The lncRNA-MALAT1/miR-126-5p Axis Promotes Growth and Metastasis of Gastric Cancer through Regulation of VEGFA

Background: It has been reported that reduction of miR-126 can promote the progression of gastric cancer (GC). However, the regulation of miR-126 in GC is still unclear. This study aims to explore the correlation between lncRNA MALAT1 and miR-126 in gastric cancer and disclose the underlying mechanisms. Methods: We analyzed the correlation of MALAT1 levels and clinical features by analysis of bioinformatic data and human samples. Then we down-regulate the expression of MALAT1 in AGS cells and examined the characteristics of cell proliferation, cycle, apoptosis, migration, invasion, and the effect on miR-126 as well as VEGFA and signaling pathway. In addition, we demonstrated the role of MALAT1/miR-126 axis in GC with dual-luciferase reporter gene assay and treatment of miR-126 inhibitor. Results: The expression of MALAT1 was higher in cancer tissues than para-cancer tissues. In addition, high MALAT1 level suggested greater malignancy and poorer prognosis. Down-regulating the expression of MALAT1 in AGS cells inhibited cell proliferation, migration, and invasion by targeting VEGFA, which is consistent with up-regulation of miR-126. According to dual-luciferase reporter gene assay and treatment of miR-126 inhibitor, we demonstrated that MALAT1 down-regulated miR-126 in GC, which leads to the up-regulation of VEGFA and activation of mTOR signaling pathway. Conclusions: MALAT1/miR-126 axis promotes growth and metastasis of gastric cancer through regulation of VEGFA via mTOR signaling pathway.


Introduction
Gastric cancer (GC) is the most common malignant tumors in the digestive system, representing one of the dominant causes of cancer-associated deaths worldwide. Despite the advanced patient management and aggressive surgical techniques, there has been unsatisfactory improvement in the 5-year overall survival rate (1). Moreover, the molecular mechanisms underlying the progress of gastric cancer is still poorly understood. Further investigation is therefore required to elucidate the mechanisms underlying the development of GC and to identify novel therapeutic targets.
Long non-coding RNAs (lncRNAs) were served as critical mediators in various biological processes, including immune responses, angiogenesis, cell proliferation, apoptosis, autophagy, cell migration and invasion (2)(3)(4). A variety of studies have demonstrated that lncRNAs present critical roles in the development of various types of cancer, such as breast cancer, colorectal cancer, hepatocellular carcinoma (2)(3)(4)(5)(6)(7). In addition, a variety of previous studies have also reported the abnormal expression and functions of lncRNAs in cancers (5,6).
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a kind of lncRNA, which is closely associated with lung cancer progression and poor clinical outcomes (8). The upregulation of MALAT1 level has been discovered in lung cancer, pancreatic cancer, bladder cancer (9)(10)(11). Currently, Xia et al. reported that MALAT1 could be served as a diagnostic marker for GC metastasis (12). In addition, it has been reported that lncRNA MALAT1 can up-regulate VEGFA and promote angiogenesis (13). However, the mechanism underlying the promoting function of MALAT1 in GC progression is still unclear.
MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that regulated gene expression by translational repression or mRNA degradation (14). microRNA-126-5p (miR-126-5p), as an endothelial-speci c microRNA essential for governing vascular integrity and angiogenesis, participates in a wide range of biological function expression (15). It has been demonstrated that miR-126-5p was down-regulated in GC, and ectopic expression of miR-126-5p could inhibit cell proliferation and metastasis (16). In addition, Chen et al. illustrated that reduced miR-126-5p expression facilitates angiogenesis of GC through its regulation on VEGFA, which may share the same target gene with MALAT1 (15).
In the present study, the effect of MALAT1 on GC and the correlation between MALAT1 and miR-126 were investigated using in situ human GC tissue, in vitro GC cell lines. We identi ed the up-regulated expression of MALAT1 in GC tissue compared to the normal gastric tissue as well as an negative correlation between MALAT1 and miR-126-5p by targeting VEGFA and its downstream signaling molecules in AGS cell line. In addition, our present evidences indicated that MALAT1 was served as the competing endogenous RNA (ceRNA) with miR-126-5p.

Patients and specimens
A total of 7 benign gastric tissue, 29 poor-differentiated and 48 well-differentiated cancerous tissue were collected under surgical resection in the rst a liated hospital of Jinan university during 2015 Jan to 2019 Nov. All of the samples were saved in liquid nitrogen until further analysis. The whole patients didn't received any chemotherapy or radiotherapy before. This research was permitted by Ethics Committee of the rst a liated hospital of Jinan university.

Bioinformatic analysis
The website of Gene Expression Pro ling Interactive Analysis (http://gepia.cancer-pku.cn) was applied to inquire and analyze the expression differences of MALA1 between gastric cancer tissues (n = 408) and para-cancer tissue (n = 211). The gastric cancer data set from kaplan-meier Plotter online database (http:/kmplot.com/analysis/) was used for online data, and the gastric cancer samples were divided into the high-expression group and the low-expression group for survival analysis.
Cell culture GES1, MKN45, MGC803, and AGS cells were purchased from the Model Culture Collection (ATCC, Manassas, VA, USA). All of the cells were cultured with RPMI1640 medium. All complete medium consisted of 10% FBS (fetal bovine serum), 1% penicillin (100 U/mL) and streptomycin (0.1 mg/mL). Total cells were cultivated at 37℃ with 5% CO2. Reagents used in cell culture were bought from Gibco

CCK 8 assay
Cell proliferation was analyzed by using CCK 8 assay kit (Beyotime, China) according to the manufacturer instructions. Treated cells were incubated in 96-well plates with or without inhibitor for indicated times and followed by the addition of 10 μL CCK 8 reagent to each well. Samples were further incubated at 37 ℃ for 1 h. OD values were measured at 450 nm. All of the experiments were performed in triplicate.
Cell apoptosis and cell cycle assay Cells were harvested after speci c treatment, washed with ice-cold PBS for 3 times, and stained with annexin V-uorescein isothiocyanate (FITC) apoptosis detection kits (KeyGEN Biotech, Nanjing, China). Cell apoptosis was analyzed in a ow cytometer (BD Biosciences).
After trypsinization, AGS cells were washed with cold PBS. Cells were participated and cold ethanol (75%) was used to dissolve the cells, followed by incubation at 4 °C for 4 h. Cells were washed with cold PBS three times. After washing, cells were stained with BD Pharmingen™ PI/RNase for 30 min at 25 °C, followed by ow cytometer at different cell cycle phases (G1, S, and G2).

Western blot
After trypsinization, AGS cells were harvested and then lysed in RIPA buffer (RIBO-BIO) with protease inhibitors (Roche, Switzerland). The concentration of protein was examined by using the BCA Protein Assay kit (Genstar, China). Protein samples were separated by 10% SDS-PAGE, and then transferred to PVDF membranes (Millipore, Boston, MA, USA). Next, the membranes were blocked with 5% milk for 1 h at room temperature and following incubation of primary antibodies (anti-VEGFA, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, anti-p-ERK, anti-ERK, and anti-GADPH) at 4 °C overnight. The PVDF membranes were incubated for another 1 h at room temperature in secondary antibody after washing three times with TBST. Strips were exposed with StarSignal Plus Chemiluminescent Assay Kit (Genstar, China).

Transwell assay
Cells were cultured for 72 h, and then in serum-free medium for another 24 h. After detachment with 0.05% trypsin-EDTA the cells were resuspended in a serum-free medium. Upper insert was lled with 100 μl of the cell suspension while reservoir chamber was lled with 600 μl of culture medium. Matrigel invasion assays were carried out in modi ed Boyden chambers with lter inserts with 8-μm pores in 24well plates (Corning, NY, USA). The surfaces of the lters were coated with 50mg/L ice-cold Matrigel (Matrigel basement membrane matrix, BD Bioscience, NJ, USA).
Migration or invasion of cells was monitored at 3, 6, and 12 h at 37 °C in 5% CO2. Crystal violet was used as the staining solution to distinguish between migrated and non-migrated cells. A cotton swab was used to remove the cells that were left in the upper chamber of the membrane. Those cells that migrated through the insert were examined and counted with bright-eld microscope.

Dual-luciferase reporter gene assay
The relationship between miR-126-5p and MALAT1 was identi ed using dual-luciferase reporter gene assay. MALAT1 and VEGFA untranslated region was arti cially synthesized and inserted into pGL3control vector (Promega, Madison) between XhoI and BamH sites. Using site-directed mutagenesis, MALAT1 (MALAT1-MUT) and VEGFA mutant (VEGF-MUT) sequence was conducted on the basis of wild-type (WT) sequence. Recombinant plasmids were co-transfected into HEK 293T cells (Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Shanghai, China) with miR-126-5p mimic and the negative control (NC) of miR-126-5p, respectively. After transfection for 48 h, the cells were lysed for determination of luciferase activity, which was measured on a Luminometer TD-20/20 (E5311; Promega, Madison, WI, USA) using a dual-Luciferase Reporter Assay System kit (Promega, Madison, WI, USA).

Statistical analysis
All assays were conducted in three separate experiments. All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed with SPSS 22.0 software (SPSS, Chicago, IL, USA). The Student' s t-test was used to determine the statistically signi cant differences between two groups. Kaplan-Meier analysis was used to determine the effects on overall survival of EC patients. Differences were considered statistically signi cant when P values less than 0.05.

MALAT1 leads to poor clinical prognosis
According to the bioinformatic analysis of database, we discovered that the expression of MALAT1 was higher in carcinoma tissue than para-carcinoma tissue ( Figure 1A). And the Kaplan-Meier curve suggested the high MALAT1 levels led to shorter overall survival ( Figure 1B). Furthermore, we analyzed the MALAT1 levels in gastric cancer tissue with different malignancy and we found that the expression of MALAT1 in carcinoma tissues was positively associated with tumor malignancy. Poor-differentiated carcinoma expressed more MALAT1 than well-differentiated carcinoma, and the gastric cancer expressed more MALAT1 than benign gastric tissue ( Figure 1C). Moreover, the Kaplan-Meier curve was consistent with above result that, poor-differentiated carcinoma with higher MALAT1 levels presented poorer prognosis ( Figure 1D).

MALAT1 promotes cell proliferation, migration and invasion.
Because we have discovered that positive association between MALAT1 levels and tumor features, we next try to demonstrate the role of MALAT1 in gastric carcinoma cells. We examined four different gastric cells and discovered that the expression of MALAT1 was highest in AGS cells (Figure 2A). The PCR analysis showed that three Lnc-shRNA exerted good down-regulatory effect, among which Lnc-sh2 was most effective ( Figure 2B).
After transfection of Lnc-sh2 in AGS cells, cell viability of AGS cells were signi cantly decreased at 48 h and 72 h than normal AGS cells and NC transfected AGS cells ( Figure 2C). After transfection of Lnc-sh2 in AGS cells, the majority of cells were arrested in G0/G1 phase, which accounts for the results in cell viability assays ( Figure 2D). Furthermore, according to cell apoptosis assay, we found that knocking down the MALAT1 levels in AGS cells could induce cell apoptosis signi cantly ( Figure 2E). In addition, cell migration and cell invasion were also inhibited signi cantly after knocking down the expression of MALAT1 ( Figure 2F and 2G).

MALAT1 regulated VEGFA via Akt signal pathway
Due to the critical role of VEGFA in tumor progression, we examined the expression of VEGFA in four different gastric cancer and we found that AGS cell expressed more VEGFA than the other cells, which is consistent with the level of MALAT1 ( Figure 3A). Following PCR and WB analysis showed that knocking down the expression of MALAT1 signi cantly inhibited the activation of ERK and AKt signal pathway and expression of VEGFA ( Figure 3B and 3C). MALAT1 exerts promoting function by regulation of miR-126-5p.
The result of relative expression of miRNA-126 in AGS cells showed that there was higher level of miRNA-126 in Lnc-sh2 transfected AGS cells, which demonstrated strong negative association between MALAT1 and miRNA-126 ( Figure 4A). According to the dual luciferase experimental results, with the increase of miR-126-5p in AGS, the ability of expressing luciferase was suppressed in cell with dual luciferase carrier of MALAT1 target or VEGFA target. On the contrary, with the treatment of miR-126-5p inhibitor, the ability of expressing luciferase was enhanced signi cantly. However, after mutation of target, the expression capacity of luciferase showed no signi cant change. Above results demonstrated that MALAT1 was served as a "spongy body" to absorb miR-126-5p, while VEGFA was the target gene of miR-126-5p ( Figure 4B and 4C).
After knocking down the expression of MALAT1, cell viability was signi cantly inhibited, which could be reversed by treatment of miR-126-5p inhibitor ( Figure 4D). The result of cell cycle showed that less proportion of cells was arrested in G0/G1 phase after treatment of miR-126-5p inhibitor ( Figure 4E). In addition, treatment of miR-126-5p inhibitor signi cantly inhibited cell apoptosis ( Figure 4F). Furthermore, treatment of miR-126-5p inhibitor showed strong promoting function on cell migration and cell invasion, and the inhibitive function of MALAT1 could be totally reversed ( Figure 4G and 4H).

MALAT1/miR-126-5p axis regulate VEGFA via Akt signal pathway
After we demonstrated that the MALAT1 could regulate the ability of miR-126-5p and VEGFA was the downstream of MALAT1/miR-126-5p axis, we tried to demonstrated the underlying mechanisms. As is shown in Figure 5, the data of PCR and WB suggested that knocking down the expression of MALAT1 in AGS cell could inhibited the expression of VEGFA and activation of Akt signal pathway, which can be reversed by treatment of miR-126-5p inhibitor. Above results demonstrated that miR-126-5p was regulated by MALAT1 and MALAT1/miR-126-5p axis regulate VEGFA via Akt signal pathway.

Discussion
A variety of studies have demonstrated that lncRNAs serve crucial roles in the development of various types of cancer. However, rare studies focused on the function of lncRNAs in GC. Identi cation of cancerrelated lncRNAs and their down-streams is crucial for exploring their roles in tumor progression and for the development of novel therapeutic strategies. The present study investigated the role of lncRNA MALAT1 in GC and its underlying mechanisms.
lncRNA MALAT1 was rstly discovered in non-small cell lung cancer. Previous studies has suggested that MALAT1 promotes GC cell proliferation, migration and invasion, and can be served as a marker of GC in clinical practice (12,17). In addition, MALAT1 presents a critical role in angiogenesis in GC, which enhanced the growth and metastasis of GC (18). In our present study, we discovered that expression of MALAT1 led to poor prognosis and promoted growth and metastasis of GC by targeting VEGFA, which are consistent with these conclusions.
MicroRNAs (miRNAs) are a type of regulatory noncoding RNAs with a length of 20 to 25 bases, which can target speci c mRNA, interact with oncogenes and tumor suppressor factors (19). It has been demonstrated that more than 50% of genes are located in cancer-associated genomic regions, which suggested that miRNA may play a key role in tumor formation and progression (20). Abnormal expression of miR-126-5p is strongly associated with human tumorigenesis. In addition, Chen et al. reported that expression of miR-126-5p associated with higher MVD and VEGFA in GC, and dysfunction of miR-126 leads to tumor growth, which suggested that miR-126-5p shared the same target with MALAT1 (15). However, the regulation of miR-126-5p expression is totally unknown in GC.
Several studies have demonstrated that MALAT1 participated in the progression and development of tumor, served as ceRNA (21)(22)(23). To investigate the correlation between MALAT1 and miR-126-5p, we analyzed the expression of miR-126-5p after down-regulation of MALAT1 and we found that miR-126-5p can be down-regulated by MALAT1. In addition, we identi ed VEGFA as the same target gene shared by MALAT1 and miR-126-5p in GC cells based on the results of the luciferase reporter assay. To further clarify it, we down-regulated the expression of MALAT1 in AGS cells with lentivirus vectors and combined with treatment of miR-126-5p inhibitor. As a result, the expression of miR-126-5p was up-regulated with down-regulation of VEGF, and its downstream signaling molecules (p-AKT, p-mTOR, and p-ERK) as well as cell proliferation reversely correlated with miR-126-5p level. Moreover, the promoting function of MALAT1 can be also reversed by treatment with miR-126-5p.
Previous studies revealed that VEGF could activate ERK and Akt, two well known kinases, signaling pathway in ovarian cancer, hepatocellular carcinoma (24,25). In our present study, the results of in vitro experiment also suggested that Akt and ERK signaling pathway were involved in MALAT1/miR-126-5p/VEGFA axis in GC.
In conclusion, increase of MALAT1 played an important role in promoting tumor growth and metastasis, and MALAT1 could signi cantly reduce the level of miR-126-5p and enhance the expression of VEGF by Akt signaling pathway, thus counteract the proliferation and invasion of GC cells both in vitro. Understanding how MALAT1 is involved in GC will be bene cial to developing potential therapeutic targets in the clinical practice.