Long non-coding RNAs (lncRNAs) have been reported to play a vital role in non-small-cell lung cancer (NSCLC). ZEB1-AS1 overexpression predicts a poor prognosis in osteosarcoma and colorectal cancers. In the current study, we determined the clinical significance and prognostic value of ZEB1-AS1 in patients with NSCLC. The expression of ZEB1-AS1 and inhibitor of differentiation-1 (ID1) was measured using qRT-PCR and Western blot. Cell growth, migration, and invasion were determined using colony formation assays, Transwell assay, and flow cytometry, respectively. Tumor growth was determined with a xenograft model. ZEB1-AS1 was significantly up-regulated in NSCLC tissues compared with normal samples. ZEB1-AS1 overexpression was significantly associated with advanced tumor, lymph node, and metastases (TNM) stage and tumor size, as well as poorer overall survival. Moreover, ZEB1-AS1 knockdown inhibited NSCLC cell proliferation and migration, and promoted cell apoptosis. In addition, a chromatin immunoprecipitation assay revealed that ZEB1-AS1 interacted with STAT3, thereby repressing ID1 expression. Furthermore, rescue experiments indicated that ZEB1-AS1 functioned as an oncogene partly by repressing ID1 in NSCLC cells. Taken together, our findings indicate that ZEB1-AS1 serves as a promising therapeutic target to predict the prognosis of NSCLC.
Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small-cell lung cancer (NSCLC) accounts for 70–85% of all lung cancers [1,2]. Metastasis has usually occurred when most patients are diagnosed as NSCLC, which is associated with a poor prognosis [3,4]. Currently, metastatic diseases are incurable; therefore, new treatments for lung cancer are urgently needed [5,6]. The molecular mechanisms underlying the occurrence, metastasis, and progression of NSCLC are largely unknown [7–9]. Less than 2% of the total genome is transcribed into protein-coding mRNA, whereas approximately 90% of the human genome is transcribed into non-coding RNAs (ncRNAs) . Therefore, ncRNA provides a promising target for the development of new cancer therapies.
Long non-coding RNAs (lncRNAs) are functional ncRNAs with a length of >200 nt, and are transcribed in the eukaryotic genome . Previous studies have shown that antisense transcripts regulate transcription and translation of sense genes. For example, XIST is a lncRNA from inactive X that plays an important role in inactivating mammalian X and is regulated by complex interactions with other lncRNAs, such as Tsix . The antisense lncRNA, ZEB1-AS1, is physically adjacent to ZEB1, which can positively regulate the expression of ZEB1, promote tumor progression, and predict the poor prognosis of prostate cancer ; however, the function and mechanism of ZEB1-AS1 in other tumors are largely unknown. Therefore, we determined the effects and mechanism of ZEB1-AS1 on ZEB1 and the downstream molecules in NSCLC cells.
In the present study, we demonstrated that the abnormal expression of ZEB1-AS1 promoted proliferation and migration of NSCLC cells, partly by down-regulating the expression of inhibitor of differentiation-1 (ID1) at the transcriptional level in combination with STAT3.
Materials and methods
Forty-eight patients with NSCLC and 26 patients with pneumonia who were diagnosed between January 2011 and October 2013 in the Department of Respiratory and Critical Medicine at the First Affiliated Hospital of Zhengzhou University were enrolled in the present study. The patients who had received radiotherapy or chemotherapy prior to surgery were excluded. Tissue samples were collected and immediately frozen in liquid nitrogen and stored at −80°C. The clinicopathologic features of the patients are summarized and recorded in Table 1, including tumor stage, lymph node, and metastasis (TNM). The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University and all patients signed the written informed consent.
|Characteristics||ZEB1-AS1 (−)||ZEB1-AS1 (+)||χ2 test||P-value|
|Lymph node metastasis|
|Smoking habits (case-years)|
|Characteristics||ZEB1-AS1 (−)||ZEB1-AS1 (+)||χ2 test||P-value|
|Lymph node metastasis|
|Smoking habits (case-years)|
Five NSCLC cell lines (PC9, H1299, H1975, H460, and A-427) and a normal human bronchial epithelial cell line (16HBE) were purchased from the Shanghai Cell Bank at the Chinese Academy of Sciences (Shanghai, China). A-427, H1975, and H1299 cells were cultured in RPMI-1640 medium (Invitrogen, Shanghai, China) and 16HBE, H1299, and PC9 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Shanghai, China) with 10% fetal bovine serum, 100 IU/ml of penicillin, and 100 μg/ml of streptomycin (Invitrogen, Carlsbad, CA, U.S.A.) in a humidified atmosphere of 5% CO2 at 37°C.
Total RNA (500 ng) was extracted from the cell lines or tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.). Total RNA was reverse-transcribed using random primers according to the standard protocol of the PrimeScript RT reagent kit (TaKaRa, Dalian, China). The expression of ZEB1-AS1 was detected according to the manufacturer’s protocol for SYBR Premix Ex Taq (TaKaRa). The primer sequences were illustrated as follows: ZEB1-AS1, F: 5′-TGAGCCGATCGGAACGTC-3′, R: 5′-GTGCAGAGTGCCGGGT-3′; ID1, F: 5′-CTCACCGGCTATCCGG-3′, R: 5′-CATGAGGCTGTTTATGTGCCA-3′; GAPDH, F: 5′-CCTGTGTGACCTGCGGACT-3′, 5′-GCTGGGGATGGATGGGTGTC-3′. The relative expression was measured by 2−ΔΔCt method. Data collection was performed on an ABI 7500 Real-time PCR System (Applied Biosystems, Foster, CA, U.S.A.).
NSCLC cells were transfected with siRNAs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and incubated for 48 h prior to qPCR and Western blot analyses. Three individual ZEB1-AS1 siRNAs (si-ZEB1-AS1-1, -2, and -3) and a scrambled negative control siRNA (si-NC) were purchased from Invitrogen.
Lipofectamine 2000 (Invitrogen, U.S.A.) was used to transfect ZEB1-AS1 siRNA into NSCLC cells. After incubation for 48 h, qPCR and Western blot analysis were carried out. Three ZEB1-AS1 siRNAs (si-ZEB1-AS-1, -2, and -3) and negative control siRNA (si-NC) were purchased from Invitrogen (U.S.A.).
After si-ZEB1-AS1 transfection for 48 h, H1299, and A-427 cells were digested with trypsin and harvested. Cell apoptosis was quantified using a FITC Annexin V Apoptosis Detection Kit II (BD Biosciences, Lake Franklin, NJ, U.S.A.). According to the instructions, double-staining of FITC Annexin V and propidium iodide (PI) was performed. The cells were divided into living, dead, early apoptotic, and apoptotic cells. Then, the relative proportion of the early apoptotic cells in each experiment was compared with the control transfection. A CycleTEST PLUS DNA kit (BD Biosciences) was used for cell cycle analysis and the cells were stained by PI according to the protocol. FACScan flow cytometry was used to analyze the cells.
The protocols were carried out as described previously . The cell protein lysates were separated by sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis and transferred to 0.22-μm PVDF membranes (Millipore, Billerica, MA, U.S.A.). Then, the membranes were incubated overnight at 4°C with anti-ID1, anti-STAT3, and GAPDH (Sigma, CA, U.S.A.). After washing, secondary antibody (Pierce, IL, U.S.A.) was added to the system. ECL chromogenic substrates were used for quantitative measurement by Quantity One software (Bio-Rad, Hercules, CA, U.S.A.). GAPDH was used as an internal reference control. Anti-ID1 and anti-STAT3, anti-SUZ12 antibodies were purchased from Cell Signaling Technology (Boston, MA, U.S.A.).
Transwell assays were conducted to evaluate cell migration and invasion abilities as described previously . Cells (2 × 105/ml) transfected for 48 h were migrate through an 8-μm pore membrane or invade through a Matrigel-coated membrane. Migrated and invasive cells were stained and counted under a light microscope.
The nuclear and cytoplasmic components were separated according to the instructions of a PARIS kit (Life Technologies, Carlsbad, CA, U.S.A.), as previously reported .
RNA immunoprecipitation was performed using an EZ-Magna RIP Kit (Millipore). According to the protocols provided by the manufacturer’s instructions, H1299, and A-427 cells were lysed using RIP lysis buffer, and a 100-μl whole cell extraction was incubated with magnetic beads conjugated with STAT3, SUZ12, or control IgG antibodies (Millipore). Finally, qRT-PCR of RNA immunoprecipitation was performed using specific primers to detect ZEB1-AS1 enrichment.
A-427 and H1299 cells were treated with formaldehyde and incubated for 10 min to produce DNA-protein cross-linking. Cell lysates were then subjected to ultrasound treatment to generate 200–300 bp chromatin fragments. Immunoprecipitation was performed with STAT3- or H3K27me3-specific antibodies (Millipore) or IgG antibody (control). The precipitated chromatin DNA was obtained and analyzed by qRT-PCR.
Tumor xenograft model in nude mice
A-427 cells (1 × 108/ml) stably transfected with sh-ZEB1-AS1 or empty vector were subcutaneously inoculated into 4-week-old male BALB/c nude mice (Chinese Academy of Sciences, Shanghai, China). The tumor volume and weight were measured every 3 days for 3 weeks. The volume of the tumor was calculated as follows: V = ab2/2, where a = length of tumor and b = width of tumor. Twenty-one days after injection, the mice were sacrificed and the tumor was measured.
In situ hybridization
In situ hybridization was performed according to the method described previously . The stained slides were scored independently by two observers who did not know the clinicopathologic information of the patients. In the case of different opinions, the agreement was reached through careful discussion among the evaluators. The percentage of positive cells was classified according to four grades (percentages), as follows: <10% (grade 0); 10–20% (grade 1); 21–50% (grade 2); and >50% (grade 3). The staining intensity was classified according to the following four grades (intensity score): no staining (grade 0); light brown (grade 1); brown (grade 2); and dark brown (grade 3). The following formula was used to determine ZEB1-AS1 staining: total score = percentage score × intensity score. Total scores <2 or ≥2 were defined as negative or positive staining, respectively.
SPSS (version 24.0; SPSS, Inc., Chicago, IL, U.S.A.) was used for data analysis. Data are presented as the mean ± S.D. of at least three independent experiments. Differences among the groups were analyzed using one-way analysis of variance followed by Tukey’s test for multiple comparisons. A P<0.05 was considered significantly different.
ZEB1-AS1 is up-regulated in human NSCLC tissues and correlates with poor prognosis of NSCLC patients
First, we used IHC to detect the expression of ZEB1-AS1 in lung tissues of patients with NSCLC and pneumonia. Brown staining was demonstrated in the cytoplasm and the nucleus of tumor cells of NSCLC tissues, suggesting that ZEB1-AS1 was positive (Figure 1A). Positive ZEB1-AS1 staining was observed in 31 of 48 NSCLC specimens, while only 3 of 26 pneumonia tissues showed positive ZEB1-AS1 staining. The expression of ZEB1-AS1 in NSCLC patients was significantly higher than the control cohort (χ2 = 11.63, P=0.01). The relationship between the expression of ZEB1-AS1 and the clinicopathologic characteristics of NSCLC patients showed that positive ZEB1-AS1 expression in NSCLC patients was related to higher TNM stage (χ2 = 6.513, P=0.030) and advanced stage (stage I, 33.5% positive rate; stage II, 40.6% positive rate; stage III, 68.7% positive rate); however, no correlation existed between ZEB1-AS1 and lymph node metastasis, tumor size, gender, age, smoking, and histology (Table 1). Kaplan–Meier survival analysis showed that OS in patients with ZEB1-AS1 positive staining was significantly shorter than that patients with ZEB1-AS1 negative staining (log-rank test, P=0.037; Figure 1B). In addition, Cox multivariate regression analysis showed that overexpression of ZEB1-AS1 suggested a poor prognosis (HR = 2.133, 95% CI: 1.035–4.086; Table 2).
ZEB1-AS1 overexpression in NSCLC predicts a poor prognosis of patients with NSCLC
|P||HR||95% CI for HR|
|Lymph node metastasis||0.071||0.483||0.246||1.056|
|P||HR||95% CI for HR|
|Lymph node metastasis||0.071||0.483||0.246||1.056|
Regulation of ZEB1-AS1 expression in NSCLC cells
To study the function of ZEB1-AS1 in NSCLC cells, qRT-PCR was performed to detect the expression of ZEB1-AS1 in five human NSCLC cell lines. The expression of ZEB1-AS1 was significantly up-regulated in H1299 and A-427 cell lines compared with the 16HBE cell line (Figure 2A). Furthermore, we transfected three different ZEB1-AS1 siRNAs into two NSCLC cell lines. After transfection for 48 h, qRT-PCR analysis showed that si-ZEB1-AS1-2 and si-ZEB1-AS1-3 had higher interference efficiency than si-ZEB1-AS1-1 (Figure 2B). Therefore, si-ZEB1-AS1-2 and -3 were chosen for the following experiments.
Impact of ZEB1-AS1 knockdown on proliferation of NSCLC cells
ZEB1-AS1 knockdown inhibits cell proliferation and induces cell apoptosis and cell cycle arrest in NSCLC cells
The MTT assay showed that the growth of H1299 and A-427 cells was dramatically decreased after si-ZEB1-AS1 transfection (Figure 2C). Similarly, colony formation assay showed that ZEB1-AS1 knockdown reduced the colony formation of H1299 and A-427 cells (Figure 2D). These results were also confirmed by (EdU [red])/DAPI (blue) immunostaining, indicating that ZEB1-AS1 knockdown significantly reduced the proliferation rate (Figure 2E). Flow cytometry analysis results showed that the apoptotic ratio was significantly higher in the ZEB1-AS1 siRNA transfection groups than the negative control group (Figure 3A,B). In addition, ZEB1-AS1 knockdown in H1299 and A-427 cells promoted cell cycle arrest (Figure 3C,D).
Effect of ZEB1-AS1 on apoptosis and migration of NSCLC cells
ZEB1-AS1 expression suppresses NSCLC cell migration
To study the effect of ZEB1-AS1 knockdown on NSCLC cell migration, we performed the Transwell assay. Our results showed that inhibition of ZEB1-AS1 decreased H1299 and A-427 cell migration compared with the control group (Figure 3E,F). These data suggested that ZEB1-AS1 promoted the migration of NSCLC cells.
ZEB1-AS1 knockdown inhibits NSCLC tumorigenesis in vivo
To further explore whether ZEB1-AS1 expression affects tumorigenesis in vivo, A-427 cells stably transfected with sh-ZEB1-AS1 or empty vector were inoculated into nude mice. Three weeks after injection, xenograft tumors occurred at the injection site in all mice, and the tumor size in the sh-ZEB1-AS1 group was significantly smaller than the control group (Figure 4A). The average tumor volume and weight in the sh-ZEB1-AS1 group were significantly less than the control group (Figure 4B,C). The expression of ZEB1-AS1 in the sh-ZEB1-AS1 group was lower than the control group (Figure 4D). Ki-67 staining intensity in A-427/sh-ZEB1-AS1 cells was lower than the empty vector-transfected group (Figure 4E).
Effect of ZEB1-AS1 on NSCLC tumorigenesis in vivo
ZEB1-AS1 affects ID1 transcription by interacting with STAT3 in NSCLC cells
Fractionation assay showed that the expression of ZEB1-AS1 in the nucleus was higher than the cytoplasm of H1299 and A-427 cells (Figure 5A), suggesting that ZEB1-AS1 may act as a transcriptional regulator. We selected STAT3 and SUZ12 to carry out the RNA immunoprecipitation assay and confirmed that ZEB1-AS1 directly bound to STAT3 in H1299 and A-427 cells (Figure 5B). To identify the potential targets associated with cell proliferation of NSCLC, we detected the expression of ID1 in H1299 and A-427 cells with ZEB1-AS1 knockdown. ID1 protein was negatively correlated with ZEB1-AS1 knockdown in H1299 and A-427 cells (Figure 5C). Inhibition of STAT3 increased the expression of ID1 mRNA and protein (Figure 5D–F). The chromatin immunoprecipitation results showed that STAT3 bound to the ID1 promoter region, while ZEB1-AS1 knockdown reduced the binding between STAT3 and the ID1 promoter region (Figure 5G). In addition, ZEB1-AS1 expression was negatively correlated with ID1 expression in 18 paired NSCLC and adjacent non-tumor lung tissues (P=0.021, Figure 5H). These data suggest that ZEB1-AS1 might partially promote NSCLC cell proliferation by binding to STAT3 to silence ID1 transcription at the epigenetic level.
ZEB1-AS1 interacted with STAT3 and regulated ID1 expression in NSCLC cells
Inhibition of ID1 may be involved in the carcinogenic function of ZEB1-AS1
To verify the effect of ID1 on NSCLC cell proliferation, we knocked down the expression of ID1 in A-427 cells (Figure 6A). Flow cytometry analysis showed that G0/G1 cell cycle arrest occurred in the A-427 cells transfected with si-ID1 (Figure 6B). Cell viability of NSCLC cells increased significantly after ID1 knockdown (Figure 6C,D). MTT and colony formation assays showed that cell proliferation in A-427 cells co-transfected with si-ZEB1-AS1 and si-ID1 were significantly increased compared with cells transfected with siZEB1-AS1 alone (Figure 6E,F). In conclusion, ZEB1-AS1 promotes NSCLC cell proliferation in part by down-regulating ID1.
Down-regulation of ID1 promotes A-427 cell proliferation and ZEB1-AS1 negatively regulates ID1 expression
Recent studies have indicated that lncRNAs, such as ANRIL, SNHG7, and TUG1, play an important role in lung cancer [17–19]. It has been reported that lncRNA ZEB1 antisense 1 (ZEB1-AS1) is up-regulated in hepatocellular carcinoma, esophageal squamous cell carcinoma, and glioma and is associated with a poor prognosis [20,21]. A previous study also showed that ZEB1-AS1 overexpression is dramatically correlated with advanced TNM stage, lymph node metastasis, and poorer overall survival in gastric cancer patients ; however, the contribution of the aberrant expression of ZEB1-AS1 to NSCLC and the underlying molecular mechanism is unclear. Accumulating evidence has focused on the role of Id1 in different tumor types [14,23]. In breast cancer, Id1 belongs to a lung cancer metastasis signature . Another study has shown that Id1 is required for tumor initiation, both in the context of primary tumor formation and during metastatic colonization of the lung microenvironment .
In the present study, we found that ZEB1-AS1 was overexpressed in NSCLC cells. The overexpression of ZEB1-AS1 in NSCLC patients was positively correlated with poor prognosis. Furthermore, our results indicated that ZEB1-AS1 expression has predictive value and could serve as a prognostic marker for NSCLC patients. In addition, we found that up-regulation of ZEB1-AS1 predicts poor prognosis of colorectal cancer and hepatocellular carcinoma. In the function loss or acquisition assay, ZEB1-AS1 down-regulation significantly inhibited cell proliferation and migration in vitro, as well as tumor growth in vivo. Conversely, ectopic expression of ZEB1-AS1 induced malignant tumor cell behavior. In conclusion, these data suggest that ZEB1-AS1 plays an important role as an oncogene in the genesis and progression of NSCLC. In general, lncRNAs affect the behavior of cancer cells by regulating the expression of target genes. In the present study, we observed the expression of important cell cycle factors and growth regulators in NSCLC cells after ZEB1-AS1 knockdown, and identified ID1 as a new target for ZEB1-AS1 in NSCLC cells.
In conclusion, the results of the present study suggest that ZEB1-AS1 is up-regulated in NSCLC tissues and associated with a poor prognosis in NSCLC patients. ZEB1-AS1 can promote cell proliferation and migration of NSCLC by silencing ID1 expression. A better understanding of the molecular etiology of ZEB1-AS1 in lung cancer will be helpful to the development of lncRNA-based cancer diagnosis and treatment. Our research provides a new perspective for ZEB1-AS1 as a non-coding oncogene in NSCLC tumorigenesis. Therefore, ZEB1-AS1 is a new early diagnostic marker and target for NSCLC treatment; however, other possible mechanisms of ZEB1-AS1 involvement in NSCLC cell function remain to be comprehensively studied.
To achieve better therapeutic outcomes and improve the survival of NSCLC patients, it is essential to understand the underlying mechanisms.
The findings described here shed light on the functional role of ZEB1-AS1, which is up-regulated in NSCLC and critically regulates progression and metastasis by repressing ID1 in NSCLC cells.
These data may provide a new molecular mechanism for the treatment of NSCLC metastasis, which may be a promising therapeutic new target in NSCLC.
J.J.J. carried out the experiments. W.H.Q. and S.J.M. carried out the experiments and drafted the manuscript. N.R. and L.Y.H. participated in the design of the study and performed the statistical analysis. W.J. conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
The authors declare that there are no competing interests associated with the manuscript.
The authors declare that there are no sources of funding to be acknowledged.
inhibitor of differentiation-1
long non-coding RNA
non-small-cell lung cancer
quantitative Real-time PCR
signal transducer and activator of transcription 3
tumor stage, lymph node, and metastasis
zinc finger E-box binding homeobox 1 antisense RNA 1