LncRNA NKX2-1-AS1 as ceRNA promotes tumor progression and angiogenesis by upregulating SERPINE1 expression and activating VEGFR-2 signaling pathway in gastric cancer

Background: Recent evidence indicated that the lncRNA NKX2-1-AS1 (NKX2-1 antisense RNA 1) plays an important role in cancer progression and metastasis. However, the associated molecular mechanisms of NKX2-1-AS1 in GC are still unclear. Methods: To determine the target of the study by bioinformatic analysis. NKX2-1-AS1 expression was measured in paired tumor and non-tumor tissues of 178 GC patients, by quantitative reverse transcription PCR. The in vitro and in vivo biological functions of NKX2-1-AS1 were examined by loss-of-function and gain-of-function experiments. The potential mechanisms of this competing endogenous RNA (ceRNA) were elucidated using dual-luciferase reporter assay, quantitative PCR, Western blot, and fluorescence in situ hybridization (FISH). Results: NKX2-1-AS1 expression was upregulated in GC cell lines and tumor tissues, which was correlated with tumor progression and enhanced angiogenesis. Functionally, NKX2-1-AS1 overexpression promoted GC cell proliferation, metastasis, invasion, and angiogenesis, while NKX2-1-AS1 downregulation reversed these effects, both in vitro and in vivo. Bioinformatics analysis and dual-luciferase assay showed that the microRNA miR-145-5p is a direct target of NKX2-1-AS1, and that NKX2-1-AS1 acts as a ceRNA that regulates angiogenesis in the context of GC. The mechanistic study revealed that miR-145-5p specifically targets serpin family E member 1 (SERPINE1), and that the complex NKX2-1-AS1/miR-145-5p activates VEGFR2 signaling, via SERPINE1, to promote tumor proliferation and angiogenesis. Conclusion: NKX2-1-AS1 upregulation is associated with tumor cell proliferation, increased angiogenesis, and poor prognosis in GC patients. NKX2-1-AS1 regulates SERPINE1 expression and VEGFR2 signaling by acting as a ceRNA for miR-145-5p. miR-145-5p and SERPINE1 detected by qRT-PCR in 178 paired GC cancer tissues and matched normal tissues. Results are presented as Δ cycle threshold (ΔCt) in tumor tissues relative to normal tissues. D-E Relative expression of NKX2-1-AS1 in GC with different TNM stage and different numbers of positive regional lymph nodes. F-G with/without peritoneum dissemination (PD), and with/without distant metastasis. Results were presented as Δ cycle threshold (ΔCt) in tumor tissues relative to normal tissues. E qRT-PCR was performed to examine NKX2-1-AS1 expression in 178 GC cancer tissues. Relative expression of NKX2-1-AS1 was presented as log2 (fold change of ΔCt value) in tumor tissue to that of matched normal tissues. GC patients were divided into high (n = 89) and low (n = 89) groups according to the median value (0.50). F H-score of SERPINE1 expression in 178 GC cancer tissues. GC patients were divided into high (n = 89) and low (n = 89) groups according to the median value (0.50).


Background
Gastric cancer (GC) is the fourth most common cancer and the third most common cause of cancer-related deaths worldwide. GC prevalence is higher in East Asia, namely in China and Japan (1). Due to the lack of early symptoms, GC diagnosis is often accompained by late stage disease (2). Despite the significant improvement in GC diagnosis and treatment in the recent decades, the prognosis of patients with advance disease is still highly unfavorable (3,4). Currently, GC-associated tumor angiogenesis is regarded as a main cause of unfavorable patient outcomes (5). Therefore, understanding the molecular mechanisms of tumor angiogenesis is key to limit GC progression and improve patient diagnosis and outcome for the improvement of GC progression, diagnosis, and prognosis.
Long non-coding RNAs (lncRNAs) are a class of RNA polymerase II transcripts of > 200 nucleotides long (6). Increasing evidence in recent years demonstrated that lncRNAs possess numerous biological functions and are involved in the regulation of multiple processes in disease, especially in cancer (7,8). Unlike microRNAs (miRNAs), the unifying molecular mechanism for lncRNAs has not yet been reported (9). However, studies have shown that some lncRNAs can "sponge" and competitively inhibit the biological functions of miRNAs (10,11). The lncRNA NKX2-1-AS1 was first reported to be highly expressed in primary lung adenocarcinomas, compared to squamous carcinomas, and to be associated with lung carcinoma cell migration (12).
Bioinformatic studies also revealed that NKX2-1-AS1 may act as a ceRNA in GC (13).
Still, changes in NKX2-1-AS1 expression, and its role in cancer is largely unknown.
Accordingly, its functions and potential regulatory mechanism in GC have not been investigated. The plasminogen activation system, which consists in the urokinase-type plasminogen activator (uPA), the cellular receptor for uPA (uPAR), and its specific inhibitor plasminogen activator inhibitor-1 (PAI-1 or SERPINE1), plays a crucial role in tumor progression and angiogenesis (14,15). Numerous studies demonstrated that SERPINE1 overexpression is associated with the tumor progression and unfavorable 4 outcome in several cancer models, including GC (16,17). Several mechanisms have been proposed that explain the correlation of SERPINE1 with cancer progression. In particular, the role of SERPINE1 in tumor angiogenesis has been intensively investigated. Studies with SERPINE1-deficient (SERPIN1 -/-) mice showed that host SERPINE1 expression is essential for tumor angiogenesis (18)(19)(20), and that its effects are likely mediated through the modulation of endothelial cell plasmin-mediated proteolysis (21), migration (22,23), and apoptosis (24). Recently, it was demonstrated that the specific inhibition of SERPINE1 by the specific PAL-1 inhibitor SK-216 limited tumor angiogenesis in vivo and suppressed VEGF-induced migration and tube formation by human umbilical vein endothelial cells (HUVECs) in vitro (25). Based on these findings, it is speculated that the association between SERPINE1 overexpression and GC progression results from increased tumor angiogenesis promoted by SERPINE1. RNA-seq data and differential gene expression analysis and DEmiRNA were generated using the "pheatmap" and "ggplot2" packages in R (27).
KEGG pathway enrichment analysis was performed on DEmRNAs using "clusterProfiler" and visualized by "ggplot2". Affected pathways were identified by P < 0.001 and fold enrichment > 1.5. The effect of differentially expressed RNAs on patient survival was analyzed by "survival" and "q-value" at P < 0.05. 6 Construction of the ceRNA network To determine lncRNA-miRNA-mRNA interactions, we considered only the pool of dysregulated lncRNAs and mRNAs. Then, we identified the miRNA seed sequences that function as shared (i.e, overlapping) binding sites for these lncRNAs and mRNAs.   1J, 1K, and data not shown). Given that the NKX2-1-AS1/miR145-5p/SERPINE1 was the only ceRNA regulatory axis that showed negative association with OS in both its 13 lncRNA and mRNA ends, and that miR145-5p was expressed at low level in cancer tissues, our data suggest that the NKX2-1-AS1/miR145-5p/SERPINE1 axis plays a potential adverse role in the prognosis of GC patients. lymph node metastases (> 6 positive lymph nodes) (Fig. 2D, E). Although NKX2-1-AS1 expression was positively correlated with larger tumor, infiltration of peritumoral tissues, peritoneum dissemination, and distant metastasis, there were no statistically significant differences among these subgroups (Additional file 2: Figure S1a-d).
Overall, the clinical data show that NKX2-1-AS1 upregulation is often observed in GC and may therefore be associated with GC progression and metastasis.
NKX2-1-AS1 and SERPINE1 overexpression is positively correlated with GC progression and poor prognosis To evaluate the prognostic value of NKX2-1-AS1 overexpression, the median score (0.50) of relative NKX2-1-AS1 expression was defined as the cutoff value for dividing all GC patients into high-and low-expression groups (Additional file 2: Figure   S1e).  Figure   S1F). Compared with patients with low SERPINE1 expression, those with high SERPINE1 expression had shorter 5-year OS (39.48% vs. 60.68%, p = 0.0073).
Altogether, these data suggest that NKX2-1-AS1 and SERPINE1 are potential prognostic predictors for GC patients.

NKX2-1-AS1 regulates GC cell proliferation and angiogenesis in vivo
To evaluate the effect of NKX2-1-AS1 on GC tissues in vivo, NKX2-1-AS1-stably expressing AGS cells were subcutaneously injected in female nude mice (Fig. 5A). As expected, xenograft tumors were larger and heavier in the AGS-NKX2-1-AS1 group than in the NC group (Fig. 5B, C). Similarly, NKX2-1-AS1 shRNA-transfected AGS was subcutaneously injected in female nude mice. In addition, it is observed that the tumors were smaller and lighter in the sh-NKX2-1-AS1 group when compared to the NC group (Fig. 5D, 5E). During necropsy, tumors appeared paler in the shNKX2-1-AS1 group than in the NC group, and it is hypothesized that NKX2-1-AS1 expression might be associated with GC tissue angiogenesis, which in turn can impact tumor growth. IHC staining revealed enhanced CD34 expression in tumors overexpressing NKX2-1-AS1, which was reversed by NKX2-1-AS1 knockdown (Fig. 5F-H).
Therefore, our in vivo results strongly suggest that NKX2-1-AS1 is positively correlated with GC tissue growth and increased angiogenesis.

NKX2-1-AS1 is a ceRNA of miR-145-5p in GC
To further elucidate the regulatory mechanism of NKX2-1-AS1 in GC, the subcellular localization of NKX2-1-AS1 was determined by FISH. As shown in Fig.   6A, the NKX2-1-AS1 transcript is primarily localized in the cytoplasm. Cytoplasmic lncRNAs are known to act as ceRNAs that attenuate negative regulation of target mRNAs by sponging miRNAs (30). Based on our TCGA data analysis and clinical sample validation, it is hypothesized that NKX2-1-AS1, miR-145-5p and SERPINE1 may be involved in a regulatory ceRNA network. It is predicted that the binding sites between NKX2-1-AS1 and miR-145-5p (Fig. 6B) and between miR-145-5p and SERPINE1 (Fig. 6C) using mircode and starbase, respectively. To validate our hypothesis, AGS and HGC-27 cells were transfected with miR-145-5p mimic or inhibitor (Fig. 6D) to up-and downregulate miR-145-5p expression, respectively.
Altogether, these findings demonstrate that the promoting effect of NKX2-1-AS1 in GC angiogenesis is primarily dependent on the miR-145-5p/SERPINE1 axis. and Src, as well as survival-related Akt. It is observed that NKX2-1-AS1 knockdown and overexpression had no significant impact on total protein expression. In contrast NKX2-1-AS1 knockdown led to reduced protein phosphorylation, whereas NKX2-1-AS1 overexpression resulted in increased protein phosphorylation, suggesting an active role of NKX2-1-AS1 in the regulation of VEGFR2 signaling. Similar results were obtained in HUVECS co-cultured with AGS cells (Fig. 8A). Overall, these findings indicated that regulation of GC cell metastasis by the NKX2-1-AS1/miR-145-5p/SERPINE1 axis is dependent on the VEGFR2 pathway.

Discussion
Dysregulated lncRNA expression was recently found in GC and other gastrointestinal cancers (32). Previous studies have shown that NKX2-1-AS1 limits human carcinoma cell migration by negatively regulating CD274/PD-L1 and cell-cell interaction genes (12). Furthermore, its close proximity to the transcription factor NKX2-1 is known to contribute to GC progression (33). In this study, it is shown that NKX2-1-AS1 is significantly upregulated in GC, promoting cancer cell proliferation and supporting angiogenesis, both in vitro and in vivo. Furthermore, in vitro experiments showed a positive correlation of NKX2-A-AS1 with metastasis and invasion potential. Altogether, the results suggest that NKX2-1-AS1 promotes GC progression. Therefore, it is proposed that NKX2-1-AS1 can be used as a potential diagnostic and prognostic biomarker in the context of gastric cancer.
An increasing number of studies has demonstrated that the biological functions of lncRNAs are largely dependent on their unique subcellular localization (34).
Cytoplasmic lncRNAs act as mRNA decoys, to regulate mRNA stability or translation, and influence signal transduction pathways (30). miR-145 was previously reported to inhibit GC cell migration and metastasis, likely due to the suppression of myosin VI (MYO6) expression (35). Using bioinformatic analysis and luciferase reporter assay, it is shown that NKX2-1-AS1 directly binds and inhibits miRNA-145-5p expression, and that miRNA-145-5p expression is significantly downregulated and negatively correlated with NKX2-1-AS1 expression in GC. Low miRNA-145-5p expression due to the ceRNA nature of NKX2-1-AS1, alleviates the downregulation of SERPINE1, thereby promoting GC progression. A similar ceRNA-related mechanism has been reported in nasopharyngeal cancer, in which MACC1 antisense RNA 1 (MACC1-AS1) acts as a molecular sponge of miR-145 (36). It was recently reported that, in cancer, miRNAs play a key role in signal transduction by degrading or inhibiting mRNAs (7).
It is well established that tumor angiogenesis is a key mechanism in promoting tumor growth and invasion (37). An increasing amount of evidence shows that miRNAs interact with several mRNA targets of the VEGF-A pathway and regulate GC-related angiogenesis (38). Conversely, miRNA-145 was shown to inhibit GC cell invasion, metastasis and angiogenesis by targeting v-ets erythroblastosis virus E26 oncogene homolog 1 (39). Additionally, inhibition of miRNA-mediated SERPINE1 limits tumor angiogenesis, regardless of the angiogenic stimuli, in malignant pleural mesothelioma 19 (40). In the present study, it is found that SERPINE1 is a direct target of miR-145-5p.
Further analysis revealed that the loss of SERPINE1 inhibition by miR-145-5p significantly enhanced VEGFR2 pathway activity. In contrast, the VEGFR2 pathway was significantly downregulated in GC cells overexpressing NKX2-1-AS1. Beyond SERPINE1, MEST mRNA, which exhibits potential miR-145-5p binding sites, was also significantly downregulated in GC samples obtained from TCGA database.
However, MEST overexpression was not associated with GC patient prognosis and no differential expression was observed between tumor and non-tumor tissues in our samples (data not shown). Thus, it is hypothesized that miR-145-5p regulates SERPINE1 expression in GC. Co-culture of GC cells with HUVECs provided direct evidence of SERPINE1 in promoting vascular endothelial cell proliferation, further confirming its importance in angiogenesis. Taken together, our findings demonstrate that the NKX2-1-AS1/miR-145-5p axis activates the VEGFR2 signaling pathway by specifically upregulating SERPINE1 expression.
VEGFR2 is mainly expressed on the cell membrane and is found on both vascular endothelial and GC cells. Conversely, SERPINE1 proteins are mainly found in the cytoplasm and are directly associated with extracellular matrix remodeling. These findings suggest that SERPINE1 proteins are transported out of GC cells, such as by exosomes, and act on the tumor microenvironment to increase GC tissue angiogenesis, enhance GC cell proliferation and metastasis (Fig. 7B). This study highlighted the role of NKX2-1-AS1 as a ceRNA in the binding of miR-145-5p and upregulation of SERPINE1. This resulted in increased activation of the VEGFR2 signaling pathway and promoted tumor angiogenesis, proliferation and metastasis. The exact drivers of SERPINE1 localization change are still to uncover. To understand such mechanism and the molecular requirements of SERPINE1-VEGFR2 interaction, will be key to better clarify the biological processes implicated in GC progression.

Conclusion
In summary, this study showed that NKX2-1-AS1 upregulation is frequent and 20 might be an independent prognostic biomarker in GC. NKX2-1-AS1 is a lncRNA that promotes in vitro and in vivo GC-cell proliferation and tumor angiogenesis. Our findings elucidate the molecular mechanisms by which NKX2-1-AS1 directly targets miR-145-5p to activate VEGFR2 signaling and consequently promote GC progression (Fig. 8B). Therefore, it is proposed that NKX2-1-AS1 might be a new prognostic biomarker and a potential therapeutic target in the context of gastric cancer.

Consent for publication
Consent to publish has been obtained from all authors.

Availability of data and materials
All data in our study are available upon request.        HUVECs cells were co-cultured with corresponding AGS cells for 7 days, the content of VEGFR-2 and p-VEGFR-2 protein was detected. GAPDH serves as an internal reference. B Schematic diagram of the regulatory mechanism of NKX2-1-AS1/miR-145-5p/SERPINE1 axis in the promotion of GC proliferation, metastasis and angiogenesis.
Additional file 1: Table S1. Primers and oligonucleotides sequences used in this study.
Additional file 2: Figure S1. The expression of NKX2-1-AS1 in GC patents with different clinical subgroups. A-D Relative expression of NKX2-1-AS1 in GC with different tumor sizes, with/without infiltration of peritumoral tissues (IPT), 31 with/without peritoneum dissemination (PD), and with/without distant metastasis.
Results were presented as Δ cycle threshold (ΔCt) in tumor tissues relative to normal tissues. E qRT-PCR was performed to examine NKX2-1-AS1 expression in 178 GC cancer tissues. Relative expression of NKX2-1-AS1 was presented as log2 (fold change of ΔCt value) in tumor tissue to that of matched normal tissues. GC patients were divided into high (n = 89) and low (n = 89) groups according to the median value (0.50).
F H-score of SERPINE1 expression in 178 GC cancer tissues. GC patients were divided into high (n = 89) and low (n = 89) groups according to the median value (0.50).