Long non-coding RNA HOXA11-AS facilitates cell migration and invasion by regulating microRNA-148a/WNT1 axis in gastric cancer

Recently, long non-coding RNAs (lncRNAs) have emerged as crucial players in the initiation and progression of multiple cancers including gastric cancer (GC). LncRNA HOXA11 antisense RNA (HOXA11AS) has been found to be an oncogenic factor in GC. However, the molecular mechanisms responsible for these oncogenic properties have not been thoroughly elucidated. In the present study, the abundances of HOXA11-AS, miR-148a and WNT1 in GC tissues and cell lines were examined by qRT-PCR. KaplanMeier survival analysis was conducted to explore the relationship between HOXA11-AS expression and GC patient prognosis. Transwell assay was performed to evaluate cell migratory and invasive capacities. Bioinformatics, dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA-pull down assays were employed to analyze the interaction between miR-148a and HOXA11-AS or WNT1. Tumor xenograft experiments were performed to further examine the infl uence of HOXA11-AS knockdown on GC tumorigenesis in vivo. The protein levels of WNT1 and β-catenin were assessed by western bolt assay. We found that HOXA11-AS and WNT1 expression was upregulated, while miR-148a expression was downregulated in GC tissues and cell lines. HOXA11-AS expression was associated with tumor size, lymph node metastasis, TNM stage, and overall survival in GC. HOXA11-AS promoted migration and invasion of GC cells through downregulating miR-148a. And, miR-148a inactivated WNT1/β-catenin signaling pathway via directly targeting WNT1. Moreover, HOXA11-AS induced the activation of WNT1/βcatenin pathway, which was abrogated by miR-148a overexpression in GC cells. Additionally, HOXA11-AS knockdown suppressed the growth of GC xenograft tumors by upregulating miR-148 and inactivating WNT1/β-catenin signaling pathway in vivo. These results suggest that HOXA11-AS overexpression promoted the tumorigenesis and progression of GC through activating WNT1/β-catenin pathway via repressing miR-148a, providing a prospective therapeutic target for GC. Research Article Long non-coding RNA HOXA11-AS facilitates cell migration and invasion by regulating microRNA-148a/WNT1 axis in gastric cancer Tao Guo1,2, Xiao Yuan3, De-Feng Liu3, Shi-Hao Peng3 and A-Man Xu1* 1Department of Gastrointestinal Surgery, The First Affi liated Hospital of Anhui Medical University, Shushan, Hefei, Anhui, 230031, China 2Department of Gastrointestinal Surgery, The Fourth Affi liated Hospital of Anhui Medical University, Baohe, Hefei, Anhui, 230041, China 3General Surgery Department, The Fourth Affi liated Hospital of Anhui Medical University, Baohe, Hefei, Anhui, 230041, China Received: 03 April, 2019 Accepted: 22 July, 2019 Published: 23 July, 2019 *Corresponding author: A Man Xu, Department of Gastrointestinal Surgery, The First Affi liated Hospital of Anhui Medical University, No.218, Shushan, Hefei, Anhui, 230031, China, Tel: +86-0551-6287-5159; +86137-0569-5470; Email:


Introduction
Gastric cancer (GC) is a serious public-health issue worldwide, ranking the fi rst in gastrointestinal cancer-related morbidity and mortality [1]. An estimated 26240 new cases and 10800 deaths from GC occurred in 2018 in the United States alone [2]. Despite the great advances in the management of GC, the prognosis remains dismal, especially for individuals diagnosed with advanced disease [3,4]. Cell migration and invasion are considered to be the major events that can drive cancer progression [5]. And, an in-depth understanding on the molecular basis involved in aggressive behaviors of GC contributes to the identifi cation and development of novel biomarkers or therapeutic targets.
LncRNAs are a group of transcripts larger than 200 nucleotides that can regulate various cellular processes, such as cell proliferation, apoptosis, invasion, and migration [6,7].
And, Li et al. pointed out that abnormal expression of HOXA11-AS was associated with tumor aggressiveness and poor clinical outcomes in multiple cancers [10]. Wang et al. revealed that HOXA11-AS expression was associated with glioma grade and poor prognosis, and HOXA11-AS overexpression promoted glioma tumorigenesis by regulating cell cycle progression [11].
Moreover, it is worth noting that HOXA11-AS level exhibits a signifi cant elevation in GC [12]. However, the roles and detail mechanisms of HOXA11-AS involved in GC tumorigenesis and progression remain unclear. A hypothesis suggests that lncRNAs can function as competing endogenous RNAs (ceRNAs) of miRNAs to regulate the expression of cancer-related target mRNAs [13]. In the present study, we performed a series of experiments to investigate the roles of HOXA11-AS in GC as well as its potential molecular mechanisms. Results showed that HOXA11-AS expression was signifi cantly upregulated in GC tissues and cell lines, and associated with the poor clinical outcome in GC. Besides, enforced expression of HOXA11-AS facilitated cell migration and invasion by miR-544a/WNT1/-catenin pathway. Our study provided a novel ceRNA network in GC and a potential biomarker or therapeutic target for GC management. The full-length fragments of HOXA11-AS were inserted into pcDNA3.1 vector to generate HOXA11-AS-overexpression plasmid (HOXA11-AS), with pcDNA3.1 vector as a negative control (vector). Small interfering RNA (siRNA) against HOXA11-AS (si-HOXA11-AS) and its control (si-NC), miR-148a mimic (miR-148a) and its negative control (miR-NC), miR-148a inhibitor (anti-148a) and its corresponding control (anti-NC) were purchased from Genepharma (Shanghai, China). SGC-7901 and MGC-803 cells were seeded into 6-well plates with a density of 1 × 10 5 cells/well, followed by the transfection of above products using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA).

RNA extraction and quantitative real-time PCR (qRT-PCR):
Total RNA was isolated from GC specimens or cultured cells using Trizol reagent (Thermo Fisher, Wilmington, DE, USA) and miRNA was extracted using mirVanaTM miRNA Isolation Kit (Thermo Fisher) referring to the manufacturer's procedures.

HOXA11-AS expression was upregulated and associated with poor prognosis in GC:
The level of wild type HOXA11-AS in GC tissues and adjacent normal tissues was fi rstly detected by qRT-PCR. Results showed that wild type HOXA11-AS level was strikingly upregulated in 30 GC tissues than that in adjacent normal tissues ( Figure 1A). Afterwards, we further explored the correlation of HOXA11-AS expression and multiple clinicopathological parameters. Results presented that HOXA11-AS expression in GC tissues was not associated with age and gender of GC patients (Table 1, P>0.05), but was related with tumor size, lymph node metastasis and TNM stage ( Table   1, P<0.05). Also, Kapan-Meier survival analysis revealed that patients with high HOXA11-AS expression had a poor overall survival compared with low HOXA11-AS expression group ( Figure 1B). Also, a notable upregulation of HOXA11-AS level was observed in SGC-7901 and MGC-803 cells compared with GES-1 cells ( Figure 1C). These results suggested that HOXA11-AS expression was upregulated and associated with poor prognosis in GC.

HOXA11-AS contributed to cell migration and invasion in GC:
To further explore the effects of HOXA11-AS on GC progression, HOXA11-AS overexpression plasmid (HOXA11-AS) and siRNA (si-HOXA11-AS) were constructed and synthesized. Following

HOXA11-AS could interact with miR-148a
Bioinformatics prediction analysis by miRcode website presents that there exists some complementary sequences between HOXA11-AS and miR-148a ( Figure 3A). pull-down assay followed by RIP is a powerful tool to confi rm the specifi c interaction between miRNA and lncRNA [14]. The results of RNA pull-down assay showed that the transfection of Bio-miR-148a-WT could induce the considerable enrichment of HOXA11-AS level in SGC-7901 cells compared with Bio-NC group, while the introduction of Bio-miR-148a-MUT had no much infl uence on HOXA11-AS level relative to Bio-NC group ( Figure 3D). In a word, these data unveiled that HOXA11-AS could bind with miR-148a by predicted binding sites.

HOXA11-AS modulated cell migration and invasion through miR-148a:
Subsequent qRT-PCR revealed that miR-148a expression was signifi cantly downregulated in GC tissues (n=30) and cells compared with their counterparts (Figure 4A,B). And, miR-148a expression was remarkably reduced in HOXA11-ASoverexpressed SGC-7901 cells, but was dramatically increased in HOXA11-AS-depleted MGC-803 cells ( Figure 4C,D), suggesting that HOXA11-AS could negatively regulate miR-148a expression in GC cells. Next, rescue experiments were performed to investigate whether the effects of HOXA11-AS on GC cell migration and invasion were mediated by miR-148a. Results showed that miR-148a overexpression suppressed the elevation of cell migratory and invasive capacities induced by HOXA11-AS in SGC-7901 cells ( Figure 4E,G). And, miR-148a knockdown reversed the detrimental effects of HOXA11-AS loss on cell migration and invasion in MGC-803 cells ( Figure 4F,H). Taken together, HOXA11-AS contributed to cell migration and invasion through downregulating miR-148a in GC.

WNT1 was an authentic target for miR-148a:
Next, TargetScan online website was used to seek for potential targets of miR-148a. Among these candidate targets, WNT1 was selected because of its oncogenic role in GC as previously reported [15] (Figure 5A). To further confi rm the interaction between miR-148a and WNT1, WNT1-WT and WNT1-MUT reporters containing wild-type or mutant miR-148a binding sites were constructed. Subsequent luciferase reporter assay showed that the luciferase activity of WNT1-WT reporter was dramatically reduced in miR-148a-overexpressed 293Tcells compared with miR-NC group ( Figure 5B). However, miR-148a overexpression  or not had no much impact on luciferase activity of WNT1-MUT reporter ( Figure 5B). That was to say, miR-148a could interact with WNT1 3'UTR by predicted sites. Next, we found that WNT1 expression was notably upregulated in GC tissues and cell lines relative to corresponding negative controls ( Figure 5C,D). Also, RT-qPCR assay demonstrated that miR-148a level was markedly elevated in SGC-7901 cells transfected with miR-148a mimic, but was remarkably downregulated in MGC-803 cells transfected with anti-miR-148a ( Figure 5E).
And, miR-148a loss alleviated the inhibitory effects of HOXA11-AS knockdown on WNT1 and -catenin expression ( Figure 6B).

HOXA11-AS knockdown inhibited GC xenograft tumor growth via the regulation of miR-148a/WNT1/-catenin pathway in vivo.
Subsequent in vivo experiments presented that HOXA11-AS knockdown resulted in the remarkable reduction of GC xenograft tumor volume ( Figure 7A) and weight ( Figure 7B), suggesting that HOXA11-AS loss hindered GC tumorigenesis in vivo. Next, qRT-PCR assay validated that HOXA11-AS expression was notably reduced in GC xenograft tumors infected with sh-HOXA11-AS lentiviruses ( Figure 7C). And, increased miR-148a expression ( Figure 7D) and reduced WNT1and -catenin expression ( Figure 7E) was observed in HOXA11-AS-depleted GC xenograft tumors. MMP-2 and MMP-9, two major members of matrix metalloproteinase (MMP) family, can lead to tumor metastasis by degrading collagen in the extracellular matrix (ECM) (16,17). Our study further demonstrated that HOXA11-AS defi ciency triggered the remarkable downregulation of MMP-2 and MMP-9 levels in GC xenograft tumors ( Figure 7E).
In a word, these data indicated that knockdown of HOXA11-AS suppressed GC xenograft tumor growth and metastasis via regulating miR-148a/WNT1/-catenin pathway in vivo.

Discussion
GC is a serious threat for human health. Till now, surgery is still the main therapeutic strategy for GC, but high recurrence rate heavily impairs the clinical outcomes after surgical resection. LncRNAs, initially thought to be transcriptional noise, are emerging as pivotal players in a variety of cancers including GC. Over the past decades, lncRNA HOXA11-AS has been found to be implicated in the initiation and progression of multiple solid tumors. For instance, HOXA11-AS overexpression accelerated cell proliferation and invasion in ovarian cancer [18]. HOXA11-AS loss repressed cell proliferation, migration, invasion, and angiogenesis, while induced cell cycle arrest and apoptosis in non-small cell lung cancer (NSCLC) [19]. And, Qu et al. suggested that HOXA11-AS expression was associated with advanced pathogenic status and poor prognosis of laryngeal squamous cell cancer (LSCC) patients, and HOXA11-AS depletion suppressed LSCC cell proliferation, migration and invasion [20].
Consistent with previous studies, we observed that HOXA11-AS level was obviously elevated in GC tissues and cell lines, and associated with tumor size, lymph node metastasis, advanced tumor stages and poor survival in GC. Previous studies used two different cell lines to investigate the effects of elevation or decrease of lncRNA HOTAIR and TINCR on cancer progression [21,22]. In this text, SGC-7901 cells were selected to conduct the gain-of-function investigations and MGC-803 cells were   [23].
Emerging evidence supports that lncRNAs can function as the molecular sponges of miRNAs to sequester miRNAs from their targets, leading to the increase of target levels [24,25].
In this study, we demonstrated that HOXA11-AS served as a ceRNA of miR-148a to relieve the inhibitory effect of miR-148a on WNT1 expression in GC cells. Functional restoration experiments revealed that miR-148a overexpression curbed the elevation of cell migratory and invasive abilities induced by HOXA11-AS in GC, suggesting that HOXA11-AS conferred the malignant phenotypes of GC cells via downregulating miR-148a. In agreement with our data, the tumor-suppressive roles of miR-148a have been elucidated in several types of cancers including GC. For instance, miR-148a level was signifi cantly reduced in GC tissues and miR-148a could regulate GC cell invasion via targeting MMP7 [26]. Enforced expression of miR-148a hindered cell growth and invasion through targeting TGF2 and SMAD2 in GC [27].
WNT1, a member of WNT gene family, has been widely reported to be involved in the regulation of cell fate and embryonic development. And, mounting evidences elucidate that WNT1 plays central roles in oncogenesis via regulation of -catenin-dependent signaling pathway [28,29]. For example, miR-148a weakened migratory and invasive capacities of breast cancer cells by targeting WNT1 and inactivating WNT/catenin signaling pathway [30]. MiR-185 functioned as a tumor suppressor by repressing WNT1 and blockading associated WNT/-catenin pathway in colon cancer [31]. Moreover, a previous report pointed out that miR-501-5p induced stem cell like phenotypes of GC by activating WNT/-catenin pathway via directly targeting WNT pathway antagonists such as DKK1, NKD1 and GSK3 [32].
In this study, we further demonstrated that WNT1 was a target of miR-148a and miR-148a inhibited WNT1 expression by direct interaction. -catenin is previously identifi ed as a key molecule of WNT/-catenin pathway [33]. Here, we also confi rmed that -catenin level was downregulated in miR-148a-overexpressed GC cells, but was upregulated in miR-148a-depleted GC cells. And, we did not fi nd the evidence to support the suppose that -catenin can be directly targeted by miR-148a. Thus, we believed that the inhibitory effect of miR-148a on -catenin expression was realized possibly by targeting WNT1. Following study further showed that miR-148a overexpression abolished the promotive effects of HOXA11-AS on WNT1 and -catenin expression, suggesting that HOXA11-AS activated WNT1/-catenin signaling pathway through modulation of miR-148a/WNT1 axis.
A large number of genes, such as transcription factor 3 (TCF3), zinc fi nger E-box binding homeobox transcription factor 1 (ZEB1), inhibitor of differentiation 2 (ID2), and SNAI2, have been found to be associated with the malignant metastatic phenotypes of tumor cells [34][35][36][37]. HOXA11-AS/ miR-148a/WNT1/-catenin reported in our study is one of multitudinous HOXA11-AS-involved regulatory mechanisms that are correlated with cell invasion and migration in GC.
In the following study, we intend to further explore whether HOXA11-AS can regulate GC initiation and progression by other signaling pathways.
In conclusion, our data indicated the correlation of HOXA11-AS expression and clinicopathological parameters of patients with GC. And, HOXA11-AS accelerated cell migration and invasion via modulation of miR-148a/WNT1/-catenin pathway. Our fi ndings uncovered a novel regulatory mechanism