Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer

Abstract Long non‐coding RNAs (lncRNAs) have been illustrated to function as important regulators in carcinogenesis and cancer progression. However, the roles of lncRNA NNT‐AS1 in gastric cancer remain unclear. In the present study, we investigate the biological role of NNT‐AS1 in gastric cancer tumorigenesis. Results revealed that NNT‐AS1 expression level was significantly up‐regulated in GC tissue and cell lines compared with adjacent normal tissue and normal cell lines. The ectopic overexpression of NNT‐AS1 indicated the poor prognosis of GC patients. In vitro experiments validated that NNT‐AS1 knockdown suppressed the proliferation and invasion ability and induced the GC cell cycle progression arrest at G0/G1 phase. In vivo xenograft assay, NNT‐AS1 silencing decreased the tumour growth of GC cells. Bioinformatics online program predicted that miR‐424 targeted the 3′‐UTR of NNT‐AS1. Luciferase reporter assay, RNA‐immunoprecipitation (RIP) and RNA pull‐down assay validated the molecular binding within NNT‐AS1 and miR‐424, therefore jointly forming the RNA‐induced silencing complex (RISC). Moreover, E2F1 was verified to act as the target gene of NNT‐AS1/miR‐424, indicating the NNT‐AS1/miR‐424/E2F1 axis. In conclusion, our study indicates that NNT‐AS1 sponges miR‐424/E2F1 to facilitate GC tumorigenesis and cycle progress, revealing the oncogenic role of NNT‐AS1 for GC.

RNAs (ncRNAs) account for a large proportion of transcriptome, which are divided into short non-coding RNAs and lncRNAs according to their length. Increasing evidences have indicated the extensive biological role of lncRNAs on multiple tumour pathological process, for example proliferation, invasion, metastasis and recrudesce. 9,10 For the tumorigenesis of GC, more and more lncRNAs have been identified to modulate the pathological process.
For instance, long intergenic non-coding RNA 01296 (LINC01296) acts as oncogenic lncRNA in GC carcinogenesis and aggravates gastric cancer cells progress via LINC01296/miR-122/MMP-9 regulatory pathway. 11 In the pre-experiments of our study, we focus on several candidate lncRNAs, and then we measured the expression levels of these candidate lncRNAs in cancer tissue using RT-PCR. After comparing, we found that the lncRNA NNT-AS1 expression level is upregulated in GC tissue samples and cells. NNT-AS1 has been found to act as oncogene in human cancer. 12 Besides, the roles and molecular mechanism of lncRNA NNT-AS1 on GC tumorigenesis are unknown and never reported. Thus, we choose lncRNA NNT-AS1 as our research object. In the present study, we aim to There are no any patients who had received preoperative chemotherapy before this study. The tissues were excised during the surgery and snap-frozen in liquid nitrogen. This study was approved by the Ethic Committee of The Affiliated Cancer Hospital of Zhengzhou University. All written informed consents had been collected from every patient before surgery.

| Cell Counting Kit-8 assay
Cell proliferation assay was performed with Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). GC cells (MGC-803, SGC-7901) were seeded into 96-well plates at a density of 1 9 10 3 per well. About 10 lL of CCK-8 solution was added into each well at indicated time points. After incubation at 37°C for 2 hours, the absorbance was measured using an automatic microplate at 450 nm. Assay was performed in triplicate.

| Western blot analysis
The expression of E2F1 protein in GC cells was detected by per-

| Luciferase reporter assay
For Luciferase assays, NNT-AS1 wild type with potential miR-424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). SGC-7901 cells (2 9 10 4 cells/well) were seeded into 96-well plates. Cells were co-transfected with luciferase plasmids and miRNA mimics or controls. Cells were cultured in 12-well plates and transfected with 5 ng plasmid and 5 ng Renilla using Dual-Luciferase Reporter Assay System (Promega). Every analysis was performed 3 times.

| RNA immunoprecipitation (RIP)
RNA immunoprecipitation (RIP) assays were performed as previously described. 13 Wild-type NNT-AS1 or mutant NNT-AS1 were synthesized and then cloned into SGC-7901 cells. SGC-7901 cells were also co-transfected with miR-NC or miR-424 mimic. Cells were lysed by lysis buffer containing a protease inhibitor cocktail and RNase inhibitor. Magnetic beads were pre-incubated with an anti-GFP antibody (Abcam) or anti-rabbit IgG (Millipore) for 1 hour at room temperature, and lysates were immunoprecipitated with beads at 4°C overnight. After 48 hours, cells were used to perform RIP assay using an anti-AGO2 antibody (Millipore) as described above.

| Statistical analysis
Statistical analyses and data calculation were performed using the SPSS19.0 (IBM, SPSS, Chicago, IL, USA) software and graphed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Overall survival curves were calculated using Kaplan-Meier analysis and the log-rank test. Pearson correlation coefficient was used to analyse the correlations. Comparisons between groups were analysed by the t tests v 2 test, one-way analysis of variance (ANOVA). The differences were considered statistically significant at P < .05.

| LncRNA NNT-AS1 was highly expressed in GC tissues and cells, and indicated poor prognosis of GC patients
To verify the expression of NNT-AS1 in GC tumour tissue and cells, RT-PCR was performed on these collected samples and cultured cells. The clinicopathologic data of GC patients were presented in Table 1. Results revealed that NNT-AS1 expression was significantly up-regulated in these collected GC tissue samples    Figure 1B). According to median value of NNT-AS1 expression, the whole group was divided into high NNT-AS1 expression group (n = 21) and low NNT-AS1 expression group (n = 27) ( Figure 1C).
Overall survival rate of GC patients was calculated using Kaplan-Meier curves and log-rank test, showing that GC patients with high NNT-AS1 levels had poor prognosis than those with low NNT-AS1 level ( Figure 1D). In summary, the data concluded that lncRNA NNT-AS1 was highly expressed in GC tissues and cells, and the ectopic overexpression indicated poor prognosis of GC patients.

| NNT-AS1 knockdown suppressed the proliferation and invasion ability in vitro, and inhibited the GC tumour growth in vivo
Subsequently, we tested the role of NNT-AS1 knockdown on GC tumour phenotype using in vitro and in vivo experiments. CCK-8 assay showed that NNT-AS1 knockdown suppressed the proliferation ability of GC cell (SGC-7901, MGC803) compared with control group (Figure 3A, B). Transwell invasion assay showed that NNT-AS1 knockdown inhibited the invasive cells compared with control groups ( Figure 3C, D). Xenograft in vivo assay was performed to verify the function of NNT-AS1 on GC tumour growth ( Figure 3E).
Results showed that NNT-AS1 knockdown induced by lentivirusmediated transfection significantly decreased the tumour volume of GC cells ( Figure 3F), besides suppressing the tumour weight after killing ( Figure 3G). In summary, the above results concluded that NNT-AS1 knockdown suppressed the proliferation and invasion ability of GC cells in vitro, and inhibited the GC tumour growth in vivo.

'sponge'
Prior assay revealed that the cellular localization of NNT-AS1 was mainly in the cytoplasm, instead of nuclear ( Figure 2A). Therefore,

| DISCUSSION
Long non-coding RNAs have recently emerged as an important regulator on transcriptional and post-transcriptional regulation of several diseases, including cancers. 14-16 LncRNA NNT-AS1 has been found to participate in the tumorigenesis of hepatocellular carcinoma, cervical cancer and colorectal cancer. [17][18][19] In the pre-experiments of our study, we found that the lncRNA NNT-AS1 expression level was up-regulated in GC tissue samples and cells.
Besides, the roles and molecular mechanism of lncRNA NNT-AS1 on GC tumorigenesis are unknown and never reported. Thus, we choose lncRNA NNT-AS1 as our research object. In the present study, our study investigates the role of lncRNA NNT-AS1 in gastric cancer (GC) tumorigenesis and identifies the underlying mechanism.
During the carcinogenesis of GC, a great deal of lncRNAs might be abnormally expressed. 20,21 Generally, the significantly dysregulated lncRNAs might involve the pathological process and exert vital roles by regulating multiple progression. In our study, we found that lncRNA NNT-AS1 was significantly high-expressed in gastric cancer Long non-coding RNAs are a type of non-coding RNA (ncRNA) without protein-coding ability. 24,25 Up to now, the most common regulatory mechanism for lncRNA is the miRNA 'sponge' theory. In our study, our data indicate that NNT-AS1 harbours miR-424 by 3 0 -UTR binding, while miR-424 also targets the 3 0 -UTR of E2F1 F I G U R E 4 NNT-AS1 targeted miR-424 at 3 0 -UTR with complementary binding sites. A, The predicted complementary binding sequences of NNT-AS1 3 0 -UTR and miR-424. B, Luciferase reporter assay showed the molecular binding within NNT-AS1 and miR-424. C, RIP assay was carried out to explore the exact mechanism of the interaction between NNT-AS1 and miR-424, indicating that both NNT-AS1 and miR-424 were significantly enriched in Ago2 immunoprecipitate compared to IgG-pellet. D, miR-424 directly binds with NNT-AS1 using pull-down assay. SGC-7901 cells were transfected with biotinylated wild-type/mutant-type miR-424, and after 48 h, NNT-AS1 expression levels were measured using RT-PCR. E, RT-PCR showed that miR- NNT-AS1 knockdown induced G0/G1 phase arrest. Based on the above findings, we conclude that NNT-AS1 exerts the cycle inhibitive function through targeting miR-424/E2F1 axis. E2F1 is found to be aberrantly high-expressed in gastric cancer, and enhanced E2F1 expression promotes proliferation, while E2F1 low expression decreased cell proliferation by blocking cell cycle in GC cells. 26 Moreover, R-424-5p is found to be up-regulated in GC tissues and cells, and its high-expression could promote the proliferation of GC cells. 27 More than NNT-AS1, other lncRNAs also modulate the cellular process of GC cells. 28 For example, lncRNA SNHG6 is overexpressed in gastric cancer tissues and cell lines, and SNHG6 could epigenetically silence p27 and competitively sponge miR-101-3p to regulate ZEB1. 29 In HCC tumorigenesis, NNT-AS1 knockdown inhibited the HCC cells progression via miR-363 thereby targeting CDK6 expression. 18 Therefore, our study firstly confirmed the oncogenic role of

CONFLI CTS OF INTEREST
All the authors declare that they have no competing interests.