lncRNA MNX1‑AS1 promotes prostate cancer progression through regulating miR‑2113/MDM2 axis.

A growing number of dysregulated long non‑coding (lnc)RNAs have been verified to serve an essential role in human prostate cancer. However, the underlying mechanisms of lncRNA MNX1 Antisense RNA 1 (MNX1‑AS1) in prostate cancer has not been explored. Therefore, the present study aimed to explore the function of MNX1‑AS1 in prostate cancer tumorigenesis and investigate the in‑depth mechanism. The expression of MNX1‑AS1, microRNA (miR)‑2113 and murine double min 2 (MDM2) in prostate cancer tissues and corresponding normal tissues were assessed by reverse transcription‑quantitative PCR. The protein expression levels of MDM2 were detected by western blotting. LNCaP and PC‑3 cells were transfected with short hairpin (sh)‑MNX1‑AS1, miR‑2113 mimics, miR‑2113 inhibitor and pCDH‑MDM2 vector using Lipofectamine® 3000. Cell proliferation, migration and invasion abilities were assessed by CCK‑8 assay, colony formation and Transwell assay, respectively. Dual luciferase reporter assay was carried out to confirm the putative targets of MNX1‑AS1 and miR‑2113. Tumor formation experiment in nude mice was applied to evaluate the tumor growth effect of MNX1‑AS1 in vivo. The expression of MNX1‑AS1 was significantly upregulated in the prostate cancer tissues and cell lines. MNX1‑AS1 knockdown suppressed the abilities of cell viability and migration and invasion in vitro and inhibited tumor growth in vivo. Additionally, luciferase reporter assay revealed that MNX1‑AS1 could target miR‑2113 and negatively interacted with miR‑2113 in prostate cancer cells. miR‑2113 directly targeted to MDM2 and negatively modulated the expression of MDM2. Rescue assays suggested that the viability, migration and invasion of impaired cells triggered by transfection with sh‑MNX1‑AS1 alone could be recovered by co‑transfection with sh‑MNX1‑AS1 + miR‑2113 inhibitor or sh‑MNX1‑AS1 + pCDH‑ MDM2 vector. The present study demonstrated that MNX1‑AS1 promoted prostate cancer progression through regulating miR‑2113/ MDM2 axis.

Introduction

Prostate cancer (PC) is one of the most life-threatening diseases for males worldwide (1.3 million cases in 2018) and the leading cause of cancer mortality among men especially in developed countries (0.36 million cases in 2015) (1,2). Even though great achievements have been made in radiotherapy, brachytherapy, radical prostatectomy, extended pelvic lymph-node dissection and post-operative radiotherapy over the past several decades, the problems of untimely diagnosis and poor prognosis remain unresolved (3). Therefore, there is an urgent need to determine reliable and consistent biomarkers to optimize patient management.

Long non-coding RNAs (lncRNAs) are RNA transcripts >200 nucleotides and lncRNAs are not able to encode proteins but post-transcriptionally regulate gene expression (4,5). Increasing evidence has indicated that aberrant lncRNAs are observed in a variety of human diseases especially in PC, accelerating or maintaining disease progression (2). For example, A positive feedback loop between androgen receptor and ARLNC1 was identified in PC progression via RNA-RNA interaction (6). Additionally, Zhang et al (7) found that lncRNA HOTAIR can bind to androgen receptor and inhibit androgen receptor degradation by interacting with E3 ubiquitin ligase murine double min 2 (MDM2), thus promoting androgen receptor transcriptional activity in castration-resistant PC. Furthermore, Gu et al (8) discovered that lncRNA HOXD-AS1 promoted proliferation and castration resistance by recruiting WDR5 to mediate H3K4me3 in PC. Mechanistically, lncRNAs PART1 and PCAT1 accelerated PC progression via activating Toll-like receptor pathway and PH domain and Leucine rich repeat Protein Phosphatases/FK506-binding protein 51/IKKα respectively (9,10). However, Wu et al (11) suggested that lncRNA MEG3 inhibited the progression of PC via the miR-9-5p/QKI-5 axis. Therefore, previous reports have proved that lncRNAs function as carcinogens or tumor suppressors in in PC progression.

Although a growing number of dysregulated lncRNAs have been observed to contribute to PC progression (2,12), among the numerous lncRNAs, lncRNA MNX1 Antisense RNA 1 (MNX1-AS1) serves an important regulatory role in numerous cancers and diseases, for instance, MNX1-AS1 is modulated by TEA domain transcription factor 4 to contribute to gastric cancer progression partly by suppressing BTG2 and activating Bcl2 (13). In addition, MNX1-AS1 can promote progression of esophageal squamous cell carcinoma by regulating miR-34a/sirtuin (SIRT)1 axis (14) and other types of cancer including glioblastoma (15), cervical cancer (16) and non-small cell lung cancer (17). In particular, Li et al (18) previously reported that knockdown of MNX1-AS1 suppresses PC cell proliferation, migration and invasion, however the underlying molecular mechanism have not been studied. Hence, The present study investigated the function and molecular mechanism of upregulation of MNX1-AS1 in PC and explored the interactions among MNX1-AS1, miR-2113 and MDM2.

Materials and methods

Cell lines and PC tissues

The LNCaP, PC-3, C4-2B, Du-145 and RWPE1 cell lines were obtained from American Type Culture Collection. All cells were maintained in high-glucose Dulbecco's modified Eagle medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere of 5% CO2. Clinical tissues of prostate cancer (n=40) and corresponding normal tissues were collected from Binhai County Hospital of TCM (Jiangsu, China). The present study conformed to the standard by the Declaration of Helsinki. Informed consent was written by all patients and donors. The research protocol was approved by the Medical Ethics Committee of Binhai County Hospital of TCM (approval no. CC-10922-3).

Cell transfection

The negative control RNA (sh-NC), shRNA-MNX1-AS1, miR-2113 mimics, miR-2113 inhibitor and pCDH-MDM2 vector (OE MDM2) were constructed by Shanghai GenePharma Co., Ltd. The cell transfection was performed using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C and collected at 72 h after transfection for subsequent experimentation. The concentration of shRNAs, mimics and inhibitor was 50 nM/1×105 cells. The concentration of plasmids was 0.8 µg/1×105 cells.

RNA isolation and reverse transcription-quantitative (RT-q)PCR

Total RNA from tissues and cell lines were extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.). Then, cDNA was synthesized using the PrimeScript RT Master Mix (Takara Bio, Inc.) according to the manufacturer's protocol. The RT-qPCR assay was carried out by the YBR Green PCR Master Mix (Vazyme Biotech Co., Ltd.). The 2−ΔΔCq method was employed to calculate the relative expression levels of genes (19). The U6 and GAPDH were used to normalize the expression level. Primer sequences were as follows: lnc MNX1-AS1 forward, 5′-AAGGTAGCCACCAAACAC-3′ and reverse, 5′-AGACTCACGTAGCACTGT-3′; GAPDH, forward, 5′-GCTCTCTGCTCCTCCTGTTC-3′and reverse, 5′-ACGACCAAATCCGTTGACTC-3′; miR-2113 forward, 5′-TGTGCTTGGCTCTGTCA-3′ and reverse, 5′-GAACATGTCTGCGTATCTC-3′ and U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. Thermocycling conditions were as follows: 95°C for 15 min, then 95°C for 30 sec, 65°C for 30 sec, 72°C for 30 sec (35–45 cycles) and 72°C for 5 min.

Dual luciferase reporter assay

StarBase 2.0 identified the miR-2113 binding site in MNX1-AS1 and the downstream target gene of miR-2113 is MDM2 (20). The MDM2 3′-untranslated region (UTR)-wild type (WT) or MDM2 3′-UTR-mutant (MUT) and MNX1-AS1-WT or MNX1-AS1-MUT reporter vectors were constructed by Beijing Transgen Biotech Co., Ltd. and miR-2113 mimics binding sequence was inserted downstream of the firefly luciferase gene in psi-CHECK2 vector to synthesis the MDM2-WT or MNX1-AS1-WT and psi-CHECK2-MDM2-MUT or MNX1-AS1-MUT plasmids, respectively. The WT and MUT plasmids subsequently were co-transfected into in LNCaP and PC-3 cells along with miR-NC and miR-2113 mimics using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. Following transfection for 48 h, the cells were lysed and the relative luciferase activity was assessed using Dual Luciferase Assay System (Promega Corporation).

Cell counting kit-8 (CCK-8) and colony formation assay

The CCK-8 (Sen Beijia Bio Tech Ltd.) assays were applied to evaluate cell viability every 24 h. The transfected cells were incubated in 96-well plates. Following incubation with CCK-8 for 2 h at 37°C, the absorbance values of each well at 450 nm were measured by ELX-800 Microplate Reader (BioTek Instruments, Inc.). For colony formation assay, cells were seeded in 6-well plates following transfection. After culture for 15 days, 4% paraformaldehyde was used to fix the cell colonies for 30 min at room temperature and 0.1% crystal violet (Ameresco, Inc.) was subsequently employed to stain the cells for 20 min at room temperature. A light microscope (magnification, ×200) and a scanner was used to visualize the number of clone spots in three randomly selected fields of view.

Cell migration and invasion assays

A Transwell system (pore size, 5 µm; Costar; Corning, Inc.) was employed to examine the cell ability of migration and invasion. For the migration assay, 1×104 transfected cells were seeded in the upper chambers supplemented with serum-free medium, while the lower chambers were filled with medium containing serum. After 36 h of incubation, the migrated cells were fixed with 10% methanol and subsequently stained with 0.1% crystal violet (Ameresco, Inc.) for 30 min at room temperature, respectively. For the invasion assay, the upper chambers were precoated Matrigel at 37°C for 30 min and the same protocols were used as in the migration assay. A total of five random visual fields per well were imaged and analyzed under a Nikon Inverted Research Microscope Eclipse Ti light microscope (magnification, ×200; Nikon Corporation).

RNA-RNA pull-down assay

For RNA pull-down assay, synthesized Biotin-MNX1-AS1 was used as a probe. Briefly, LNCaP and PC-3 cells were trypsinized using trypsin (Gibco; Thermo Fisher Scientific, Inc.) and lysed using lysis buffer (Thermo Fisher Scientific, Inc.). The streptavidin-coated magnetic beads (Thermo Fisher Scientific, Inc.) were coated by biotinylated MNX1-AS1 (Bio-MNX1-AS1) and Bio-Oligo and then transfected into 1×106 LNCaP and PC-3 cells at 50 nM as a final concentration for 48 h before harvesting. One part of the lysate served as the input control. The lysate was incubated at 4°C with magnetic streptavidin-coated magnetic beads (Thermo Fisher Scientific, Inc.) for 48 h. The bound RNA complexes were washed with wash buffer (Thermo Fisher Scientific, Inc.) and subsequently collected by centrifugation at 9,600 × g for 10 min at room temperature and then the miR-2113 enrichment level was detected with RT-qPCR.

RNA immunoprecipitation (RIP) assay

RIP assay was utilized to explore the interaction of MNX1-AS1 with miR-2113. Magna RIP kit (EMD Millipore) was used for the immunoprecipitation experiments according to the manufacturer's guidelines. For the first step, LNCaP and PC-3 cells were lysed by RIP lysis buffer (MilliporeSigma) and then cultured with the conjugated Ago2-specific antibody (1:1,000; cat. no. 2897; Cell Signaling Technology, Inc.) or IgG which served as respective control (1:5,000; cat. no. 2985; Cell Signaling Technology, Inc.) magnetic beads in RIP buffer for 4 h at 4°C. The precipitated RNA was isolated and quantified by RT-qPCR to obtain the enrichment value.

Western blotting

Proteins were collected and extracted using Beyotime RIPA assay (Beyotime Institute of Biotechnology) and BCA kit (EMD Millipore) was used for determination of protein concentration. Samples (0.5 µg/lane) were separated by 10% SDS-PAGE and then transferred to PVDF membranes (EMD Millipore). Following blocking with 5% skimmed milk for 2 h at room temperature, membranes were incubated with primary antibodies against MDM2 (1:1,000 dilution; cat. no. 3521) and GAPDH (1:1,000 dilution; cat. no. 8884) (Cell Signaling Technology, Inc.) overnight at 4°C. Subsequently, the membranes were incubated with the HRP-conjugated secondary antibodies [anti-Rabbit IgG(H+L), 1:2,000 dilution; cat. no. A0208; Beyotime Institute of Biotechnology] at room temperature for 1 h. Finally, the membranes were visualized using Chemiluminescence Assay (Epizyme, Inc.). ImageJ 1 software (National Institutes of Health) was used for the quantification of band densities.

Immunohistochemistry (IHC)

IHC was performed as previously (21). Ultrasensitive™ S-P kit (Maixin-Bio, China) was used. In brief, Sections from paraffin-embedded tumor tissues from transplanted nude mice were fixed with 4% paraformaldehyde at room temperature for 20 min and embedded in paraffin, then cut into slices. The thickness of paraffin sections was 4 mm. Tissues were incubated with primary antibodies for MDM2 (1:5,000; cat. no. 86934; Cell Signaling Technology, Inc.) at 4°C overnight. The immunocomplex was visualized with DAB kit (Thermo Fisher Scientific, Inc.) at room temperature for 10 min, and the nucleus were counterstained with 1% hematoxylin at room temperature for 1 min. Images were captured with a light microscope (×200 magnification; Leica GmbH). Two pathologists who were blinded to the experiment evaluated the results separately.

Tumor formation experiment in nude mice

A total of 12 male five-week-old BALB/C nude mice (20–25 g) were purchased from Cancer Institute of the Chinese Academy of Medical Science. The stably transfected cells (2×106) were injected subcutaneously in the axillae of each mice. The padding material in the cages was changed twice a week. The temperature was maintained at 18–22°C and the humidity was 50–60%. Drinking water and a food supplement were provided 3–4 times a week. The behavior and food intake of the mice were monitored every day to maintain their health. Tumor volume was calculated every week. The maximum tumor weight was 8.5% of mouse body weight. Following inoculation for 24 days, in strict accordance with the principles of animal welfare, anesthesia was used to relieve the pain during sacrifice. Briefly, the sodium pentobarbital was mixed with sterile saline to make a 3% solution and injected intraperitoneally at a dose of 50 mg/kg (1–1.25 mg per mouse). The mice were sacrificed by cervical dislocation and mortality confirmed by cessation of heartbeat. Each tumor was isolated, weighed and measured. Based on Institutional Animal Care and Use Committee guidelines, tumor size <20 mm (2.0 cm) was considered the humane endpoint (22). The research protocol was approved by the Medical Ethics Committee of Binhai County Hospital of TCM Hospital (approval no. CC-10922-3).

Bioinformatics analysis

The data of MNX1-AS1 expression in PC patients were visualized in the Gene Expression Profiling Interactive Analysis v1 (GEPIA, http://gepia.cancer-pku.cn/), which contains datasets from The Cancer Genome Atlas. The DIANA tool of LncBase Predicted v.2 (http://carolina.imis.athenainnovation.gr/diana_tools/web/index.php?r=lncbasev2/index-predicted) was used to predict the targets of MNX1-AS1 and miR-2113.

Statistical analysis

The data were visualized by the GraphPad Prism 9.0. The results were described as mean ± standard deviation. All statistical significance was analyzed by SPSS 20.0 (IBM Corp.). The difference between tumor tissues and adjacent tissues was analyzed using the paired t-test; group differences were compared by one-way ANOVA with LSD post hoc test. The survival analysis was conducted with the Kaplan-Meier plots and the log-rank test. Correlations were analyzed by Pearson's correlation test. All experiments were performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

Results

MNX1-AS1 is upregulated in PC tissues and cells

To assess the role of MNX1-AS1 in PC, expression level in PC samples downloaded from the GEPIA database was first examined and the result showed that MNX1-AS1 was frequently overexpressed in PC tissues compared with normal tissues (Fig. 1A). PC tissues (n=40) and paired normal tissues were collected. RT-qPCR demonstrated that the expression level of MNX1-AS1 in PC tissues was markedly higher compared with matched normal tissues (Fig. 1B). The relationship between MNX1-AS1 expression and overall survival was assessed by KM-Plot analysis, and the upregulation of MNX1-AS1 indicated a worse prognosis in patients with PC (Fig. 1C). Furthermore, four PC cell lines (LNCaP, PC-3, C4-2B and Du-145) and one normal human prostate cell line (RWPE1) were chosen to measure the expression of MNX1-AS1 by RT-qPCR, demonstrating that MNX1-AS1 expression was statistically increased in PC cell lines compared to normal human prostate cell line (RWPE1; Fig. 1D). These results suggested that MNX1-AS1 might function as an oncogene in PC.

Figure 1.

MNX1-AS1 is upregulated in PC tissues and cells. (A) MNX1-AS1 was over-expressed in PC samples downloaded from the Gene Set Enrichment Analysis database. (B) Relative MNX1-AS1 expression in 40 PC tissue samples and 40 corresponding normal tissues using reverse transcription-quantitative PCR. (C) Kaplan Meier analysis was used to evaluate overall survival in PC patients. Patients with high MNX1-AS1 expression had worse overall survival rates compared with those with low MNX1-AS1 expression. (D) Relative MNX1-AS1 expression in different PC cell lines (LNCaP, PC-3, C4-2B and Du-145) and normal human prostate cell line RWPE1. *P<0.05; **P<0.01 vs. RWPE1. MNX1-AS1, lncRNA MNX1 Antisense RNA 1; PC, prostate cancer.

MNX1-AS1 promotes PC cell proliferation, migration and invasion

Control sh-NC, sh-MNX1-AS1#1 and sh-MNX1-AS1#2 were respectively transfected into LNCaP and PC-3 cell lines and the knockdown efficiency results indicated that MNX1-AS1 expression was clearly decreased (Fig. 2A). CCK-8 and clone formation assays were performed to determine the proliferation and clone formation abilities, respectively. As shown in Fig. 2B and C, the cellular proliferation and growth were significantly suppressed in LNCaP and PC-3 cell lines following sh-MNX1-AS1 transfection. In addition, the results of Transwell assay showed that the migration and invasion abilities were considerably reduced in LNCaP and PC-3 cell lines after sh-MNX1-AS1 transfection (Fig. 2D and E).

Figure 2.

MNX1-AS1 promotes PC cell proliferation, migration and invasion. (A) Reverse transcription-quantitative PCR analysis of knockdown efficiency of MNX1-AS1 in LNCaP and PC-3 cells transfected with sh-NC, sh-MNX1-AS1#1 and sh-MNX1-AS1#2, respectively. (B) CCK-8 assay results of cell viability in LNCaP and PC-3 cells transfected with sh-NC, sh-MNX1-AS1#1 and sh-MNX1-AS1#2, respectively. (C) Clone formation assays showing proliferation ability in LNCaP and PC-3 cells transfected with sh-NC, sh-MNX1-AS1#1 and sh-MNX1-AS1#2. Transwell assays showing (D) migration and (E) invasion ability in LNCaP and PC-3 cells transfected with sh-NC, sh-MNX1-AS1#1 and sh-MNX1-AS1#2; original magnification, ×200. Scale bars=50 µm. **P<0.01 vs. sh-NC. MNX1-AS1, lncRNA MNX1 Antisense RNA 1; PC, prostate cancer; sh, short hairpin; NC, negative control.

MNX1-AS1 can negatively interact with miR-2113 in PC

As predicted by the bioinformatics prediction website, MNX1-AS1 had a complementarity for the miR-2113 (Fig. 3A). To verify the complementarity between MNX1-AS1 and miR-2113, the dual-luciferase reporter assay was carried out and it was found that the relative luciferase activities were significantly suppressed in LNCaP and PC-3 cells following co-transfection with MNX1-AS1-WT and miR-2113 mimics compared with those co-transfected with MNX1-AS1-MUT and miR-2113 mimics (Fig. 3B). The relative miR-2113 enrichment in both LNCaP and PC-3 cells was significantly higher in bio-MNX1-AS1 group compared with bio-Oligo group as assessed with RNA pull-down assay (Fig. 3C). Similarly, the data of RIP-RT-qPCR assay demonstrated that both relative MNX1-AS1 and miR-2113 enrichment were increased in the AgO2 group in comparison with that in IgG group (Fig. 3D). In addition, the miR-2113 expression was markedly increased by MNX1-AS1 inhibition (Fig. 3E). PC tissues and corresponding normal tissues collected from PC patients were also used to evaluate the relative miR-2113 expression via RT-qPCR, suggesting that miR-2113 was downregulated in PC tissues (Fig. 3F). In addition, Pearson's correlation analysis found that a negative correlation existed between the expression of miR-2113 and MNX1-AS1 in PC (Fig. 3G). RT-qPCR was used to detect the expression of miR-2113 in both LNCaP and PC-3 cells transfected with miR-2113 mimics (Fig. S1A). The results showed that in cells transfected with miR-2113 mimics, the level of miR-2113 increased significantly compared with the control. The above results indicated that miR-2113 exhibits a negative correlation with MNX1-AS1 in PC.

Figure 3.

MNX1-AS1 can target miR-2113 and negatively interact with miR-2113 in PC. (A) MNX1-AS1 was predicated as a molecular sponge for miR-2113 using lncBASE. (B) Luciferase reporter assays showing relative luciferase activity in cells co-transfected with MNX1-AS1 WT + miR-2113 mimics and MNX1-AS1 MUT + miR-2113 mimics in LNCaP and PC-3 cells. (C) Relative enrichment of miR-2113 using RNA pull-down experiments in LNCaP and PC-3 cells. (D) Relative enrichment of miR-2113 and MNX1-AS1 using RNA immunoprecipitation-RT-qPCR experiments in LNCaP and PC-3 cells. (E) Relative miR-2113 expression in LNCaP and PC-3 cells after MNX1-AS1 knockdown. (F) Relative miR-2113 expression in 40 PC tissue samples and 40 corresponding normal tissues using RT-qPCR assay. (G) The association of the levels of miR-2113 and MNX1-AS1 was analyzed using Pearson's correlation analysis. **P<0.01. MNX1-AS1, lncRNA MNX1 Antisense RNA 1; miR, microRNA; WT, wild type; MUT, mutant; RT-qPCR, reverse transcription-quantitative PCR.

miR-2113 directly targets MDM2

Potential miR-2113 binding sites in the 3′-UTR of MDM2 were also predicted from a bioinformatics website (DIANA tool of LncBase Predicted v.2; Fig. 4A). To verify the complementarity between miR-2113 and MDM2, dual-luciferase reporter assay was performed and it was found that the relative luciferase activities were significantly suppressed in both LNCaP and PC-3 cells following co-transfection with MDM2-WT and miR-2113 mimics in contrast to those co-transfected with MDM2-mut and miR-2113 mimics (Fig. 4B). Furthermore, a significant reduction of MDM2 was detected in LNCaP and PC-3 cell lines following MNX1-AS1 inhibition via western blot analysis (Fig. 4C). Additionally, to assess the correlation between MDM2 and MNX1-AS1 in PC, expression level in PC samples downloaded from GEPIA database were examined and the result showed that MDM2 was over-expressed in PC tissues compared with that in normal tissues (Fig. 4D). In addition, a clear increase in MDM2 expression was observed in 40 PC tissues compared with corresponding normal tissues (Fig. 4E). Spearman correlation analysis found that a positive correlation existed between the expression of MDM2 and MNX1-AS1 in PC (Fig. 4F). The above data clarified that miR-2113 directly targeted MDM2.

Figure 4.

miR-2113 directly targets MDM2. (A) The putative binding sites between miR-2113 and MDM2 predicated by lncBASE. (B) Luciferase reporter assays showing relative luciferase activity in cells co-transfected with MDM2 WT + miR-2113 mimics and MDM2 MUT + miR-2113 mimics in LNCaP and PC-3 cells. (C) Relative MDM2 protein expression in LNCaP and PC-3 cells after MNX1-AS1 inhibition. (D) The expression of MDM2 in PC was provided based on the data from The Cancer Genome Atlas dataset utilizing Gene Expression Profiling Interactive Analysis. (E) Relative MDM2 expression in 40 pairs of PC tissue samples and corresponding adjacent normal tissues using reverse transcription-quantitative PCR. (F) The association of the levels of miR-2113 and MNX1-AS1 was analyzed using Pearson's correlation analysis. **P<0.01 vs. sh-NC. miR, microRNA; MDM2, murine double min 2; WT, wild type; MUT, mutant; PC, prostate cancer; NC, negative control.

MNX1-AS1 regulates PC cell proliferation, migration and invasion by regulating miR-2113/MDM2 axis

RT-qPCR was used to detect the expression of miR-2113 in LNCaP and PC-3 cells transfected with miR-2113 inhibitor (Fig. S1B). The results showed that in cells transfected with miR-2113 inhibitor, the level of miR-2113 was significantly decreased, indicating that the transfection was successful. In addition, the MDM2 protein expression was detected by western blotting in cells transfected with OE MDM2 (Fig. S1C). The results demonstrated that after MDM2 was overexpressed, its expression level increased significantly, which also indicated that its transfection was successful. MNX1-AS1 knockdown inhibited both mRNA and protein expression levels of MDM2 in LNCaP and PC-3 cells. In the present study, while co-transfection of miR-2113 inhibitor or pCDH-MDM2 vector reversed the effects of MNX1-AS1 silencing on MDM2 expression (Fig. 5A and B), CCK-8 and clone formation assays indicated that cell viability was inhibited in cells transfected with sh-MNX1-AS1, while this effect was abrogated by co-transfection with sh-MNX1-AS1 + miR-2113 inhibitor or sh-MNX1-AS1 + pCDH-MDM2 vector (Fig. 5C and D). Furthermore, Transwell assay confirmed that MNX1-AS1 knockdown inhibited cell migration and invasion ability, while this effect was reversed when miR-2113 inhibition or MDM2 over-expression at the same time (Fig. 5E and F). These data demonstrated that MNX1-AS1 modulated the abilities of proliferation, migration and invasion in PC cells via sponging miR-2113/MDM2 axis.

Figure 5.

MNX1-AS1 regulates PC cell proliferation, migration and invasion by sponging miR-2113. (A) Relative MDM2 mRNA expression in LNCaP and PC-3 cells transfected with sh-NC, sh-MNX1-AS1, sh-MNX1-AS1 + miR-2113 inhibitor and sh-MNX1-AS1 + pCDH- MDM2 vector, respectively. (B) Relative MDM2 protein expression in LNCaP and PC-3 cells transfected with sh-NC, sh-MNX1-AS1, sh-MNX1-AS1 + miR-2113 inhibitor and sh-MNX1-AS1 + pCDH- MDM2 vector, respectively. (C) CCK-8 assay showing cell viability in LNCaP and PC-3 cells transfected with sh-NC, sh-MNX1-AS1, sh-MNX1-AS1 + miR-2113 inhibitor and sh-MNX1-AS1 + pCDH- MDM2 vector, respectively. (D) Colony formation assay showing cell proliferation ability in LNCaP and PC-3 cells transfected with sh-NC, sh-MNX1-AS1, sh-MNX1-AS1 + miR-2113 inhibitor and sh-MNX1-AS1 + pCDH- MDM2 vector, respectively. Transwell assays showing (E) migration and (F) invasion ability in LNCaP and PC-3 cells transfected with sh-NC, sh-MNX1-AS1, sh-MNX1-AS1 + miR-2113 inhibitor and sh-MNX1-AS1 + pCDH- MDM2 vector; original magnification, ×200. Scale bars=50 µm. **P<0.01. MNX1-AS1, lncRNA MNX1 Antisense RNA; PC, prostate cancer; MDM2, murine double min 2; sh, short hairpin; NC, negative control; OD, optical density.

MNX1-AS1 promotes PC proliferation in in vivo experiments

The oncogenic effect of MNX1-AS1 through MDM2 we reconfirmed in in vivo experiments. As shown in Fig. 6A and B, the results suggested that both the tumor volume and weight were significantly reduced in the sh-MNX1-AS1 group compared with the sh-NC group. MDM2 expression was also detected by IHC (Fig. 6C) and the result revealed that MDM2 expression was decreased in the sh-MNX1-AS1group compared with the control. In summary, MNX1-AS1 could promote PC progression through regulating miR-2113/MDM2 axis.

Figure 6.

MNX1-AS1 promotes PC proliferation in vivo. (A) Tumor volume in nude mice implanted subcutaneously with PC-3 cells transfected with sh-NC and sh-MNX1-AS1#1. (B) Tumor weight in nude mice implanted subcutaneously with PC-3 cells transfected with sh-NC and sh-MNX1-AS1#1. (C) MDM2 protein levels were detected in the xenografts by immunohistochemistry. Original magnification, ×200. Scale bar, 50 µm. ***P<0.001. MNX1-AS1, lncRNA MNX1 Antisense RNA; PC, prostate cancer; sh, short hairpin; NC, negative control; MDM2, murine double min 2.

Discussion

MNX1-AS1 is located in chromosome 7q36.3 (13). Previous studies have revealed that lncRNA MNX1-AS1 exerts oncogenic functions in various human cancers, including glioblastoma, lung cancer, esophageal squamous cell, cervical cancer, ovarian cancer, bladder cancer, hepatocellular carcinoma and breast cancer (14,15,23–27). For example, Liu et al transition (17,28) indicated that MNX1-AS1 facilitates the progression of non-small cell lung cancer and gastric cancer through engaging in epithelial-mesenchymal transition (17,28). In addition, MNX1-AS1 is proved to respectively activate MAPK pathway and AKT/mTOR pathway in cervical and breast cancer (16,29). Furthermore, MNX1-AS1 acts as a sponge of miR-4443 in glioblastoma cells and directly targets miR-34a/SIRT1≈axis in esophageal squamous cells (14,15). Recently, it was reported that MNX1-AS1, as a prognostic indicator in gastric cancer, can sponge miR-6785-5p by suppressing the transcription of BTG2 and activating Bcl2 (13). However, the molecule mechanism of MNX1-AS1 in PC has not been fully studied until the present study. The present study demonstrated that MNX1-AS1 expression was statistically increased in both PC tissues and cell lines. Additionally, MNX1-AS1 triggered the abilities of cell proliferation and migration and invasion, which was consistent with a previous report (18).

miR-2113 is located in chromosome 6q16.1 (30). Zhang et al (31) revealed the role of miR-2113 in human cancer and their results suggested that miRNA-2113 was downregulated in hepatocellular carcinoma tissues and could bind to eukaryotic initiation Factor 4A-III to accelerate malignant progression and epithelial-mesenchymal transition process. The present study identified that MNX1-AS1 targeted miR-2113 and modulated its expression negatively.

MDM2 is a negative regulator of p53 by engaging its amino-terminal transactivation domain with N-terminal hydrophobic pockets (32). Previous studies demonstrated that MDM2-p53 interaction was negatively regulated by miR-509-5p and mir-660, triggering tumorigenesis including cervical cancer, hepatocellular carcinoma and lung cancer (33,34). Thus, MDM2 is related to various types of malignancies (35). For instance, upregulation MDM2 has been detected in non-small cell lung cancer, gastric cancer and bladder cancer, acting as a sponge of miR-1305, miR-410 and miR-379-5p, respectively (36–38). Additionally, the 3′-UTR of MDM interacts with miRNA-509-5p and miR-129 to modulate the progression of prostate cancer and glioma (39,40). The data from the present study confirmed that MDM2 was upregulated in PC tissues and the 3′-UTR of MDM2 is a direct target of miR-2113. Furthermore, the upregulation of MDM2 and knockdown of miR-2113 completely reversed the suppressive effect of MNX1-AS1 inhibition in PC cell lines.

To summarize, the present study identified a potential therapeutic biomarker: MNX1-AS1. The data indicated that MNX1-AS1 is over-expressed in PC patients, posing promotive effects on proliferation, migration and invasion via miR-2113/MDM2 axis. Together, these findings offer an improved understanding of the pathological function of MNX1-AS1 in PC and partially provide a new sight to explore lncRNA- based therapeutics against cancers.