve as a potential biomarker for diagnosis of ESCC [17]. Shi et al reported that lncRNA PCAT-1 was correlated with advanced clinical stage and poor prognosis of ESCC patients [18]. Li et al found that lncRNA UCA1 was associated with poor prognosis of ESCC, and in vitro analysis revealed that decreased expression of lncRNA UCA1 inhibited ESCC cell proliferation, migration, and invasion [19]. Hu et al provided that over expression of lncRNA PlncRNA-1 was correlated with advanced tumor stage and lymph node metastasis, and might serve as a potential prognostic marker and therapeutic target for ESCC [20]. However, there were no reports about the clinicopathologic and prognostic significance of lncRNA ZEB1-AS1 expression in human ESCC.
In the present study, we found that lncRNA ZEB1-AS1 expression was up-regulated in ESCC tissues. The relationships of ZEB1-AS1 with clinical features of ESCC were further explored. Our results showed that ZEB1-AS1 expression was associated with tumor grade, depth of invasion, and lymph node metastasis, indicating that ZEB1-AS1 might be involved in the carcinogenesis and metastasis of ESCC. More importantly, we proved that ESCC patients with a high expression of ZEB1-AS1 had a shorter overall survival and disease-free survival than those with low ZEB1-AS1 expression group. In a multivariate Cox model, our data demonstrated that ZEB1-AS1 expression was an independent poor prognostic factor for both overall survival and disease-free survival of ESCC patients, suggesting that lncRNA ZEB1-AS1 could be a promising non-invasive biomarker for prognosis of ESCC patients. However, the precise molecular mechanisms behind the altered expression of ZEB1-AS1 in ESCC and its function is still unclear. Li et al showed that over expression of ZEB1-AS1 could promote HCC cell proliferation and invasion both in vitro and in vivo [11]. Thus, additional studies are needed to more clearly and comprehensively articulate the molecular mechanisms of both the cause and the effects of altered expression of ZEB1-AS1 in the progression of ESCC.
In conclusion, our results revealed that lncRNA ZEB1-AS1 was significantly up-regulated in ESCC and correlated with poorer patients’ prognosis and it might be a new and potential prognostic biomarker for ESCC.
Disclosure of conflict of interest
None.
References
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