Increased expression of long-noncoding RNA ZFAS1 is associated with epithelial-mesenchymal transition of gastric cancer

Results

ZFAS1 was up-regulated in gastric cancer tissues as compared to the paired normal tissues

To evaluate the expression of fourteen candidate lncRNAs in GC tissues, we performed real-time RT-PCR to quantify these lncRNAs in GC tissues and the paired normal tissues derived from sixty-six GC patients. The result showed that among these lncRNAs, only ZFAS1 expression level was significantly up-regulated in gastric cancer tissues compared with paired normal tissues, while the expression levels of other lncRNAs did not differ significantly (Fig. 1A). Next, we tested the correlation between the ZFAS1 expression level and the clinicopathological characteristics of the 66 gastric cancer patients, we found that ZFAS1 was highly correlated with TNM stage (Fig. 1B), cancer invasion (Fig. 1C), lymph node metastasis (Fig. 1D) and cancer diameter (Fig. 1E), while there was no significant correlation between ZFAS1 expression level and gender, age, cancer differentiation, or CEA and CA199 levels (summarized in Table 1).

Figure 1. LncRNA ZFAS1 was up-regulated in gastric cancer tissues and was associated with TNM stage, cancer invasion, lymph node metastasis and cancer diameter. (A) ZFAS1 was highly expressed in GC tissues compared to paired non-tumor tissues (P < 0.0001). (B) ZFAS1 expression levels in different clinical stage of gastric cancer (P = 0.0053). (C) Up-regulation of lncRNA ZFAS1 was associated with tumor invasion (P = 0.0043). (D) Up-regulation of lncRNA ZFAS1 was associated with lymph node metastasis (P = 0.0059). (E) Up-regulation of lncRNA ZFAS1 was associated with cancer diameter (P = 0.0209). The relative expression level was calculated using 2^-ΔCT method. **, P < 0.01, *, P < 0.05. Statistical analysis were conducted by Wilcoxon test (A) and Mann-Whitney U test (B,C,D and E).

Table1. Correlation between ZFAS1 level in tissues and clinicopathologic of GC

Feature		No. of cases	Mean ± SD	P
					Age
≥60	34		0.0787 ± 0.0526	0.2621
<60	32		0.1196 ± 0.1174
Gender
Male	38		0.0814 ± 0.0668	0.5168
Female	28		0.0676 ± 0.1158
Cancer location				0.9979
Distal	18		0.1008 ± 0.0976
Middle	33		0.0885 ± 0.0861
Proximal	35		0.0953 ±0.0998
Diameter (cm)
≥5	25		0.1220 ± 0.0959	0.0209
<5	41		0.0842 ± 0.0871
Differentiation
Well	6		0.0707 ± 0.0443	0.2012
Moderate	18		0.0707 ± 0.0443
Poor	42		0.1140 ± 0.1079
TNM stage
0,Iand II	24		0.0790 ± 0.1060	0.0053**
III,IV	42		0.1097 ± 0.0817
Invasion
T0-T3	6		0.0223 ± 0.0273	0.0043**
T4	60		0.0293 ± 0.0249
Lymph-node metastasis
N0	20		0.0823 ± 0.1145	0.0059**
N1-N3	46		0.1061 ± 0.0792
Distant metastasis
M0	61		0.0972 ± 0.0930	0.4917
M1	5		0.1144 ± 0.0802
CEA
Positive	11		0.0855 ± 0.0485	0.8359
Negative	55		0.1011 ± 0.0982
CA19-9
Positive	15		0.0947 ± 0.0707	0.8886
Negative	51		0.0996 ± 0.0976

ZFAS1 was highly expressed in the plasmas of preoperative patients than paired postoperative plasmas and the healthy controls

In addition to expression in tissues, we also examined ZFAS1 expression in seventy-seven paired plasma samples derived from GC patients and sixty plasma samples from healthy controls. There were no significant differences between the GC cases with the healthy controls in the frequency of age distribution (P = 0.2114). The mean age was 57.6 in the patient group and 54.9 in the healthy group. In consistent with our findings in tissues, we found that the ZFAS1 expression in preoperative GC plasmas was up-regulated as compared with the paired postoperative ones and the healthy controls (Fig. 2A). Similar to the results in tissues, circulating ZFAS1 level was highly correlated with TNM stage (Fig. 2B), lymph node metastasis (Fig. 2C) and distant metastasis (Fig. 2D), while there was no correlation with other clinicopathologic factors, such as age, gender, cancer differentiation, or CEA and CA199 levels (summarized in Table 2), indicating ZFAS1 might serve as a potential diagnostic maker for GC.

Figure 2. Relative expression level of lncRNA ZFAS1 in 77 paired GC plasmas and 60 healthy controls. (A) ZFAS1 was highly expressed in GC patients compared to healthy controls (P < 0.0001) and patients after surgical treatment (P < 0.0001). (B-D) ZFAS1 was highly expressed in patients with high TNM stage (B), depth invasion (C), and distant metastasis (D). The relative expression level was calculated using 2^-ΔCT method. **, P < 0.01, *, P < 0.05. Statistical analyses were conducted by Wilcoxon test (A) and Mann-Whitney U test (B,C,D and E).

Table 2. Correlation between ZFAS1 level in plasma and clinicopathologic of GC

Feature	No. of cases	Mean ± SD	P
				Age
≥60	37	0.0227 ± 0.0242	0.3841
<60	40	0.0278 ± 0.0282
Gender
Male	46	0.0258 ± 0.0271	0.9486
Female	31	0.0248 ± 0.0255
Cancer location
Distal	17	0.0314 ± 0.0298	0.5767
Middle	25	0.0221 ± 0.0307
Proximal	35	0.0264 ± 0.0227
Diameter(cm)
≥5	18	0.0318 ± 0.0324	0.3493
<5	59	0.0234 ± 0.0242
Differentiation
Well	7	0.0214 ± 0.0159	0.135
Moderate	24	0.0185 ± 0.0214
Poor	46	0.0296 ± 0.0293
TNM stage
0,Iand II	31	0.0216 ± 0.0278	0.0052*
III,IV	36	0.0297 ± 0.0242
Invasion
T0-T3	43	0.0223 ± 0.0273	0.0285*
T4	34	0.0293 ± 0.0249
Lymph-node metastasis
N0	44	0.0223 ± 0.0243	0.2937
N1-N3	33	0.0279 ± 0.0279
Distant metastasis
M0	73	0.0238 ± 0.0254	0.0154*
M1	4	0.0548 ± 0.0292
CEA
Positive	13	0.0265 ± 0.0229	0.5121
Negative	64	0.0251 ± 0.0271
CA19-9
Positive	11	0.0283 ± 0.0360	0.4397
Negative	66	0.0249 ± 0.0247

ZFAS1 was relatively stable in peripheral blood and plasma for conventional detection methods

To investigate the potential application for using ZFAS1 as a potential plasma biomarker for GC, we first tested the stability of ZFAS1 in plasma and the whole blood. The whole blood was aliquoted and incubated in different conditions. Plasma was subjected to six cycles of freezing and thawing to mimic potential clinical detection scenario. Based on real-time RT-PCR analysis, the expression level of ZFAS1 was relatively stable when incubate at room temperature for 6h (P = 0.6668), while significant reduction was observed when stored over 12h (P = 0.0071). When we stored plasma samples at 4°C, we found no statistical differences when the samples were incubated for 12h (P = 0.9318) or 24h (P = 0.0713), until incubated for 3d (P < 0.0001) (Fig. 3A). Plasma ZFAS1 level was stable when stored at -80°C and the freeze-thaw process had no obvious effects on the stability of ZFAS1 (Fig. 3B). Finally, we tested whether ZFAS1 could be used as a marker of gastric cancer. We used the healthy plasma as a control to produce a ROC curve, which has been established as a standard to determine the value of biomarker. The area under the ROC curve was 0.727 (95% CI=0.642–0.813, P < 0.001; Fig. 3C), with a sensitivity of 0.766 and specificity of 0.639. The Youden index was 0.406. Thus, our results provide evidence that plasma ZFAS1 level can be used to distinguish GC patients from controls.

Figure 3. ZFAS1 is suitable for biomarker testing and may serve as a novel biomarker for GC. (A) The expression level of lncRNA ZFAS1 remained stable when incubated at room temperature for 6h and at 4°C for 12h. ZFAS1 was stable when stored at -80°C. (B) ZFAS1 remained relatively stable when treated with freeze-thaw cycles. (C) The ROC curve analysis for the diagnostic value of lncRNA ZFAS1 in GC (AUC = 0.727, sensitivity: 0.766; specificity: 0.639).

ZFAS1 was up-regulated in GC cell lines compared in normal gastric cell line

To study the cellular and molecular mechanisms underlying ZFAS1 mediated GC progression, we investigated the expression levels of ZFAS1 in three GC cell lines (AGS, SGC-7901 and BGC-823) and one normal cell line GES-1. The result showed that ZFAS1 was highly expressed in GC cell lines than that in normal cell line (P < 0.05) (Fig. 4), thus, we can use these cell lines to perform more mechanistic studies.

Figure 4. Evaluation of ZFAS1 in three GC cell lines compared to a normal gastric cell line GES-1. The expression level of ZFAS1 in gastric cancer cell lines (AGS, SGC-7901, and BGC-823) were higher than that in human gastric epithelial cell line GES-1, *, P < 0.05. The relative expression level was calculated using 2^-ΔΔCt method.

ZFAS1 was down-regulated in simulated CTCs compared to un-captured cells and was positively correlated to the mRNA expression levels of mesenchymal markers and negatively correlated to epithelial markers

EMT and CTCs have been shown to play an important role in gastric cancer progression and metastasis [21]. EpCAM (epithelial cell adhesion molecule) is a transmembrane glycoprotein which is over expressed in numbers of solid tumor cells and is absent from hematologic cells. To explore the potential role of ZFAS1 in CTCs and EMT process, we simulated CTCs by mixing tumor cells with peripheral blood. By using biotinylated epithelial cell adhesion molecule antibody (anti-EpCAM) and streptavidin coated magnetic particles, CTCs were divided into two groups (bind and un-bind groups). The captured cells were the so-called simulated CTCs with high expression of EpCAM, while uncaptured cells express no or low EpCAM. To determine the molecular signatures of two groups of cells, we tested the levels of ZFAS1 and the EMT markers, CDH1, CDH2, Snail, ZEB1, Vimentin, MMP14 and Twist by qRT-PCR. We found a decreased expression of the epithelial markers CDH1, EpCAM, and increased expression of the mesenchymal markers CDH2, Vimentin, ZEB1, Snail, MMP14 and Twist in un-captured cells in gastric cancer cell lines AGS, SGC7901 and BGC-823 (Fig. 5). More importantly, expression of ZFAS1 is upregulated in un-captured cells as well, indicating ZFAS1 is enriched in mesenchymal type of cancer cells.

Figure 5. Evaluation of lncRNA ZFAS1 and EMT markers expression level in CTCs simulated by three GC cell lines. ZFAS1 level was positively correlated with mesenchymal markers and negatively correlated with epithelial markers in three GC cell lines simulated by AGS (A), SGC-7901 (B), and BGC-823 (C).

Correlation between ZFAS1 and Twist in cancer tissues derived from GC patients

To further investigate the relationship between ZFAS1 and the EMT associated factors, we randomly selected 40 GC tissues to detect the expression level of Twist, a key EMT factor, with immunohistochemical staining. Then, we analyzed the correlation between ZFAS1 and Twist, and found that ZFAS1 expression is positively correlated with Twist. Spearman correlation analysis showed the correlation coefficient was 0.394(P < 0.05) (Fig. 6). Thus, we demonstrated, in human cancer tissues, that expression of ZFAS1 is closely correlated with EMT process of GC, which can potentially be targeted for treatment of GC.

Figure 6. The correlation between ZFAS1 level and Twist. ZFAS1 level was positively correlated to the expression level of Twist. The correlation coefficient was 0.394 (P < 0.05).

Discussion

LncRNAs play important roles in the development of gastric cancer [22]and can be used as a marker for GC diagnosis [23]. In this study, fourteen lncRNAs were selected from the database and literatures to test their potential roles in GC. We found that ZFAS1 is up-regulated in the GC tissues compared with the paired normal tissues. Furthermore, we found the correlation between the ZFAS1 level and the clinicopathological characteristics of the 66 gastric cancer patients and found that the expression level of ZFAS1 is positively correlated to the TNM stage, cancer invasion, lymph node metastasis and cancer diameter.

Circulating biomarkers including lncRNAs are one of the most promising means of diagnosis for the easy access of serum or plasma, and recent studies have showed that lncRNAs are quite stable in plasma or serum [13,23], while the precise mechanism is still unclear. A possible explanation is that they could be packaged and protected by several microparticles, such as exsomes [24]. Consistently, our results indicated that lncRNA ZFAS1 was sufficiently stable in plasma. More importantly, we detected the expression level of ZFAS1 in 77 paired plasma samples of GC patients obtained before and after surgery. We found that the expression level of ZFAS1 was higher in preoperative plasmas than postoperative plasmas and the healthy controls. Also plasma ZFAS1 level was associated with TNM stage, cancer invasion, lymph node metastasis and distant metastasis. There exist litter differences in the subgroup analyses between tissue and plasma because some samples were too small and were all used for clinical application. The results indicated that ZFAS1 expression level had a significant predictive value for gastric cancer, and the AUC value for discriminating GC patients from healthy controls was 0.760.

As tumor cells proliferate, they promote angiogenesis and invade into the bloodstream, survive, spread, extravagate, escape into the parenchyma and develop into distant metastasis [25]. CTCs released from primary solid tumors into the vascular circulation are now considered a real-time “liquid biopsy” reflecting the progression of the disease [17]. CTCs invaded into the bloodstream is one of the leading causes for the distant metastasis of cancer [26], and these CTCs may act as a prognostic marker [27].

Recent studies have shown that circulating nuclear acids including miRNAs and lncRNAs may act as a marker for CTCs. Madhavan’s [19] study showed that circulating miRNAs can predict the CTC status in patients with metastatic breast cancer. Ortega’s [28] study showed that miRNA-21 may act as a good marker for EMT phenotype CTCs. Also, Yuan’s [16] study showed that lncRNA-ATB may increase CTC numbers, indicated that lncRNA may also act as a CTC marker.

EMT plays a crucial role in the progress of cancer invasion and metastasis through a variety of mechanisms [15]. Numbers of transcription factors may induce EMT, including zinc finger proteins of the SNAIL superfamily Snail1 and Snail2, zinc finger and E-box binding proteins of the ZEB family ZEB1 and ZEB2, and he TWIST bHLH proteins (TWIST and TWIST2) [29]. These factors may repress the gene transcription by binding to the promoter region of genes involved in cell-cell adhesion such as E-cadherin (CDH1) [30–33], and allow dissociation of cancer cells from the epithelial matrix [14]. Also, the ZEB protein may increase the expression of matrix metalloproteinases (MMPs) [34] which may promote the invasion and metastasis by degrading the extracellular matrix (ECM) proteins.

Recent studies have shown that EMT markers such as Twist [35] are overexpressed in CTCs, while CTCs expressed low but detectable level of epithelial markers such as keratins, MUC1, EpCAM and/or CDH [36,37]. Also Lindsay’s study [37] showed that patients with Vimentin-positive CTCs has a reduction in OS (overall survival). Angela’s study [38] showed that patients with EMT-positive CTCs had a short PFS (perfect forward secrecy).

During EMT, cells lose their epithelial characteristics and transfer to mesenchymal phenotype [15], change their shape, motility, and adhesion through numerous mechanisms [39], resulting in increased invasive and migratory capability. Thus, elevated numbers of the tumor cells leave the primary parenchyma and enter into the systemic circulations during cancer metastasis [40]. During the progress, several epithelial markers such as EpCAM and CDH1 decreased, while the mesenchymal makers such as CDH2, Snail, ZEB1, Vimentin and Twist increased [14,15]. EpCAM is up-regulated in numerous solid tumor cells and is absent from hematologic cells [41], so that EpCAM is used as a surface marker to capture circulating tumor cells [42,43]. The epithelial surface markers EpCAM and CDH1 may down-regulated after EMT [44,45]. In this study, three representative cell lines of GC were chosen and ZFAS1 was investigated with a non-tumor gastric cell line as control. The data showed that ZFAS1 was highly expressed in GC cell lines (AGS, SGC7901 and BGC-823) compared in non-tumor gastric cell line GES-1. To investigate the mechanism, we simulated CTCs by mixing tumor cells into peripheral blood. Cells were divided into two groups by biotinylated EpCAM antibody and streptavidin coated magnetic particles. Cells captured by magnetic particles expressed a high level of EpCAM. We compared the expression level of ZFAS1 and EMT markers in the two groups, we found that the expression level of ZFAS1 was positively related with the mesenchymal markers CDH2, Vimentin, ZEB1, Snail, MMP14 and Twist, and was negatively related with the epithelial marker CDH1 and EpCAM. These results indicated that ZFAS1 may play an important role in the progress of EMT, which may induce the metastasis of gastric cancer, and ZFAS1 may act as a diagnostic marker for gastric cancer and a mesenchymal marker for EMT and CTCs (Fig.7).

Figure 7. The potential role of lncRNA ZFAS1 in GC. ZFAS1contributed to the regulation of EMT in GC progression and thus may serve as a potential diagnostic marker for GC.

A previous study [46] showed that ZFAS1 is down-regulated in breast tumors compared with normal tissues and the silencing of ZFAS1 in mammary epithelial cell line increased cellular proliferation and differentiation, indicated that ZFAS1 may act as a tumor suppressor gene and the mechanisms were unknown. While another study [47] showed that ZFAS1 transcripts are highly expressed in most HCC tissues compared with the paired non-tumor tissues and the upregulation of ZFAS1 promoted the metastasis through a miR-150 dependent manner. ZFAS1 act as a miR-150 sponge and inhibited miR-150, leading to the activating of ZEB1, MMP14 and MMP16. Recent study showed that ZFAS1 may destabilize p53 and interact with CDK1/cyclin B1 complex leading to cell cycle progression and inhibition of apoptosis [48]. Our results showed that ZFAS1 is up-regulated in GC tissues compared with the paired normal tissues, and plasma ZFAS1 level is down-regulated after operation, and the expression level of ZFAS1 is positively related with mesenchymal markers and negatively related with epithelial markers. Thus, it is possible that ZFAS1 plays different roles in different type of cancers, and the mechanism requires further investigation.

Author Contributions

Hu zhou, Fubing Wang and Jiancheng Tu conceived and designed the experiments. Shengwei Ye, Qian Tan, Hao Chen, Shanshan Chen and Wei Jing contributed to the sample collection. H performed the research, conducted the data analyses and wrote the manuscript. Chunzi Liang revised the manuscript. Jiancheng Tu revised the article and coordinated the research team. Mingxia Yu contributed the technical assistance. All authors have read and approved the final manuscript.

Acknowledgements

Taken together, the expression of ZFAS1 was significantly increased in GC tumor tissues and plasma, and may decrease after surgery. ZFAS1 may act as a marker for gastric cancer diagnosis, and a marker for EMT and CTCs. ZFAS1 may promote cancer invasion and metastasis by regulating EMT and CTCs. However, the molecular mechanisms still require further investigation.

Funding

This study was supported by the National Program on Key Basic Research Project of China (973 Program, 2012CB720605) and the Natural Science Foundation of Hubei Province of China (2012FFB01703). This work was also funded by “351 talent project (Luojia Young Scholars)” of Wuhan University and Wuhan City health and family planning medical talented youth development project.

Conflicts of Interest

The authors declare no conflict of interest.

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