Long non-coding RNA MAGI2-AS3 inactivates STAT3 pathway to inhibit prostate cancer cell proliferation via acting as a microRNA-424-5p sponge

Aberrant expression of long non-coding RNAs (lncRNAs) that results in sustained activation of cell growth promoting pathways is an important mechanism in driving prostate cancer progression. In the present study, we explored differentially expressed lncRNAs in two microarray datasets of prostate benign and malignant tissues. We found that MAGI2-AS3 was one of the most downregulated lncRNAs in prostate tumors, which was further confirmed in our collected clinical samples. The function assays showed that MAGI2-AS3 overexpression decreased cell viability and led to obvious cell apoptosis in PC-3 and DU145 prostate cancer cells. Elevation of MAGI2-AS3 decreased the activity of STAT3 in PC-3 and DU145. In addition, microRNA-424-5p (miR-424-5p), a positive regulator of STAT3 pathway, was predicted as a target of MAGI2-AS3, furthermore, the interaction between MAGI2-AS3 and miR-424-5p was confirmed via reverse-transcript polymerase chain reaction (RT-qPCR), dual luciferase reporter assay and RNA immunoprecipitation (RIP). MAGI2-AS3 upregulated miR-424-5p and downregulated COP1 in PC-3 and DU145. More importantly, IL6-induced activation of STAT3 pathway could attenuate the biological effect of MAGI2-AS3 in PC-3 and DU145. In clinical samples, MAGI2-AS3 levels were negatively correlated with miR-424-5p expression, while positively correlated with COP1 mRNA expression. Altogether, the current study revealed MAGI2-AS3 as a novel negative regulator of prostate cancer development.


Introduction
Prostate cancer is the most prevalent cancer type for males worldwide, accounting for nearly 19% of newly diagnosed cancer cases in United States, 2018 [1]. In China, statistics suggest that the morbidity and mortality of prostate cancer has been gradually increased in recent years [2]. As the primary treatment approach for patients with prostate cancer, androgen deprivation therapy has greatly improved the overall survival of the patients. However, acquired and de novo resistance to the inhibition of androgen receptor signaling occur in these patients, which is closely associated with the death of patients with prostate cancer [3]. Therefore, it is important to study the molecular mechanisms of prostate cancer progression and provides novel targets and biomarkers for the treatment of patients.
Long non-coding RNAs (lncRNAs) are defined as single-stranded transcripts (more than 200 nucleotides) with no protein coding potential. Previously known as "junk" RNAs, recent studies have revealed that they play central roles in mediating normal physiology processes and the development of human diseases. Mechanistically, lncRNAs can act as microRNA (miRNA) sponge, directly interact with miRNAs, leading to downregulation of miRNAs and upregulation of miRNA targets. Most recent reports Ivyspring International Publisher have identified several oncogenic or tumor suppressor lncRNAs in prostate cancer via the miRNA sponge method [4][5][6]. For example, UCA1 (urothelial carcinoma associated 1) promotes prostate cancer progression via binding to miR-143 and increase MYO6 expression [4]. MEG3 enhances expression of QKI-5 via interaction with miR-9-5p in prostate cancer cells, therefore triggering cell apoptosis and inhibiting cell proliferation and motility [5]. The expression of MAGI2-AS3, which locates on chromosome 7q21.11, is associated with several cancer types such as breast cancer and bladder cancer [7][8][9]. A recent bioinformatic analysis shows that, MAGI2-AS3 in combination with 2 lncRNAs and 6 mRNAs, are signatures of prognostic prediction of patients with prostate cancer [10]. The precise molecular mechanisms and functions of MAGI2-AS3 have not been studied in prostate cancer yet.
In current study, we discovered that MAGI2-AS3 is a significantly downregulated lncRNA in prostate cancer according to previously published datasets, which was further confirmed in our collected clinical samples. In prostate cancer cell lines, we showed that MAGI2-AS3 inhibited cell proliferation and promoted cell apoptosis. In addition, MAGI2-AS3 sponged miR-424-5p, a miRNA with oncogenic potential, and inactivated STAT signaling in the studied cells. The data collectively demonstrated a tumor suppressor role of MAGI2-AS3 in prostate cancer.

Collection of patient samples
109 pairs of prostate tumors and adjacent non-tumor tissues were collected during surgery in China-Japan Union Hospital of Jilin University from July 2016 to June 2019. Inclusion criteria: Patients diagnosed with prostate cancer and has provided informed consents. Exclusion criteria: Patients received chemotherapy or other therapy before the surgery. These patients were aged from 45 to 82 with the average age of 66. The protocol of the current study was reviewed and approved by the Ethical Committee of China-Japan Union Hospital of Jilin University. These tissues were stored in -80℃ before subjected to RNA extraction.

Cell culture and treatment
The immortalized prostate myofibroblast stromal cell line WPMY-1, prostate cancer cell lines PC-3 and DU145 were obtained from ATCC (Manassas, VA). These cells were maintained in DMEM (Gibco; Thermo Fisher Scientific) supplemented with 10% FBS (Hyclone; Thermo Fisher Scientific). The culture condition was 5% CO2 and 37℃. Cells were treated with a mixture of IL6 (Sigma Aldrich) (10 ng/mL) or equal amount of vehicle (sterilized water) and the culture medium for 24 h. MAGI2-AS3 (Gene ID: 100505881) was inserted into pcDNA3.1 for overexpression. miR-424-5p mimic and miR-NC mimic were purchased from GenePharma (Suzhou, China). Plasmids and miRNA mimic were transfected into cells by Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific).

RNA extraction and realtime PCR (RT-qPCR)
Nuclear and cytoplasm RNA was isolated by Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Ontario, Canada) following manufacturer's protocol. Tissues and cells were both treated with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Carlsbad, CA) for the extraction of total RNA. RNA was reverse transcribed into cDNA via M-MLV Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific). The RT-qPCR was carried out using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The protocol was as following: We selected GAPDH and U6 as internal controls of mRNA and miRNA, respectively. The relative expression of each gene was calculated using the comparative ΔΔCt method.

RNA immunoprecipitation (RIP)
The RIP assay was applied to study the interaction between MAGI2-AS3 and miR-424-5p using the EZMagna RIP RNA-binding protein immunoprecipitation kit (Millipore) according to the manufacturer's instructions. Briefly, prostate cancer cells were lysed with RNA lysis buffer supplemented with protease and RNase inhibitors. The lysate was incubated with magnetic beads pre-coated with Ago2 antibody (Cat. 10686-1-AP, ProteinTech) or a control IgG (Cat. ab133470, Abcam) at 4°C overnight. Then, the RNAs were subjected to RT-qPCR to detect MAGI2-AS3 and miR-424-5p expression.

Determination of cell proliferation and cell apoptosis
The cell proliferation was assessed by the CCK-8 kit (AbMole, Houston, TX). Medium was mixed with CCK-8 solution, incubated with cells for 2 hours, then transferred to a new 96-well plate. OD 450 of each well was read by the Microplate Reader to manifest the cell number.
The percentage of apoptotic cells was detected by Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China). Cells were collected and stained with Annexin V-FITC and PI sequentially. After that, cells were subjected to flow cytometry analysis and the data were analyzed with FlowJo software. Cells were distributed in 4 phases according to the PI and FITC status. FITC+/PI+ and FITC+/PIcells were the apoptotic cells.

Statistical analysis
All data were calculated and the graphs were generated by the GraphPad Prism 6.0. Student's t test was used to compare the differences between 2 groups. For the differences among 3 groups, they were analyzed by one-way ANOVA followed by Tukey test. Pearson analysis was selected to study the association between gene expression. All experiments were repeated three times. P value less than 0.05 was considered as statistically significant.

MAGI2-AS3 was a downregulated lncRNA in prostate cancer
To explore the differentially expressed lncRNAs in prostate cancer, we firstly retrieved expression profiles of prostate cancer and benign prostate from GSE46602 and GSE55945. MAGI2-AS3 was found to be one of the most significantly downregulated lncRNA in prostate cancer from both GSE46602 and GSE55945 (Fig. 1A-B). For validation, we also analyzed MAGI2-AS3 expression in TCGA-PRAD dataset. Consistently, compared with 52 normal prostate tissues, MAGI2-AS3 was significantly decreased in 492 prostate cancers (Fig. 1C). We further collected patient samples. According to RT-qPCR results, MAGI2-AS3 was approximately 3-fold lower in prostate cancer than in the adjacent normal tissues (Fig. 1D). Decreased MAGI2-AS3 expression was associated with high Gleason score (Fig. 1E). MAGI2-AS3 expression was not associated with age or tumor stage ( Table 1).

Overexpression of MAGI2-AS3 inhibited prostate cancer cell proliferation
We next detected MAGI2-AS3 expression in two prostate cancer cell lines (PC-3 and DU145) and the immortalized prostate myofibroblast stromal cell line WPMY-1. MAGI2-AS3 was decreased in PC-3 and DU145 compared with WPMY-1 ( Fig. 2A). We cloned full length of MAGI2-AS3 into pcDNA3.1 plasmid. After transfection, it was found that MAGI2-AS3 expression was increased 9-fold and 15-fold in PC-3 and DU145 cells respectively (Fig. 2B). Via separating nuclear and cytoplasm fractions of cells, we found that MAGI2-AS3 was mainly localized in cytoplasm of PC-3 and DU145 cells (Fig. 2C). Function assays showed that elevation of MAGI2-AS3 inhibited cell proliferation of both PC-3 and DU145 cells (Fig. 2D-E). In addition, we detected the percentage of apoptotic PC-3 and DU145 cells by flow cytometry analysis. We found that MAGI2-AS3 overexpression led to significant cell apoptosis in PC-3 and DU145 cells (Fig.  2F-G).

MAGI2-AS3 regulated prostate cancer cell proliferation via inactivation of STAT3 signaling
To study whether STAT3 signaling was pivotal for the function of MAGI2-AS3, we combined IL6 treatment with recombinant MAGI2-AS3 in PC-3 and DU145 cells. As we expected, IL6 administration attenuated the effect of MAGI2-AS3 overexpression on cell proliferation and apoptosis (Fig. 6A-D).

MAGI2-AS3 was correlated with miR-424-5p, COP1, Bcl-2 and Mcl-1 expression in clinical samples
RT-qPCR showed that miR-424-5p levels were upregulated in collected prostate tumors and its expression was negatively correlated with MAGI2-AS3 expression (Fig. 7A-B). We further found that COP1 mRNA levels were downregulated in collected prostate tumors, which was positively correlated with MAGI2-AS3 expression (Fig. 7C-D). In TCGA-PRAD dataset, it was revealed that MAGI2-AS3 expression was positively correlated with COP1 mRNA levels (Fig. 7E). In conclusion, the current data suggested that MAGI2-AS3 acted as a sponge for miR-424-5p to elevate COP1 expression and inactivated STAT3 signaling in prostate cancer cells (Fig. 7F).

Discussion
In recent years, a large number of aberrant expressed lncRNAs have been found to be associated with initiation, uncontrolled cell proliferation, metastasis and drug resistance of cancer [12]. In particular, researchers found that several lncRNAs could mediate resistance to cell death to maintain cell proliferation in prostate cancer cells [13,14]. According to WGCNA analysis of datasets (TCGA, GSE17951, GSE7076), Cai et al. found that MAGI-AS3 was one of the downregulated lncRNAs in prostate cancer and was one of prognosis-related genes for patients with prostate cancer [10]. In the present study, we also retrieved TCGA and GEO data (GSE46602), consistently, significant downregulation of MAGI2-AS3 was found. Moreover, we collected patient samples for validation. In consistent with the microarray and RNA sequencing data, RT-qPCR also showed that MAGI2-AS3 expression was decreased in prostate cancer. However, the role of MAGI2-AS3 in cancer was controversial. In non-small cell lung cancer, breast cancer and esophageal cancer, MAGI2-AS3 played a key role in inhibiting cell proliferation and serving as a radiotherapy sensitizer [15][16][17]; on the contrary, MAGI-AS3 functioned as an oncogenic potential in gastric cancer and colorectal cancer [9,18]. Herein, we found that in prostate cancer cells, forced elevation of MAGI2-AS3 inhibited prostate cancer cell proliferation and induced cell apoptosis, supporting a tumor suppressor role of MAGI2-AS3 in prostate cancer.

STAT3
signaling was one of the well-characterized cancer driver pathways in prostate cancer, which was critical for maintaining uncontrolled cell proliferation [19]. The activity of STAT3 signaling was tightly controlled by its interactors and several of them were excellent drug targets [20]. The hyper-activity of STAT3 signaling was partially due to the dysregulation of non-coding RNAs including lncRNAs in prostate cancer cells [21,22]. The activated STAT3 upregulated Bcl-2, Mcl-1 and several other apoptosis-related genes to mediate resistance to cell apoptosis [23]. As we showed in our findings, MAGI2-AS3 promoted prostate cancer cell apoptosis, and repressed the activity of STAT3 signaling. The findings added new sights into understanding the regulation of STAT3 signaling in prostate cancer.
Many lncRNAs exerted their function via acting as miRNA sponges. Mainly localized in cytoplasm, MAGI2-AS3 also sponged several miRNAs to regulate cancer progression. It was revealed that MAGI2-AS3 could sponge miR-141/200a, miR-25 and miR-3163 in different cell backgrounds [9,16,18]. Similar to MAGI2-AS3, miR-424-5p could act as an oncogene or tumor suppressor in different cancer types. miR-424-5p facilitated cancer progression of prostate cancer and inhibited hepatocellular carcinoma development [11,24]. Interestingly, we found a binding site for miR-424-5p in the middle area of MAGI2-AS3 sequence, we further confirmed that MAGI2-AS3 and miR-424-5p mutually repressed the expression of each other. Their direct interaction was confirmed by dual luciferase activity assay and RIP assays. miR-424-5p targeted E3 ligase COP1 to stabilize STAT3, thus promoting cell proliferation of prostate cancer cells [11]. Our data also showed that COP1 was upregulated and STAT3 was downregulated in prostate cancer cells with overexpression of MAGI2-AS3, moreover, the effects of MAGI2-AS3 on COP1 was reversed by miR-424-5p mimic. These results indicated that MAGI2-AS3 activated STAT3 signaling via sponge miR-424-5p in prostate cancer cells.
Collectively, the current study defined MAGI2-AS3 as a novel tumor suppressor in prostate cancer and suggested MAGI2-AS3 as a biomarker and drug target for the patients with prostate cancer.