Long noncoding RNA MAGI2-AS3 inhibits bladder cancer progression through MAGI2/PTEN/epithelial-mesenchymal transition (EMT) axis

Abstract

BACKGROUND:

Long noncoding RNA (lncRNA) are critical regulators of tumor progression.

OBJECTIVE:

To determine how the lncRNA membrane associated guanylate kinase, WW and PDZ domain-containing 2 (MAG12) antisense RNA 3 (MAGI2-AS3) and the phosphatase and tensin homolog (PTEN) gene function in regulating bladder cancer (Bca) progression.

METHODS:

Total RNA from 80 Bca tissues and 30 paired para-cancerous tissues from patients was sequentially extracted, quantified, purified, and reverse transcribed using RT-PCR. A library was constructed and sequenced. Four Bca cell lines and a normal urothelial cell line were transfected with lentiviral plasmids, and cell migration and invasion were assayed in vitro. An orthotopic mouse model of Bca was created for in vivo studies.

RESULTS:

MAGI2-AS3 expression was significantly downregulated in Bca, compared with normal tissues, and negatively associated with tumor stage and a poor prognosis. MAGI2-AS3 and its sense RNA MAGI2 showed significant and positive correlation. The expression of MAGI2 and its downstream gene, PTEN, increased in Bca cells overexpressing MAGI2-AS3, and interference by MAGI2 expression reversed the migration and invasion inhibited by MAGI2-AS3 overexpression.

CONCLUSION:

MAGI2-AS3 overexpression inhibited Bca cell progression by regulating the MAGI2/PTEN/epithelial-mesenchymal transition, offering novel insights into the mechanism of Bca progression.

Keywords

Bladder cancer lncRNA MAGI2-AS3 progression

1. Introduction

Bladder cancer (Bca) is one of the most prevalent genitourinary tumors in men [1], among which approximately 75% are non-muscle invasive (NMIBC) and approximately 25% are muscle-invasive (MIBC) Bca [2]. The standard treatment for MIBC and high risk NMINC is radical cystectomy (RC) [2]. However, tumors in approximately 50% of the patients with MIBC who undergo RC progress with local recurrence or distant metastases [3]. Moreover, the median survival rate for patients with metastatic Bca rarely exceeds 6 months [4]. Thus, the overall prognosis of patients with Bca remains unsatisfactory. Therefore, a better understanding of the molecular mechanisms and the identification of biomarkers involved in Bca progression are urgently needed.

Long non-coding (lnc) RNA are $>$ 200 base pairs long and do not encode proteins. The aberrant expression of lncRNA in many solid tumors is critical for tumor progression (for example, lncRNA HULC in hepatocellular carcinoma [5], lncRNA SNHG4 in cervical cancer [6], and lncRNA SNHG3 in breast cancer [7]). Several lncRNA similarly promote Bca. For example, upregulated lncRNA urothelial cancer associated-1 (UCA1) promotes cell migration and invasion in Bca via the hsa-miR-145-ZEB1/2-FSCN1 pathway [8]. However, some poorly expressed lncRNA in Bca (e.g. lncRNA HCG22) can serve as protective regulators that suppress metastasis [9]. MAGI2-AS3 was originally discovered in breast cancer [10]. Subsequently, its anticancer effects, such as the inhibition of tumor proliferation, migration, and invasion, as well as the induction of apoptosis, were confirmed in non-small cell lung cancer (NSCLC) [11], hepatocellular carcinoma (HCC) [12], and high-grade serous ovarian carcinoma (HGSC) [13]. Although differential expression and roles of MAGI2-AS3 in Bca have been determined [14], the specific mechanisms of MAGI2-AS3, particularly for Bca progression, remain poorly understood.

Here, we analyzed the expression profiles of MAGI2-AS3 and its sense RNA MAGI2 in Bca tissues, and analyzed the correlations between the MAGI2-AS3 expression and clinical-pathological variables as well as prognosis. Moreover, we explored the functional importance and the detailed mechanisms of MAGI2-AS3 that influence Bca progression, and determined the significant correlation between MAGI2-AS3 and MAGI2 expression.

2. Materials and methods

2.1 Clinical samples

The Ethics Committee at the Affiliated Hospital of Jining Medical University approved the study, which conformed to The Code of Ethics of the World Medical Association (Declaration of Helsinki, 2013 amendment). All patients provided written, informed consent to collect resected tissue samples for research use. In all, 80 Bca tissues and 30 paired para-cancerous tissues, respectively, were obtained from patients diagnosed with Bca between March 2010 and May 2015, at the Department of Urology, Affiliated Hospital of Jining Medical University. All samples were immediately stored at $-$ 80 ${}^{\circ}$ C. Two pathologists diagnosed the tissues based on the World Health Organization 2004 [15], and the UICC 2009 guidelines [16].

2.2 High-throughput transcriptome sequencing

Total RNA was sequentially extracted, quantified, and purified, and the integrity was confirmed. Total RNA was enriched using magnetic beads with oligo (dT) (New England BioLabs, Inc., Ipswich, MA, USA) after digesting DNA with DNase I (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Short fragments that dissociated from the total RNA were sublimated and resolved in Elution buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Appropriate templates were amplified using OneTaq ${}^{\text{\textregistered}}$ DNA polymerase (New England BioLabs, Inc., MA, USA). Amplicons were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Technologies GmbH., Waldbronn, Germany) and a library was created using the ABI StepOnePlus Real-Time PCR System (Agilent Technologies, Inc.). The library was sequenced using a HiSeq™ 2000 system (Illumina, Inc., San Diego, CA, USA).

2.3 Cell culture and transfection of lentiviral plasmids

The Bca cell lines, T24, J82, 5637, RT4, and the normal urothelial cell line, SV-HUC, (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were respectively cultured in RPMI-1640 and L-15 media (both: Invitrogen, Carlsbad, CA, USA containing 1% ampicillin (100 units/mL), streptomycin (100 units/mL), and 10% (v/v) fetal bovine serum (FBS) at 37 ${}^{\circ}$ C under a 5% CO ${}_{2}$ atmosphere. The MAGI2-AS3 (LV-MAGI2-AS3) and negative control (LV-NC) lentiviral plasmids (Genechem Co., Ltd., Shanghai, China) were transfected into the cells as described by the manufacturer. When the cells reached 60%–70% confluence, the media were replaced with FBS-free media supplemented with the infection enhancers, HiTransG A or HiTransG (both from Shanghai GeneChem Co., Ltd., Shanghai, China). Thereafter, the cells were incubated with lentiviral plasmids at various concentrations and multiplicities of infection (MOI), added to the media for 12 h. These infective media were then replaced with complete media after 12 h of culture. During the first 48 h, the media contained 2 g/mL puromycin to kill uninfected cells and achieve maximal infection.

2.4 Quantitative real-time PCR

Total RNA was extracted from bladder tissues and cell samples using TRIzol™ Reagent (Thermo Fisher Scientific Inc., MA, USA) as described by the manufacturer. Vimentin, MAGI2-AS3, Snail1, E-cadherin, and N-cadherin expression was quantified using RT-qPCR and SYBR select master mix kits (Thermo Fisher Scientific Inc.) Expression levels of these genes were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Table S1 lists the primers for all genes. Data are presented as means of fold change compared with an internal reference.

2.5 Migration and invasion assays

Cell migration and invasion were determined using Transwell chambers. Chambers for invasion assays were inserted into Matrigel (Millipore Sigma Co., Ltd., Burlington, MA, USA), and activated under pure media at 37 ${}^{\circ}$ C under a 5% CO ${}_{2}$ atmosphere for 30 min. Media (approximately 800 $\mu$ L) were added to the lower chambers, then 5 $\times$ 104 cells were mixed in 500 $\mu$ L of media, and seeded in triplicate into the upper chambers in 24-well plates. After 24 h, the cells in the upper chamber were discarded, and the cells that had migrated or invaded the lower chamber were fixed in 4% formalin and stained with 0.1% crystal violet for 30 min. The chambers were then washed twice with phosphate buffered saline (PBS), and images were acquired using a camera attached to a microscope.

2.6 Mouse model of orthotopic Bca

Six 6-week-old female NOD/SCID mice (Shanghai Laboratory Animal Center, SLAC, Shanghai, China) were anesthetized via an intraperitoneal injection of 10% Nembutal (45 mg/kg), then an indwelling catheter was placed in the mice with paraffin oil. Residual urine in bladders was removed, then the bladders were washed twice with PBS, and flushed with 30 $\mu$ L of HCl (0.1 N) for 20 s via the indwelling catheters. Thereafter, 30 $\mu$ L NAOH (0.1 N) was injected into the bladders to neutralize the acid, and the bladders were washed twice with PBS. T24 cells (1 $\times$ 107/100 $\mu$ L) were injected into the bladders, and the urethras were ligated for 2 h. The mice were monitored weekly via whole-body, live-animal bioluminescence optical imaging using D-luciferin (150 mg/kg) assay kits (Gold Biotechnology, Inc., Olivette, MO, USA) and an IVIS Lumina II imaging system (PerkinElmer, Life and Analytical Sciences Inc., Waltham, MA, USA). The mice were sacrificed at 6 weeks after tumor-cell implantation and their bladders were weighed. Major organs and whole bladders were harvested and fixed in 10% formalin (Thermo Fisher Scientific Inc.) at 3-day intervals for 8 weeks.

2.7 Western blotting

Protein expression was quantified by western blotting. All samples were adjusted to a protein concentration of 1 g/L using the bicinchoninic acid (BCA) method, then 5 $\mu$ g portions were separated by SDS-PAGE and subsequently transferred to PVDF membranes (Pall Corporation, Port Washington, NY, USA). The membranes were washed with tris-buffered saline (TBS), then non-specific binding was blocked with non-fat milk diluted in TBS buffer, followed by an overnight incubation with primary antibodies (all from Abcam, Cambridge, UK) diluted 1: 1,000: anti-GAPDH, anti-MAGI2, anti-PTEN, Snail1, E-cadherin, N-cadherin, and vimentin (1:500). All membranes were then immersed in horseradish peroxidase (HRP) conjugated anti-rabbit secondary antibodies (1:5,000, Invitrogen). Target proteins were visualized and analyzed on a ChemiDoc MP System (Bio-Rad Laboratories Inc.).

2.8 Immunohistochemistry (IHC)

Paraffin-embedded tissue samples from orthotopic Bca models were assessed by immunohistochemistry (IHC) using polyclonal antibodies against MAGI2 (diluted 1:500; Thermo Fisher Scientific Inc.) and PTEN (1:50; Abcam), followed by a standard IHC protocol. Three pathologists independently evaluated staining intensity and the proportions of positively stained tumors.

2.9 Statistical analyses

All data were statistically analyzed using GraphPad Prism version 5.0 (GraphPad Software In., San Diego, CA, USA) and SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as means $\pm$ standard error of the means (SEM) from at least three independent assays. Differences between groups were compared using Student t-tests, and quantitative data were analyzed by chi square tests. Correlations between MAGI2-AS3 expression and common clinicopathological parameters were analyzed using Spearman rank tests. Differences in overall survival (OS) between high and low MAGI2-AS3 expression were determined using Kaplan-Meier curves and log rank tests. Values with two-tailed $P<$ 0.05 were considered statistically significant.

Figure 1.

Long non-coding RNA MAGI2-AS3, MAGI2, and PTEN expression in bladder cancer tissues determined by RNA-seq. (a) Heatmap shows differentially expressed lncRNA in RNA-seq data. Red box, expression profile of n410887 (MAGIS-AS3). Histograms show significant differences in MAGI2-AS3 (b) and MAGI2 (c) and PTEN (d) among three tissues. ** $P<$ 0.01, **** $P<$ 0.0001.

3. Results

3.1 Expression of lncRNA MAGI2-AS3 is downregulated in Bca tissues and cell lines

The results of RNA-seq data from 10 paired of Bca and para-cancerous tissues, and 5 base tissues, showed that 36 lncRNA were significantly downregulated by at least 3-fold in Bca, compared with para-cancerous and corresponding base tissues. These downregulated lncRNA included LINC01082 (ln375190), ADAMTS9-AS2 (ln384265), and ADAMTS9-AS1(ln339386).MAGI2-AS3, defined as n410887 in the NONCODE database was also differentially expressed (Fig. 1a and b). Like that of MAGI2-AS3, expression of the sense RNA, MAGI2 (Fig. S1), and its downstream gene, PTEN, was also downregulated in Bca tissues (Fig. 1c and d). Thus, we speculated that MAGI2-AS3 correlated with its sense RNA MAGI2 in Bca. Consequently, further investigated MAGI2-AS3.

We confirmed the expression profiles of lncRNA MAGI2-AS3 in 80 Bca and 30 paired para-cancerous normal tissues by qPCR. The expression levels of MAGI2-AS3 were significantly lower in Bca, than in normal tissues (Fig. 2a). Similarly, the expression of MAGI2 and PTEN in tissues was consistent with the RNA-seq data; expression was downregulated in cancer, compared with normal tissues (Fig. 2b and c). Furthermore, expression of the three genes was obviously higher in NMIBC, than in MIBC tissues (Fig. 2d–f).

Figure 2.

Expression profiles of lncRNA MAGI2-AS3, MAGI2, and PTEN in enlarged bladder cancer tissues validated by qRT-qPCR. Expression of (a) MAGI2-AS3, (b) MAGI2 and (c) PTEN is significantly decreased in Bca, compared with adjacent non-tumorous bladder tissues. Expression of (d) MAGI2-AS39, (e) MAGI2 and (f) PTEN is much lower in NMIBC, than in MIBC tissues. * $P<$ 0.05, ** $P<$ 0.01, *** $P<$ 0.001, **** $P<$ 0.0001.

3.2 Expression of lncRNA MAGI2-AS3 is negatively associated with less differentiation, advanced stage, and poor clinical prognosis

We classified the 80 patients with Bca into groups with high ( $n=$ 40) and low ( $n=$ 40) MAGI2-AS3 expression based on the median value. We compared age, gender, tumor number, tumor size, tumor grade, lymph node invasion, and TNM stage, and found that 31 (38.8%) of them had $<$ stage T2 cancer, whereas 49 (61.2%) 0 had stages T2–T4. We also found that 15 (15.0%) of the 80 patients had positive lymph node invasion. Therefore, we analyzed associations between MAGI2-AS3 expression and clinicopathological parameters using chi square tests. The results significantly associated downregulated MAGI2-AS3 with the number of tumors ( $P=$ 0.021), tumor stage ( $P=$ 0.001), and tumor grade ( $P=$ 0.000), but not with age ( $P=$ 0.073), gender ( $P=$ 0.499), tumor size ( $P=$ 0.501), and lymph node invasion ( $P=$ 0.102). Table 1 shows details of this correlation analysis. Potential prognostic differences between the two groups were assessed as overall survival (OS) using Kaplan-Meier curves. The first and third OS rates were 89.5% and 69.9%, respectively, and 79.4% and 39.7%, respectively, in the groups with high and low MAGI2-AS3 expression (Significantly different at $P=$ 0.029 log rank test; Fig. 3).

Figure 3.

Associations between lncRNA MAGI2-AS3 expression and prognosis of patients with bladder cancer. Kaplan-Meier curves show differences in overall survival between patients with high and low lncRNA MAGI2-AS3 expression ( $P<$ 0.05, log rank test).

3.3 Progression of Bca influenced by lncRNA MAGI2-AS3 positively modulating MAGI2 and downstream gene PTEN expression

The expression of MAGI2-AS3 and MAGI2 mRNA significantly correlated (Fig. 4a). Moreover, expression of the latter positively correlated with that of PTEN mRNA (Fig. 4b). Subsequent qPCR results verified these correlations among the three genes (Fig. 4c and d). Analysis using the GEPIA webtool in TCGA datasets revealed that MAGI2-AS3 expression positively correlates with MAGI2 expression (Fig. 4e). The correlation between MAGI2 and PTEN was not statistically significant, as trends in expression were the same for both genes (Fig. 4f). Thus, we investigated whether MAGI2-AS3 influences Bca progression by regulating the sense strand RNA MAGI2 and its downstream gene PTEN expression.

Table 1
Correlations between clinical-pathological characteristics and relative expression of MAGI2-AS3 in Bca tissues from 80 patients

Characteristics	Total	High expression ( $N=$ 40, 50.0%)	Low expression ( $N=$ 40, 50.0%)	$P$ value
Age
$<$ 65 year	38(47.5%)	15(39.5%)	23(60.5%)	0.073
$\geqslant$ 65 year	42(52.5%)	25(59.5%)	17(40.5%)
Gender
Male	70(87.5%)	34(48.6%)	36(51.4%)	0.499
Female	10(12.5%)	6(60.0%))	4(40.0%)
Tumor size
$<$ 3 cm	43(40.0%)	23(53.5%)	20(46.5%)	0.501
$\geqslant$ 3 cm	37(60.0%)	17(45.9%)	20(54.1%)
Tumor number
$<$ 3	50(62.5%)	30(70.0%)	20(30.0%)	0.021
$\geqslant$ 3	30(37.5%)	10(16.7%)	20(83.3%)
Tumor stage
$<$ T2	31(38.8%)	23(74.2%)	8(25.8%)	0.001
T2–T4	49(61.2%)	17(34.7%)	32(65.3%)
Lymph node invasion
No	68(85.0%)	37(54.4%)	31(45.6%)	0.102
Yes	12(15.0%)	3(25.0%)	9(75.0%)
Tumor grade
Low	32(40.0%)	28(87.5%)	4(12.5%)	0.000
High	48(60.0%)	12(25.0%)	36(75.0%)

Figure 4.

Correlations between expression of MAGI2-AS3 and MAGI2, and MAGI2-AS3 and PTEN. (a) Significantly positive correlation between MAGI2-AS3 and MAGI2 expression ( $r=$ 0.749, $P=$ 0.000) in RNA-seq data. (b) MAGI2 expression also positively correlates with PTEN expression in RNA-seq data ( $r=$ 0.774, $P=$ 0.000). Positive correlations between expression of (c) MAGI2 and MAGI2-AS3 and (d) PTEN verified by qRT-PCR ( $r=$ 0.433, $P=$ 0.001, and $r=$ 0.564, $P=$ 0.000, respectively). Correlations between expression of MAGI2 and MAGI2-AS3 (e) and PTEN (f) revealed using GEPIA webtool in TCGA datasets ( $r=$ 0.33, $P=$ 9.3*e-12, and $r=$ 0.086, $P=$ 0.085, respectively).

We applied western blotting and qPCR assays to determine changes in MAGI2 and PTEN expression while overexpressing MAGI2-AS3 in T24 and J82 Bca cells in vitro. WE found that MAGI2 and PTEN expression was upregulated when MAGI2-AS3 expression was increased (Fig. 5a and b). Metastatic sites occurred in the abdomen of orthotopic Bca models after 10 weeks in the T24-LV-NC, but not in the T24-LV-MAGI2-AS3 group (Fig. 5c). The IHC findings of the resected tumors revealed more MAGI2 and PTEN protein in the T24-LV-MAGI2-AS3, than the T24-LV-NC group (Fig. 5d). Furthermore, transwell assays showed significantly higher migration and invasion capacity in T24 Bca cells overexpressing MAGI2-AS3 together with interfering MAGI2, compared with T24 Bca cells overexpressing only MAGI2-AS3 (Fig. 6a). These findings indicated that the expression of interfering MAGI2 reversibly inhibited the migration and invasion capacity induced by overexpressing MAGI2-AS3. Finally, the results of western blotting and qPCR assays showed significantly downregulated MAGI2 and PTEN expression in the co-transfected group compared with the group overexpressing only MAGI2-AS3 (Fig. 6b and c).

Figure 5.

MAGI2-AS3 regulates MAGI2 and PTEN expression. (a) Western blots show more MAGI2 and PTEN protein expression in LV-MAGI2-AS3 than in LV-NC groups. (b) Obviously upregulated expression of MAGI2 and PTEN in LV-MAGI2-AS3, compared with LV-NC groups validated by RT-qPCR. (c) Distant metastatic sites are more likely to arise in T24-LV-NC, than T24-LV-MAGI2-AS3 mouse models of orthotopic bladder cancer. (d) MAGI2 and PTEN proteins are more intensely stained according to IHC assays in LV-MAGI2-AS3, than LV-NC models of orthotopic bladder cancer (magnification: $\times$ 200). * $P<$ 0.05, ** $P<$ 0.01, *** $P<$ 0.001.

Figure 6.

Functional correlation between MAGI2-AS3 and MAGI2 in terms of migration and invasion capacity of T24 Bca cells. (a) Findings of transwell migration and invasion assays show that fewer cells overexpressing (OE) MAGI2-AS3 combined with interfering MAGI2-AS3+si MAGI2 passed through polycarbonate membrane those OE MAGI2-AS3. (b) Western blots show significantly downregulated MAGI2 and PTEN expression in OE MAGI2-AS3+si MAGI2 than OE MAGI2-AS3. (c) Findings of RT-qPCR show significantly downregulated MAGI2 and PTEN expression in OE MAGI2-AS3+si MAGI2, than in OE MAGI2-AS3 group. * $P<$ 0.05, ** $P<$ 0.01, *** $P<$ 0.001.

3.4 Overexpression of lncRNA MAGI2-AS3 inhibits EMT pathway in Bca cells

Inactivating PTEN induces the EMT process and tumor metastasis [17]. Furthermore, gene ontology (GO) enrichment analysis of RNA-seq data revealed that differentially expressed genes associated with MAGI2-AS3 were significantly enriched on the extracellular matrix (Fig. S2), and that protease recruitment was mostly caused by EMT activation [18]. Hence, lncRNA MAGI2-AS3 probably inhibits Bca progression by inducing EMT inactivation. We therefore selected the vital genes Vimentin, Snail1, E-cadherin, and N-cadherin that are involved in the EMT pathway, and measured their protein levels expressed in LV-MAGI2-AS3 and LV-NC groups in both cell lines by western blotting. The expression of vimentin, Snail1, and N-cadherin was much lower in the LV-MAGI2-AS3, than the LV-NC groups, whereas that of E-cadherin was obviously enhanced (Fig. 7a). The mRNA expression of the corresponding genes detected using qRT-PCR showed much more Vimentin, Snail1, and N-cadherin mRNA expression in the LV-MAGI2-AS3, than the LV-NC groups, while E-cadherin mRNA expression was significantly downregulated in the LV-MAGI2-AS3 groups (Fig. 7b and c). Changes in gene expression on the background of MAGI2-AS3 overexpression suggested inhibition of the EMT pathway.

Figure 7.

Protein and mRNA expression of EMT-associated genes in LV-MAGI2-AS3 and LV-NC groups detected by western blotting and RT-qPCR. (a) Band intensities of vimentin, Snail1, and N-cadherin proteins are significantly lower in LV-MAGI2-AS3, than LV-NC groups in both cell lines, whereas E-cadherin protein expression is obviously enhanced. (b) Relative Vimentin, Snail1, and N-cadherin mRNA expression is significantly decreased in the T24-LV-MAGI2-AS3 compared with T24-LV-NC group. More E-cadherin mRNA is expressed in the T24-LV-MAGI2-AS3 group. (c) Relative expression of vimentin, Snail1, and N-cadherin mRNA expression is significantly decreased in J82-LV-MAGI2-AS3 than J82-LV-NC group. More E-cadherin mRNA is also expressed in J82-LV-MAGI2-AS3 group.

4. Discussion

Long non-coding RNA can regulate changes in gene expression in cancer development by recruiting chromatin-modifying complexes and directly targeting transcriptional or posttranscriptional changes [19]. Indeed, many upregulated and downregulated lncRNA play crucial roles in carcinogenesis and Bca metastasis. For example, high levels of UCA1 are expressed in Bca tissues, but not in non-neoplastic cancers [21, 20]. In contrast, the imprinted maternally expressed gene 3 (MEG3) encodes an lncRNA that not found in Bca tissues; it might serve as a tumor suppressor to inhibit Bca proliferation and invasion through MEG3-induced apoptosis and increased PHLPP2 protein translation [23, 22]. MAGI2-AS3 was initially identified in Bca as a tumor suppressor [10], as its overexpression inhibited the proliferation, migration and invasion of BCa cells [14]. The present study investigated how MAGI2-AS3 regulates Bca progression. We found significantly downregulated MAGI2-AS3 expression in Bca tissues compared with adjacent non-tumorous tissues, and that its expression negatively correlates with number of tumors, grade, stage, and prognosis. We discovered that MAGI2-AS3 overexpression might improve the expression of the sense strand RNA MAGI2 and its downstream gene, PTEN, and subsequently enhance the active EMT process, leading to the inhibition of Bca progression.

Indeed, the function of MAGI2-AS3 in tumors is inconsistent. Some investigators have indicated that MAGI2-AS3 functions in promoting cervical squamous cell carcinoma (CSCC) and gastric cancer (GC); a higher expression level of which reflects an increased capacity for more aggressive malignant progression and, to some extent, a shorter OS [26, 24, 25]. However, antagonistic roles have been validated in most solid tumors, including Bca [28, 27, 10], HCC [29, 12], and NSCLC [11, 30]. The low expression of MAGI2-AS3 was closely associated with tumor progression and metastasis, and overexpression obviously inhibited cancer cell proliferation, migration, and invasion. Recent studies have generally focused on the MAGI2-AS3 pathway in which suppressing the ceRNA sponging of some miRNA could interfere with the expression of tumor-associated genes. For example, the MAGI2-AS3/miR-374b-5p/SMG1 axis is suppressed during HCC progression [12], and MAGI2-AS3 regulates CCDC19 expression by sponging miR-15b-5p, thus, suppressing Bca progression [15]. The present study also found that MAGI2-AS3 plays an anti-cancer role in Bca. More importantly, based on the correlation between MAGI2-AS3 and MAGI2, we discovered that MAGI2-AS3 overexpression positively regulates the expression of MAGI2 and its downstream gene, PTEN, which is an important regulator for EMT activation. Such positive regulation of PTEN might lead to the inhibition of migration and invasion of Bca cells.

The EMT has been regarded as the critical process for the development and metastasis of various tumors including Bca [31]. Several lncRNA stimulate the EMT process [32]. MAGI2-AS3 was originally identified as an EMT-related lncRNA in gastric cancer. Ectopic MAGI2-AS3 promotes tumor progression by sponging miR-141/200a and maintaining ZEB1 overexpression [26]. However, whether MAGI2-AS3 regulates the EMT as a tumor suppressor remains poorly understood. Studies initially confirmed that suppressing MAGI2 in tumors destabilizes PTEN in cancer cells [34, 35, 33]. The inactivation of PTEN induces EMT and tumor metastasis [17, 36, 38, 37]. Overexpressed MAGI2-AS3 inhibits breast cancer progression by decreasing miR-374a and enhancing PTEN expression, and the MAGI2-AS3/miR-374a/PTEN axis is important for regulating tumor progression [28]. Furthermore, the target of miRNA-23a-3p is PTEN [30], and miRNA-23a-3p is the target gene of MAGI2-AS3, indicating that MAGI2-AS3 upregulation suppresses the proliferative and invasive capacity of NSCLC via miRNA-23a-3p/PTEN axis. Thus, although the exact regulation axis seemed non-conforming, PTEN was a downstream target gene of MAGI2-AS3. Therefore, we believe that the MAGI2-AS3/MAGI2/PTEN axis regulates Bca progression by influencing EMT activation. Combined with the changes in mRNA and protein expression of EMT-associated genes, including vimentin, Snail1, E-cadherin, and N-cadherin, we affirmed that the EMT pathway is crucial for the MAGI2-AS3-mediated regulation of Bca cell migration and invasion.

Although this study revealed some important insights into the detailed mechanism of MAGI2-AS3 in Bca, it has several limitations. Our findings were based on relatively few clinical samples from a single center. More comprehensive and objective findings should be independently validated in a larger cohort derived from several centers. Models of metastatic Bca do not exist; thus we could not provide sufficient objective evidence that MAGI2-AS3 inhibits Bca metastasis in vivo. Thus, optimized orthotopic models are urgently required. The specific mechanisms involved in the MAGI2-AS3 regulation of the MAGI2 and EMT pathways are unknown. Further studies should investigate the regulatory mechanism between MAGI2-AS3 and MAGI2 using chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), and Luciferase reporter assays.

In summary, the present study revealed that MAGI2-AS3 is a tumor suppressor in Bca, and that low MAGI2-AS3 expression positively correlates with numbers of tumors, grade, stage, and a poor prognosis. MAGI2-AS3 overexpression induced the expression of the sense strand RNA MAGI2 and its downstream gene, PTEN, which interfered with EMT activation, and led to the inhibition of Bca progression. These findings offer novel insights into the mechanism of Bca progression.

Footnotes

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-201421.

References

Ferlay

Colombet

Soerjomataram

Dyba

Randi

Bettio

Gavin

Visser

and Bray

, Cancer incidence and mortality patterns in Europe: Estimates for 40 countries and 25 major cancers in 2018, Eur J Cancer 103 (2018), 356–387.

Witjes

J.A.

Compérat

Cowan

N.C.

, De Santis M Gakis

Lebret

and Ribal

M.J.

, Van der Heijden AG Sherif

, EAU guidelines on muscle-invasive and metastatic bladder cancer: Summary of the 2013 guidelines, Eur Urol 65 (2014), 778–792.

Compérat

Larré

Roupret

Neuzillet

Pignot

Quintens

Houéde

Roy

Durand

Varinot

Vordos

Rouanne

and Bakhri

M.A.

Bertrand

Jeglinschi

S.C.

Cussenot

Soulié

Pfister

, Clinicopathological characteristics of urothelial bladder cancer in patients less than 40 years old, Virchows Arch 466 (2015), 589–594.

Sternberg

C.N.

and Vogelzang

N.J.

, Gemcitabine, paclitaxel, pemetrexed and other newer agents in urothelial and kidney cancers, Crit Rev Oncol Hematol 46 (2003), S105–115.

Zhang

Liao

Liu

Zhu

Tao

Liang

Zhang

and Zhang

, Long noncoding RNA HULC promotes hepatocellular carcinoma progression, Aging (Albany NY) 11 (2019), 9111–9127.

Hong

and WSMA

, Long non-coding RNA SNHG4 promotes cervical cancer progression through regulating c-Met via targeting miR-148a-3p, Cell Cycle 18 (2019), 3313–3324.

Lin

Chen

and Hu

, LncRNA SNHG3 promotes cell proliferation and invasion through the miR-384/hepatoma-derived growth factor axis in breast cancer, Hum Cell 33 (2020), 232–242.

Xue

Pang

Pan

and Chen

, Long non-coding RNA urothelial cancer-associated 1 promotes bladder cancer cell migration and invasion by way of the hsa-miR-145-ZEB1/2-FSCN1 pathway, Cancer Sci 107 (2016), 18–27.

Jiang

Zhang

Yang

Mai

Guo

and Liu

, Long noncoding RNA HCG22 suppresses proliferation and metastasis of bladder cancer cells by regulation of PTBP1, J Cell Physiol 235 (2020), 1711–1722.

10.

Yang

Zhang

Sun

and Dong

, Du S Liu

, Long non-coding RNA (lncRNA) MAGI2-AS3 inhibits breast cancer cell growth by targeting the Fas/FasL signalling pathway, Hum Cell 31 (2018), 232–241.

11.

Pang

and Xu

, LncRNA MAGI2-AS3 upregulates cytokine signaling 1 by sponging miR-155 in non-small cell lung cancer, Cancer Biother Radiopharm 35 (2020), 72–76.

12.

Yin

Yan

Shi

Zhang

Hou

and Jian

, LncRNA MAGI2-AS3 inhibits hepatocellular carcinoma cell proliferation and migration by targeting the miR-374b-5p/SMG1 signaling pathway, J Cell Physiol 234 (2019), 18825–18836.

13.

Gokulnath

, de Cristofaro T Manipur

P.T.

Soriano

A.A.

Guarracino

M.R.

and Zannini

, Long non-coding RNA MAGI2-AS3 is a new player with a tumor suppressive role in high grade serous ovarian carcinoma, Cancers (Basel) 11 (2019).

14.

Wang

Zhu

Yang

Huang

Xie

and Li

, Long noncoding RNA MAGI2-AS3 regulates CCDC19 expression by sponging miR-15b-5p and suppresses bladder cancer progression, Biochem Biophys Res Commun 507 (2018), 231–235.

15.

Patrick

D.J.

Fitzgerald

S.D.

Sesterhenn

I.A.

Davis

C.J.

and Kiupel

, Classification of canine urinary bladder urothelial tumours based on the World Health Organization/International Society of Urological Pathology consensus classification, J Comp Pathol 135 (2006), 190–199.

16.

Edge

S.B.

and Compton

C.C.

, The American Joint Committee on Cancer: The 7th edition of the AJCC cancer staging manual and the future of TNM, Ann Surg Oncol 17 (2010), 1471–1474.

17.

Perumal

Y.K.

Sun

Seung-Hyun

Suji

Jieying

and Yeun-Jun

, PTEN inactivation induces epithelial-mesenchymal transition and metastasis by intranuclear translocation of β-catenin and snail/slug in non-small cell lung carcinoma cells, Lung Cancer 130 (2019), 25–34.

18.

Bydoun

Sterea

ICG

Bharadwaj

A.D.

and Waisman

D.M.

, A novel mechanism of plasminogen activation in epithelial and mesenchymal cells, Sci Rep 8 (2018), 14091.

19.

Zhang

and Wang

, The complexity of bladder cancer: Long noncoding RNAs are on the stage, Mol Cancer 12 (2013), 101.

20.

Quan

Pan

Zhao

Dai

Yan

Liu

and Lai

, LncRNA as a diagnostic and prognostic biomarker in bladder cancer: A systematic review and meta-analysis, Onco Targets Ther 11 (2018), 6415–6424.

21.

H.J.

Sun

X.M.

Z.K.

Yin

Q.W.

Pang

Pan

J.J.

and Chen

, LncRNA UCA1 promotes mitochondrial function of bladder cancer via the MiR-195/ARL2 signaling pathway, Cell Physiol Biochem 43 (2017), 2548–2561.

22.

Feng

S.Q.

Zhang

X.Y.

Fan

H.T.

Sun

Q.J.

and Zhang

, Up-regulation of LncRNA MEG3 inhibits cell migration and invasion and enhances cisplatin chemosensitivity in bladder cancer cells, Neoplasma 65 (2018), 925–932.

23.

Huang

Liao

Jin

Xie

Zheng

Zhou

Jiang

X.R.

and Huang

, MEG3, as a competing endogenous RNA, binds with miR-27a to promote PHLPP2 protein translation and impairs bladder cancer invasion, Mol Ther Nucleic Acids 16 (2019), 51–62.

24.

Liu

Wang

Zhang

and Liu

, LncRNA MAGI2-AS3 is involved in cervical squamous cell carcinoma development through CDK6 up-regulation, Infect Agent Cancer 14 (2019), 37.

25.

Zhang

Jiang

Xie

Leng

and Song

, Comprehensive analysis of lncRNAs associated with the pathogenesis and prognosis of gastric cancer, DNA Cell Biol 39 (2020), 299–309.

26.

Wang

Zhang

She

Qiu

Zhang

Liu

and Qin

, LncRNA MAGI2-AS3 is regulated by BRD4 and promotes gastric cancer progression via maintaining ZEB1 overexpression by sponging miR-141/200a, Mol Ther Nucleic Acids 19 (2020), 109–123.

27.

Zhang

Zhuang

Liu

Ding

and Sun

, Integrative transcriptome data mining for identification of core lncRNAs in breast cancer, PeerJ 7 (2019), e7821.

28.

Zhao

Zhou

Wen

Zhao

and Liu

, Long non-coding RNA MAGI2-AS3 inhibits breast cancer cell migration and invasion via sponging microRNA-374a, Cancer Biomark 24 (2019), 269–277.

29.

Wang

Wei

Shao

Luo

and Lu

, lncRNA MAGI2-AS3 prevents the development of HCC via recruiting KDM1A and promoting H3K4me2 demethylation of the RACGAP1 promoter, Mol Ther Nucleic Acids 18 (2019), 351–362.

30.

Hao

X.Z.

and Yang

, LncRNA MAGI2-AS3 suppresses the proliferation and invasion of non-small cell lung carcinoma through miRNA-23a-3p/PTEN axis, Eur Rev Med Pharmacol Sci 23 (2019), 7399–7407.

31.

Derynck

and Weinberg

R.A.

, EMT and Cancer: More than meets the eye, Dev Cell 49 (2019), 313–316.

32.

Zhong

Huang

Tian

Jiang

and Chen

, lncRNA H19 regulates epithelial-mesenchymal transition and metastasis of bladder cancer by miR-29b-3p as competing endogenous RNA, Biochim Biophys Acta Mol Cell Res 1864 (2017), 1887–1899.

33.

Fan

Yin

Zhao

Liu

Yao

Cai

Wang

and Zha

, MAGI2 enhances the sensitivity of BEL-7404 human hepatocellular carcinoma cells to staurosporine-induced apoptosis by increasing PTEN stability, Int J Mol Med 32 (2013), 439–447.

34.

Kitamura

Seike

Okano

Matsuda

Miyanaga

Mizutani

Noro

Minegishi

Kubota

and Gemma

, MiR-134/487b/655 cluster regulates TGF-β-induced epithelial-mesenchymal transition and drug resistance to gefitinib by targeting MAGI2 in lung adenocarcinoma cells, Mol Cancer Ther 13 (2014), 444–453.

35.

Jiang

Yan

Guan

Zhang

Chen

Sun

Yao

Zhao

Jin

and Wei

, MiR-487a promotes TGF-β1-induced EMT, the migration and invasion of breast cancer cells by directly targeting MAGI2, Int J Biol Sci 12 (2016), 397–408.

36.

Wang

Zhao

Cao

Zhang

Wang

and Xu

, Metastasis suppressor protein 1 regulated by PTEN suppresses invasion, migration, and EMT of gastric carcinoma by inactivating PI3K/AKT signaling, J Cell Biochem 120 (2019), 3447–3454.

37.

Yang

Fan

Zhang

Gao

and Ma

, Role of miRNA-21/PTEN on the high glucose-induced EMT in human mesothelial peritoneal cells, Am J Transl Res 10 (2018), 2590–2599.

38.

Kim

Kang

H.S.

Lee

Y.J.

Lee

H.J.

Yun

Shin

J.H.

Lee

C.W.

Kwon

B.M.

and Hong

S.H.

, EGR1-dependent PTEN upregulation by 2-benzoyloxycinnamaldehyde attenuates cell invasion and EMT in colon cancer, Cancer Lett 349 (2014), 35–44.

Long noncoding RNA MAGI2-AS3 inhibits bladder cancer progression through MAGI2/PTEN/epithelial-mesenchymal transition (EMT) axis

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Clinical samples

2.2 High-throughput transcriptome sequencing

2.3 Cell culture and transfection of lentiviral plasmids

2.4 Quantitative real-time PCR

2.5 Migration and invasion assays

2.6 Mouse model of orthotopic Bca

2.7 Western blotting

2.8 Immunohistochemistry (IHC)

2.9 Statistical analyses

3.1 Expression of lncRNA MAGI2-AS3 is downregulated in Bca tissues and cell lines

Table 1 Correlations between clinical-pathological characteristics and relative expression of MAGI2-AS3 in Bca tissues from 80 patients

Footnotes

Supplementary data

References

Table 1
Correlations between clinical-pathological characteristics and relative expression of MAGI2-AS3 in Bca tissues from 80 patients