lncRNA OXCT1-AS1 Promotes Metastasis in Non-Small-Cell Lung Cancer by Stabilizing LEF1, In Vitro and In Vivo

Long noncoding RNAs (lncRNAs) play nonnegligible roles in the metastasis of non-small-cell lung cancer (NSCLC). This study is aimed at investigating the biological role of lncRNA OXCT1-AS1 in NSCLC metastasis and the underlying regulatory mechanisms. The expression profiles of lncRNA OXCT1-AS1 in different NSCLC cell lines were examined. Then, the biological function of lncRNA OXCT1-AS1 in NSCLC metastasis was explored by loss-of-function assays in vitro and in vivo. Further, the protective effect of lncRNA OXCT1-AS1 on lymphoid enhancer factor 1 (LEF1) was examined using RNA pull-down and RNA immunoprecipitation assays. Additionally, the role of LEF1 in NSCLC metastasis was investigated. Results indicated that lncRNA OXCT1-AS1 expression was significantly increased in NSCLC cell lines. Functional analysis revealed that knockdown of lncRNA OXCT1-AS1 impaired invasion and migration in vitro. Additionally, the ability of lncRNA OXCT1-AS1 to promote NSCLC metastasis was also confirmed in vivo. Mechanistically, through direct interaction, lncRNA OXCT1-AS1 maintained LEF1 stability by blocking NARF-mediated ubiquitination. Furthermore, LEF1 knockdown impaired invasion and migration of NSCLC in vitro and in vivo. Collectively, these data highlight the ability of lncRNA OXCT1-AS1 to promote NSCLC metastasis by stabilizing LEF1 and suggest that lncRNA OXCT1-AS1 represents a novel therapeutic target in NSCLC.


Introduction
Non-small-cell lung cancer (NSCLC) is the main type of lung cancer and one of the most frequently occurring cancers worldwide [1]. According to the Global Cancer Statistics 2018, lung cancer cases account for about 11.6% of the total number of cancer diagnoses; furthermore, among the total number of cancer-related deaths, 18.4% are attributable to lung cancer [2]. Although progress has been made in NSCLC treatment, metastatic NSCLC is still associated with a high rate of mortality [3,4], indicating the critical need for the identification of new therapeutic targets. Metastasis is a complex multistep process, and the invasion and migration capability of cancer cells determines the metastasis of NSCLC [5,6]. Furthermore, a gain of mesenchymal characteristics as well as loss of epithelial features frequently accompany the attainment of metastatic ability in NSCLC; this epithelialmesenchymal transition involves numerous molecules such as long noncoding RNA (lncRNA), transcription factor, and circular RNA [7,8]. However, the precise mechanisms underlying NSCLC metastasis have not yet been identified.
lncRNA is a class of RNA without protein-coding functions. Emerging researches have shown that many lncRNAs are dysregulated in NSCLC, and that their dysregulation contributes to metastasis in cancer [9,10]. Recently, Liao et al. [11] found that the lncRNA CCHE1 increased the proliferation, metastasis, and invasion of NSCLC cells, and was predictive of poor survival in NSCLC patients. Chen et al. [12] identified a novel lncRNA OXCT1-AS1 through microarray experiments, and showed that its expression was elevated in lymph node metastasis. Furthermore, these authors found that lncRNA OXCT1-AS1 suppresses miR-455-5p to promote bladder cancer proliferation and invasion. However, the exact role of lncRNA OXCT1-AS1 in the metastasis of NSCLC, and the mechanisms involved, remain poorly understood.
Growing evidence indicates that lncRNAs participate in the regulation of gene expression at the transcription or posttranscription levels, as well as that of pathological and cellular physiological processes [13,14]. In addition, lncRNAs regulate protein stability [15], as competing endogenous RNAs [16], and also regulate mRNAs' stability [17]. Jiang et al. [18] recently reported that lncRNA SNHG15 interacts with and stabilizes Slug by blocking its ubiquitination to promote colon cancer progression. Furthermore, Xue et al. [15] found that lncRNA LINRIS blocked ubiquitination of IGF2BP2 in colorectal cancer, maintaining its stability. Therefore, we speculated that lncRNA OXCT1-AS1 may play a potential role in migration, invasion, and metastasis of NSCLC by influencing the stability of target protein.
Lymphoid enhancer-binding factor 1 (LEF1), which is characterized by a high expression in tumors, is a key transcription factor of the WNT pathway and participates in the regulation of cell metastasis and proliferation [19]. Nguyen et al. [20] found that the WNT/TCF pathway through HOXB9 and LEF1 mediates metastasis in lung adenocarcinoma. Furthermore, results from Zhao et al.'s study [21] revealed that in esophageal squamous cell carcinoma, elevated level of LEF1 was obviously associated with lymph node metastasis, histologic grade, TNM stage, and poor prognosis. Therefore, we speculate that lncRNA OXCT1-AS1 may promote NSCLC metastasis by stabilizing LEF1.
To the best of our knowledge, the present study is the first to profile the expression of lncRNA OXCT1-AS1 in NSCLC cells. Additionally, we explored the role of lncRNA OXCT1-AS1 in regulating migration, invasion, and metastasis of NSCLC and investigated the interactions between lncRNA OXCT1-AS1 and LEF1, with the aim of identifying novel therapeutic targets for metastatic NSCLC.

Methods
2.1. Cell Culture. Human normal epithelial cell line BEAS-2B and human NSCLC cell lines (A549, H1299, H23, and HCC827) were obtained from the American Type Culture Collection (Manassas, VA, USA). NSCLC cell lines were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) adding 10% fetal bovine serum (FBS; Thermo Fisher Scientific). BEAS-2B cells were cultured in BEGM medium (Thermo Fisher Scientific) adding 10% FBS. All cells were incubated in a humidified incubator at 37°C with 5% CO 2 and saturated humidity. When cell confluence had reached 70%-80%, the cells were detached with trypsin (0.25%) to obtain a single-cell suspension.

Isolation of Nuclear and Cytoplasmic Fractions.
In accordance with the manufacturer's protocol of the Nuclear and Cytoplasmic Extraction Reagent Kit (Beyotime Institute of Biotechnology), nuclear-cytoplasmic fractionation was conducted. Briefly, after washing in PBS, cells were suspended in cytoplasmic extraction reagent I (0.2 mL), followed by the addition of cytoplasmic extraction reagent II (11 μL). The suspension was then incubated on ice for 60 s, followed by centrifugation (16,000 g, 5 min). The cytoplasmic extract was the supernatant fraction, and the crude nuclei formed the pellet fraction.  2.8. RNA Pull-Down Assays. Biotinylated lncRNA OXCT1-AS1, antisense lncRNA OXCT1-AS1, and lncRNA OXCT1-AS1 fragments were transcribed in A549 and 293T cells. The RNA products treated with RNase-free DNase I (Thermo Fisher Scientific) were purified using a Total RNA Purification Kit (Sangon Biotech Co., Ltd.). Then, 4 μg of biotin-labelled RNAs were denatured (65°C, 300 s) in PA buffer and slowly cooled. Next, the folded RNA was supplemented with 2 U/mL RNasin (Solarbio) and streptavidin Dynabeads (Thermo Fisher Scientific) and then incubated for 60 min at 4°C. After washing with wash buffer (4 × 5 min) and preclearing using streptavidin Dynabeads, the protein lysate was incubated with the folded RNA-bead complex and 20 μg/mL yeast tRNA at 4°C for 210 min. After washing, beads were boiled in 40 μL 1x SDS loading buffer for 600 s. Then, lncRNA OXCT1-AS1 interacting proteins were subjected to western blot analysis.  BioMed Research International 2.9. RNA Immunoprecipitation Assay. A549 cells were lysed in 500 μL of RNA immunoprecipitation buffer (Beyotime Institute of Biotechnology) containing protease inhibitors and an RNAase inhibitor. After centrifugation (8,000 g, 600 s). The supernatants were incubated with anti-Flag, anti-LEF1, anti-mouse IgG, or anti-rabbit IgG antibody for 120 min at 4°C (gentle rotation). Then, protein A/G beads (40 mL) were added and incubated for 60 min (4°C). After washing in RIPA buffer (3 times) and PBS (once), RNA was extracted and qRT-PCR was performed to detect the level of gene expression.
2.11. In Vivo Tumorigenesis in Nude Mice. Animal studies were implemented following the principles and procedures of the National Institutes of Health Guide for the Care and Use of Animals. Four-to-five-week-old BALB/c-nude mice (50% male and 50% female) were obtained from the Laboratory Animal Center of Jilin University (SCXK (Ji) 2016-0001) (Changchun, Jilin, China). The research procedures were reviewed and approved by the Medical Ethics Committee of China Medical University. Mice were allowed free access to food as well as water and maintained at 20 ± 2°C. For in vivo assay of cancer cell metastasis (ten mice per group), negative control or lncRNA OXCT1-AS1-(or LEF1-) knockdown A549 cell suspensions (1 × 10 6 cells, 100 μL) were injected into the tail veins of nude mice. BALB/c-nude mice were euthanized as soon as the following symptoms were detected: (a) inability to obtain water or food; (b) general lack of moving activities; (c) severe cachexia (weight loss approaching 25%); (d) pale appearance, body coat looking scruffy and unhealthy; (e) infection at the injection site; and (f) breathing problem. After 3 weeks, BALB/c-nude mice were anesthetized with 1% isoflurane and sacrificed by decapitation. Their lungs were harvested for hematoxylin and eosin (H&E) staining. The lung nodules were quantified microscopically by three observers. The body weights of nude mice were measured regularly.
2.12. Statistical Analysis. Each experiment was repeated in triplicate independent experiments. The data are shown as the mean ± SEM. GraphPad software 8 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS (IBM Corp., Armonk, NY, USA) were used for statistical analyses. Student's t-test was performed to evaluate the difference between two groups. The significance of the difference between multiple groups was analyzed by one-way analysis of variance followed by   BioMed Research International Tukey's post hoc test. Values of P < 0:05 were considered to indicate statistical significance. * P < 0:05 and * * P < 0:01 compared with BEAS-2B or sh-NC cells. # P < 0:05 and ## P < 0:01 compared with MG132 treatment.

lncRNA OXCT1-AS1 Is Upregulated in NSCLC Cell Lines.
In order to obtain lncRNA OXCT1-AS1 expression profiles in NSCLC, lncRNA OXCT1-AS1 levels were measured in different NSCLC cell lines via qRT-PCR assays. As shown in Figure 1(a), lncRNA OXCT1-AS1 levels were significantly elevated in NSCLC cells (H23, HCC827, H1299, and A549) compared with that in BEAS-2B cells; among these cancer cell lines, H1299 and A549 cells showed the highest upregulation of lncRNA OXCT1-AS1 expression. sh-lncRNA was synthesized to knock down the expression of lncRNA OXCT1-AS1, and successful interference was observed in H1299 and A549 cells relative to sh-NC (P < 0:01), as shown in Figure 1(b). Cell proliferation curves were drawn at different time points, and CCK-8 assays demonstrated that the viability of H1299 cells transfected with shRNA was obviously lower than that of the sh-NC group cells (P < 0:01; Figure 1(c)). Similar results were obtained in A549 cells (P < 0:01; Figure 1(d)). ). Interestingly, the body weights of mice were measured and were shown to be stable (Figure 3(d)). These results demonstrate that lncRNA OXCT1-AS1 might act as a key factor to promote metastasis in NSCLC.

lncRNA OXCT1-AS1 Interacts with LEF1 and Upregulates
Its Expression. To identify the mechanism underlying lncRNA OXCT1-AS1-induced metastasis in NSCLC, the distribution of lncRNA OXCT1-AS1 was first investigated in H1299 and A549 cells. The results shown in Figures 4(a) and 4(b) indicated that lncRNA OXCT1-AS1 was enriched in the cytoplasm. Then, downstream targets of lncRNA OXCT1-AS1 were predicted using bioinformatics databases. As shown in Figure 4(    To confirm the interaction between lncRNA OXCT1-AS1 and LEF1 protein, RNA pull-down assays were performed in A549 cells. The results manifested that lncRNA OXCT1-AS1 pulled down LEF1 but not GAPDH (Figures 4(h) and 4(i)). Additionally, RNA pulldown assays in 293T cells transfected with Flag-LEF1, lncRNA OXCT1-AS1, or its antisense lncRNA revealed an ability of lncRNA OXCT1-AS1 to pull down Flag-LEF1 (Figure 4(j)).
To further validate this interaction between lncRNA OXCT1-AS1 and LEF1, RNA immunoprecipitation assays were performed. As Figure 4(k) displays, LEF1 was associated with lncRNA OXCT1-AS1 but not control GAPDH mRNA. Deletion-mapping analysis indicated that the lncRNA OXCT1-AS1 fragments 1201 nt and 1731 nt were involved in the interaction with LEF1 (Figures 4(l) and 4(m)). We conclusively demonstrate that lncRNA OXCT1-AS1 interacts with LEF1 in NSCLC cells.
3.5. lncRNA OXCT1-AS1 Stabilizes LEF1 by Blocking NARF-Mediated Ubiquitination. To elucidate the potential mecha-nisms underlying the role of lncRNA OXCT1-AS1 in regulating LEF1 stability, we investigated the ubiquitination sites of LEF1 using the Protein Lysine Modifications Database (http://plmd.biocuckoo.org/index.php). The results of Figure 5(a) show that LEF1 had a ubiquitination site at the 54 position, which was close to the binding site of lncRNA OXCT1-AS1 on LEF1 at the 55 position. A549 cells were treated with MG132 to suppress LEF1 degradation. As shown in Figures 5(b) and 5(c), LEF1 levels were significantly reduced following lncRNA OXCT1-AS1 knockdown in the presence of MG132. This suggests that an enhancement of LEF1 stability by lncRNA OXCT1-AS1 is related to proteasomal degradation. Subsequently, we explored the role of lncRNA OXCT1-AS1 on LEF1 expression in the presence of CHX, as shown in Figures 5(d) and 5(e). We found that lncRNA OXCT1-AS1 knockdown shortened the half-life of LEF1. Additionally, lncRNA OXCT1-AS1 overexpression in 293T cells decreased the levels of LEF1 ubiquitination ( Figure 5(f)), which was also confirmed in A549 cells ( Figure 5(g)). To further investigate the mechanism by which lncRNA OXCT1-AS1 regulates LEF1 ubiquitination, E3 ubiquitin-ligase NARF was knocked down and overexpressed in A549 cells. As shown in Figures 5(h)   BioMed Research International (P < 0:01); however, NARF overexpression resulted in a remarkable decrease (P < 0:01). Additionally, lncRNA OXCT1-AS1 overexpression weakened the interaction between LEF1 and NARF in A549 cells, including NARF coimmunoprecipitating less LEF1 and LEF1 coimmunoprecipitating less NARF (Figures 5(k) and 5(l)). Interestingly, downregulation of lncRNA OXCT1-AS1 did not affect the levels of NARF (P > 0:05), as shown in Figures 5(m) and 5(n). Therefore, these results confirm that lncRNA OXCT1-AS1 abrogates NARF-mediated LEF1 ubiquitination by blocking the interaction between LEF1 and NARF, preventing LEF1 proteasomal degradation.
3.6. LEF1 Is Involved in Migration and Invasion of NSCLC Cells. To evaluate whether LEF1 is functionally involved in NSCLC cell metastasis, A549 and H1299 cells with LEF1 knockdown were established. The transfection efficiency of sh-LEF1 was determined by western blot assay (Figures 6(a)  and 6(b)). CCK-8 assays showed that the cell viability of H1299 cells with sh-LEF1 transfection was significantly decreased compared with that in the sh-NC group cells (P < 0:01, Figure 6(c)). Similar results were obtained in A549 cells (P < 0:01, Figure 6(d)). In functional experiments, LEF1 knockdown demonstrated a stronger ability to decrease the migration of H1299 and A549 cells compared with the control cells (Figures 6(e) and 6(f)). Importantly, sh-LEF1 markedly inhibited the invasion of H1299 and A549 cells (Figures 6(g) and 6(h)). Further, we investigated the correlation between LEF1 and proteins associated with epithelialmesenchymal transition. Western blot assay revealed that LEF1 knockdown significantly downregulated the expression of N-cadherin, vimentin, and Snail, while significantly upregulating E-cadherin expression, in H1299 and A549 cells (Figures 6(i)-6(l)).   (Figures 7(a)-7(c)). H&E staining indicated that the number of metastatic nodules in the lung was obviously lower in the sh-LEF1 group than in the sh-NC group (Figures 7(b) and 7(c)). Interestingly, the body weights of mice were shown to be stable (Figure 7(d)). These results collectively suggest that LEF1 plays a role in NSCLC cell metastasis.

Discussion
The 5-year survival rate of NSCLC is lower than 15%, primarily because of the occurrence of distant metastatic disease [23,24]. Protein modification is closely associated with metastasis in NSCLC [25]. Among the various complex regulatory networks of cancer metastasis, lncRNAs also play a key role in regulating tumor fate [26]. Despite the rapidly increasing number of lncRNAs with growing knowledge about their underlying mechanisms, studies of lncRNAs in NSCLC metastasis remain inadequate. Here, we characterize, for the first time, the functions of lncRNA OXCT1-AS1 in NSCLC metastasis and the mechanisms involved.
Our findings identify potential novel therapeutic targets for cancer metastasis.
In this study, we demonstrated that knockdown of lncRNA OXCT1-AS1 inhibited the invasion and migration in NSCLC cells. Additionally, the function and pathological significance of lncRNA OTUD6B-AS1 in NSCLC metastasis were also confirmed in vivo. Subsequently, we investigated the potential molecular mechanism by which lncRNA OXCT1-AS1 maintains LEF1 stability, and found that this involves the blockade of NARF-mediated LEF1 ubiquitination and proteasomal degradation in vitro. Moreover, we found that LEF1 stimulated cell migration and invasion in vitro and in vivo (Figure 8).
An increasing number of studies have demonstrated that lncRNA is involved in tumorigenesis and development of NSCLC. For example, results from Jiang et al.'s study [27] revealed that the lncRNA HOTAIR contributes to tumorigenesis and metastasis in NSCLC by upregulating miR-613 expression. Therefore, the identification of lncRNA signatures may be of clinical value in the diagnosis, treatment, and prognostic prediction of NSCLC. Recently, a study by Chen et al. [12] highlighted the important roles of lncRNA OXCT1-AS1 in the complex molecular processes that contribute to bladder cancer cell aggressiveness. lncRNA OXCT1-AS1 imprinted 1.7 kb is highly expressed in bladder cancer, metastastic lymph node tissues, and multiple bladder cancer cell lines [12]. A key finding of our study was that the expression of lncRNA OXCT1-AS1 was significantly enhanced in NSCLC cells compared with that in normal lung epithelial cells, especially in the H1299 and A549 cell lines, suggesting the clinical significance of this lncRNA in NSCLC. This observation is additionally consistent with Chen et al.'s report [12].
Extensive research has demonstrated the crucial roles of lncRNAs in NSCLC metastasis [28,29]. lncRNA PCAT6, a well-studied lncRNA, contributes to tumorigenesis and metastasis in NSCLC by binding to EZH2 [30]. Furthermore, results from He et al.'s study [31] showed that lncRNA AFAP1-AS1, an oncogene, promotes cell migration by increasing AFAP1 expression in NSCLC. In this study, lossof-function experiments revealed that depletion of lncRNA OXCT1-AS1 strongly inhibited NSCLC cell migration and invasion in vitro. Furthermore, in vitro function assays showed that lncRNA OXCT1-AS1 knockdown significantly reduced the number of metastatic nodules in the lung. These data confirmed that lncRNA OXCT1-AS1 induces NSCLC metastasis. Silencing lncRNA OXCT1-AS1 may represent a promising strategy to inhibit NSCLC metastasis.
lncRNAs have been shown to exert their functions through a variety of mechanisms, by directly interacting with proteins, mRNAs, and/or microRNAs [12,32]. In the present study, we performed cell nuclear and cytoplasmic RNA isolation and found that lncRNA OXCT1-AS1 was enriched in the cytoplasm, which suggests that lncRNA OXCT1-AS1 plays a role in metastasis in a posttranscriptionaldependent manner. However, the mechanisms by which lncRNA OXCT1-AS1 induces metastasis remain largely unknown, with numerous studies proposing that protein regulation is involved [33,34]. Using bioinformatics databases, 429 downstream targets were predicted. Considering that transcription factor LEF1 is reported to be involved in lung cancer metastasis [20], and there is a steric hindrance effect that the binding of lncRNA OXCT1-AS1 to the 55 position of LEF1 affects the ubiquitination of LEF1 at the 54 position, we selected LEF1 as a target. Interestingly, we found that lncRNA OXCT1-AS1 knockdown resulted in the attenuation of LEF1 protein expression. LEF1, a downstream factor of the WNT/β-catenin pathway, regulates gene transcription independently [35]. Extensive research has shown that LEF1 plays an essential role in the epithelial-mesenchymal transition by activating the transcription of N-cadherin, vimentin, and Snail [34,35]. Bleckmann et al. [36] reported that LEF1 overexpression correlates with poor prognosis in cerebral metastasis of lung adenocarcinomas. Therefore, we hypothesized that lncRNA OXCT1-AS1 maintains LEF1 stability to contribute to NSCLC metastasis. To verify this hypothesis, validation experiments were conducted, and showed that lncRNA OXCT1-AS1 directly interferes with LEF1 through its fragments 800 nt and 1201 nt, supporting our hypothesis. These findings also suggest that LEF1 stability is maintained by lncRNA OXCT1-AS1 at the posttranslational level.
Accumulating evidence shows that the stability of LEF1 can be controlled by its ubiquitination [37] and sumoylation [38]. Interestingly, recent research indicates that lncRNAs are capable of regulating protein ubiquitination [34,39]. For example, Tang et al. [40] found that the lncRNA GLCC1 stabilizes c-Myc by blocking ubiquitination via direct interaction with the HSP90 chaperone protein. In this study, we found that lncRNA OXCT1-AS1 blocked LEF1 ubiquitination, thereby preventing LEF1 proteasomal degradation. Previous studies have indicated that nemo-like kinaseassociated ring finger protein NARF regulates the ubiquitylation and degradation of LEF1 [41]. Therefore, we explored the role of NARF in lncRNA OXCT1-AS1-mediated LEF1 stability, and found that lncRNA OXCT1-AS1 knockdown facilitates the interaction between NARF and LEF1. These findings show that lncRNA OXCT1-AS1 blocks NARFmediated LEF1 ubiquitination by inhibiting the interaction between NARF and LEF1, thereby preventing LEF1 proteasomal degradation. Our findings are largely consistent with previous studies, in which constitutively activated STAT5a was shown to recruit the E3 ubiquitin-ligase NARF to LEF1 and lead to LEF1 ubiquitination and degradation [42]. These findings highlight the important role of lncRNA OXCT1-AS1 in NSCLC metastasis. Although lncRNA OXCT1-AS1 stabilized LEF1 in NSCLC, the other regulatory mechanisms for this lncRNA remain undefined.
An increasing number of studies indicate that LEF1 is essential for invasion, migration, and epithelial-mesenchymal transition in cancer cells [43]. For example, Rosmaninho et al. [44] found that Zeb1 potentiates LEF1 transcription to promote glioblastoma cell invasion. In the present study, we observed that LEF1 knockdown markedly reduced the migration and invasion of NSCLC cells. These functional roles of LEF1 were further confirmed by loss-of-function assays in vivo. In line with previous findings in esophageal squamous cell carcinoma [21], these results confirm that lncRNA OXCT1-AS1 promotes NSCLC metastasis by stabilizing LEF1.
In conclusion, the present study reveals, for the first time, that upregulation of the lncRNA OXCT1-AS1 induces NSCLC metastasis in vitro and in vivo. Mechanistically, our findings demonstrate that lncRNA OXCT1-AS1 blocks NARF-mediated LEF1 ubiquitination by inhibiting the interaction between LEF1 and NARF, thereby preventing LEF1 proteasomal degradation. Furthermore, LEF1 was confirmed to be involved in NSCLC metastasis. These findings establish a foundation for the application of lncRNA OXCT1-AS1 in diagnosis, prognostic evaluation, and therapy of NSCLC metastasis. Although these findings may lack clinical evidence, our study still have implications for treatment strategies of NSCLC metastasis.

Data Availability
All data generated or analyzed during this study are included in this published article. The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.