ZNF674-AS1 antagonizes miR-423-3p to induce G0/G1 cell cycle arrest in non-small cell lung cancer cells

ZNF674-AS1, a recently characterized long noncoding RNA, shows prognostic significance in hepatocellular carcinoma and glioma. However, the expression and function of ZNF674-AS1 in non-small cell lung cancer (NSCLC) are unclear. In this work, we investigated the expression of ZNF674-AS1 in 83 pairs of NSCLC specimens and adjacent noncancerous lung tissues. The clinical significance of ZNF674-AS1 in NSCLC was analyzed. The role of ZNF674-AS1 in NSCLC growth and cell cycle progression was explored. Our data show that ZNF674-AS1 expression is decreased in NSCLC compared to normal tissues. ZNF674-AS1 downregulation is significantly correlated with advanced TNM stage and decreased overall survival of NSCLC patients. Overexpression of ZNF674-AS1 inhibits NSCLC cell proliferation, colony formation, and tumorigenesis, which is accompanied by a G0/G1 cell cycle arrest. Conversely, knockdown of ZNF674-AS1 enhances the proliferation and colony formation of NSCLC cells. Biochemically, ZNF674-AS1 overexpression increases the expression of p21 through downregulation of miR-423-3p. Knockdown of p21 or overexpression of miR-423-3p blocks ZNF674-AS1-mediated growth suppression and G0/G1 cell cycle arrest. In addition, ZNF674-AS1 expression is negatively correlated with miR-423-3p in NSCLC specimens. ZNF674-AS1 suppresses NSCLC growth by downregulating miR-423-3p and inducing p21. This work suggests the therapeutic potential of ZNF674-AS1 in the treatment of NSCLC.

Long noncoding RNAs (lncRNAs) are a class of transcripts lacking protein-coding potential, with a length of over 200 nucleotides [5]. Accumulating evidence indicates that lncRNAs are aberrantly expressed in various cancer types and play a critical role in tumor progression [6,7]. For example, lncRNA JPX is upregulated in metastatic lung cancer and shows the capacity to promote lung cancer growth and metastasis [6]. It has been suggested that lncRNAs regulate gene expression through interaction with micro-RNAs (miRNAs), which are small noncoding RNAs 22-24 nucleotides in length [8,9]. Specially, lncRNAs may act as a sponge of miRNAs to release miRNA targets [10,11]. Also, lncRNAs can suppress the expression of miRNAs [12].
Although several lncRNAs such as JPX [6], UCA1 [7], and JHDM1D-AS1 [13] have been reported to coordinate lung cancer progression, most aberrantly expressed lncR-NAs in lung cancer remain uncharacterized. The lncRNA ZNF674 antisense RNA 1 (ZNF674-AS1) is downregulated in hepatocellular carcinoma (HCC) [14]. Moreover, ZNF674-AS1 downregulation is significantly correlated with distant metastasis, clinical stage, histopathologic grade, and poor prognosis in patients with HCC [14]. Another study analyzing the Chinese Glioma Genome Atlas microarray dataset revealed that ZNF674-AS1 serves as an unfavorable prognostic factor for glioma [15]. These preliminary results imply that ZNF674-AS1 may play a cell context-dependent role in tumor progression. However, the role of ZNF674-AS1 in NSCLC has not yet been clarified.
In the current study, we investigated the expression and clinical significance of ZNF674-AS1 in NSCLC. We performed gain-and loss-of-function experiments to determine the impact of ZNF674-AS1 on NSCLC aggressive phenotype. In addition, we identified the key miRNAs involved in the action of ZNF674-AS1 in NSCLC.

Patients and tissues
A total of 83 paired tumor samples and corresponding normal lung tissues were collected from NSCLC patients who underwent surgery at our hospital. Clinicopathological information of the patients is summarized in Additional file 1: Table S1. None was given any anticancer treatment before surgery. Tissue specimens were immediately frozen in liquid nitrogen and stored at − 80 °C.

Cell culture
NSCLC cell lines A549, H1299, H358, and PC9 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) at 37 °C with 5% CO 2 atmosphere. BEAS-2B cells were cultured in growth factor-supplemented medium (BEGM; Lonza, Walkersville, MD, USA). These cell lines were purchased from the Type Culture Collection of the Chinese Academy of Science (Shanghai, China). No mycoplasma infection was detected in the cell lines used in this study.

Colony formation assay
Colony formation assay was performed as described previously [18]. Cells were seeded onto 6-well plates (600 cells per well). After incubation for 10 days, cells were fixed and stained with crystal violet (Sigma-Aldrich). The number of colonies per well was counted.

Animal studies
Male BALB/c nude mice, 5 weeks of age, were acclimated to the facility environment (12-h light/dark cycle, 23 ± 2 °C, and 50% humidity) for 1 week. Xenograft tumors were generated by injecting stably transfected A549 cells (2 × 10 6 ) into the flanks of the mice. Tumor volume was measured every week. After 4 weeks mice were euthanatized, and tumors were weighed.

Immunohistochemistry
Xenograft tumors were fixed and sectioned. The sections were deparaffinized, probed with anti-Ki-67 antibody (Sigma-Aldrich) in a humidified chamber, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Signals were developed using 3,3′-diaminobenzidine solution (Sigma-Aldrich). The sections were counterstained with hematoxylin.

Cell cycle analysis
For analysis of cell cycle progression, cells were fixed with 70% ethanol and stained with 50 μg/mL propidium iodide (PI) in the presence of 50 μg/mL RNase A (Sigma-Aldrich). Stained cells were analyzed by flow cytometry.

Western blot analysis
Cells were lysed in ice-cold RIPA lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein samples (30 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were incubated overnight at 4 °C with primary antibodies recognizing p21 and GAPDH. These antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The membranes were then incubated with secondary antibodies conjugated to HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. Enhanced chemiluminescent reagents (Millipore, Billerica, MA, USA) were used to visualize bound antibodies.

Statistical analysis
Data are expressed as mean ± standard deviation. Significant differences were analyzed by Student's t test or one-way analysis of variance. The relationship of ZNF674-AS1 with clinicopathological parameters was analyzed using the chi-square test. Survival analysis was performed by the Kaplan-Meier method. Pearson correlation analysis was conducted to determine the correlation between ZNF674-AS1 and miR-423-3p. P < 0.05 was considered statistically significant.

ZNF674-AS1 is downregulated and predicts poor prognosis in NSCLC
To explore the expression of ZNF674-AS1 in NSCLC, we analyzed the expression of ZNF674-AS1 in 83 pairs of NSCLC specimens and adjacent noncancerous lung tissues.
The results showed that ZNF674-AS1 expression was significantly decreased in NSCLC relative to normal tissues (P = 0.0069; Fig. 1a). We then analyzed ZNF674-AS1 levels in tumors grouped by TNM staging. Notably, downregulation of ZNF674-AS1 was significantly correlated with advanced TNM stage (P = 0.0010; Fig. 1b). Consistently, Kaplan-Meier analysis indicated that NSCLC patients with low ZNF674-AS1 levels had a shorter overall survival than those with high ZNF674-AS1 levels (P < 0.0001; Fig. 1c). In addition, analysis of ZNF674-AS1 expression in 504 lung adenocarcinoma specimens using KM Plotter (http://kmplo t.com/analy sis/) confirmed that low ZNF674-AS1 expression was associated with decreased overall survival (Fig. 1d). These 504 samples are derived from The Cancer Genome Atlas. These data suggest that ZNF674-AS1 downregulation may contribute to NSCLC progression.

ZNF674-AS1 suppresses NSCLC cell proliferation and colony formation
Consistent with the clinical findings, ZNF674-AS1 expression was downregulated in the NSCLC cell lines tested compared to BEAS-2B bronchial epithelial cells (Fig. 2a).
To determine the role of ZNF674-AS1 in NSCLC cell growth and invasion, ZNF674-AS1 was ectopically overexpressed in both A549 and H1299 cells (Fig. 2b), which had low levels of endogenous ZNF674-AS1. The growth of NSCLC cells was significantly suppressed by overexpression of ZNF674-AS1 (Fig. 2c). Moreover, ZNF674-AS1mediated growth suppression was confirmed in colony formation assays (Fig. 2d). However, ZNF674-AS1 overexpression had no impact on the invasion ability of NSCLC cells (Additional file 1: Figure S1). We further investigated the effect of ZNF674-AS1 on A549 cell tumorigenesis in a nude mouse model. Tumor volume measurements demonstrated that ZNF674-AS1overexpressing A549 xenografts were significantly smaller than control xenografts (Fig. 3a). At 4 weeks after cell inoculation, tumor weight in the ZNF674-AS1-overexpressing group was ~ fourfold lower than that in the control group (Figs. 3b and c). Histological analysis confirmed that there were fewer ki-67-positive proliferating cells in ZNF674-AS1-overexpressing xenografts than in control xenografts (Fig. 3d). Collectively, these data indicate that ZNF674-AS1 can exert growth-suppressing activity in NSCLC.

ZNF674-AS1 causes a G0/G1 cell cycle arrest through upregulation of p21
Next, we asked whether ZNF674-AS1-mediated growth suppression was ascribed to induction of cell cycle arrest. Flow cytometric analysis of cell cycle distribution revealed that overexpression of ZNF674-AS1 increased the number of cells in the G1 phase and decreased the number of cells in the S phase (Fig. 5a), indicating a partial cell cycle arrest at the G0/G1 phase. We further examined the expression of a number of cell cycle-related genes. The results demonstrated that overexpression of ZNF674-AS1 led to a marked increase of p21 protein in both A549 and H1299 cells (Fig. 5b). Other cell cycle-related genes including cyclin D1, p27, p16 and Ki67 remained unaffected (data not shown). Moreover, the level of p21 mRNA was elevated in ZNF674-AS1-overexpressing NSCLC cells (Fig. 5c). Therefore, we speculated that p21 was involved in the growth-suppressing activity of ZNF674-AS1. To validate the hypothesis, we performed p21 knockdown experiments (Fig. 5d). As shown in Fig. 5e and f, p21 knockdown rescued ZNF674-AS1-mediated growth suppression and G0/ G1 cell cycle arrest. The results suggest that upregulation of p21 at least partially contributes to the ZNF674-AS1 phenotypes observed.

Discussion
Previous studies have identified a number of lncRNAs that are dysregulated in NSCLC and serve as potential prognostic biomarkers for this malignancy [13,26]. For instance, upregulation of lncRNA SNHG3 by E2F1 is associated with a low overall survival rate of patients with NSCLC [26]. In this study, we report a novel prognostic factor for NSCLC. We show that ZNF674-AS1 is downregulated in NSCLC relative to corresponding normal lung tissues. The downregulation of ZNF674-AS1 is associated with advanced TNM stage and reduced overall survival of NSCLC patients. The potential of ZNF674-AS1 as a prognostic biomarker has also been described in HCC [14] and glioma [15]. Biochemically, ZNF674-AS1 overexpression inhibits the proliferation and tumorigenesis of NSCLC cells. In contrast, the invasive capacity of NSCLC cells is not affected by ZNF674-AS1 overexpression. Our data provide the first evidence for the role of ZNF674-AS1 in modulating tumor progression. The tumor-suppressive activity of ZNF674-AS1 may provide an explanation for the clinical relationship between ZNF674-AS1 downregulation and reduced overall survival of NSCLC patients. Multiple lncRNAs have exhibited the capacity to regulate cell cycle progression [27,28]. For instance, knockdown of lncRNA EPIC1 leads to growth suppression and G0/G1 cell cycle arrest in pancreatic cancer cells [27]. The lncRNA EPIC1 has been shown to promote cell cycle progression in cancer cells through interaction with MYC [28]. Our data indicate ZNF674-AS1 as a negative regulator of cell cycle progression in NSCLC cells. In particular, ectopic expression of ZNF674-AS1 arrests NSCLC cells at the G0/G1 phase. Therefore, ZNF674-AS1 inhibits NSCLC growth, at least partially through inducing G0/G1 cell cycle arrest.
Mechanistically, ZNF674-AS1 selectively stimulates the expression of p21 in NSCLC cells. p21 belongs to the Cip and Kip family of CDK inhibitors [29]. It exerts biological activities primarily by inhibiting the kinase activity of CDKs [30,31]. Induction of p21 has been shown to impair the G1/S cell cycle transition in NSCLC cells by inhibiting the activation of CDK2 complexes [30]. Interestingly, we show that knockdown of p21 prevents growth suppression of NSCLC cells induced by ZNF674-AS1. Therefore, ZNF674-AS1-mediated antiproliferative activity in NSCLC cells depends on the upregulation of p21.

Conclusion
We identify ZNF674-AS1 as a new growth suppressor in NSCLC. Induction of p21 due to miR-423-3p downregulation is responsible for ZNF674-AS1-mediated growth suppression. Our data suggest that restoration of ZNF674-AS1 represents a potential therapeutic strategy to treat NSCLC.