MAGI2‐AS3 suppresses MYC signaling to inhibit cell proliferation and migration in ovarian cancer through targeting miR‐525‐5p/MXD1 axis

Abstract Ovarian cancer (OV) is one of the most lethal gynecological malignance in females, and usually diagnosed at advanced stages. Long noncoding RNAs (lncRNAs) exhibit their crucial functions in modulatory mechanisms of cancers. Substantive studies have proven the anti‐tumor role of MAGI2‐AS3 in multiple cancers, but the physiological functions of MAGI2‐AS3 in OV need more detailed explanations. The current study corroborated that overexpression of MAGI2‐AS3 executed inhibitory activity in OV via hindering cell proliferation, cell cycle, migration as well as invasion while promoted apoptosis. Moreover MAGI2‐AS3 bound with miR‐525‐5p and negatively regulated the expression of miR‐525‐5p. Further studies testified that MXD1 was a downstream target of miR‐525‐5p and the competing relationship between MAGI2‐AS3 and MXD1 were confirmed by RNA pull down. Based on the combination between MAX and MYC, we analyzed the effects of MAGI2‐AS3 on MXD1 and MYC, unveiling the competing relationship between MXD1 and MYC for binding to MAX. Finally, we constructed rescue assays to certify that MAGI2‐AS3 suppressed the course of OV via enhancing MXD1 expression. In summary, MAGI2‐AS3 repressed the progression of OV by targeting miR‐525‐5p/MXD1 axis, offering a novel insight into understanding OV at the molecular level.


| INTRODUCTION
Ovarian cancer (OV) is a malignant tumor in gynecologic pathology with a high morbidity and induces increasing deaths among the females across the global. 1,2 The primary factors contributed to late diagnosis are that there are no distinct symptoms in early stage and lack of advanced screening. 3,4 The majority of OV patients always missed the golden opportunity for surgery until the exact diagnosis. Although great improvements have been achieved in therapies for OV patients, the survival of advanced OV patients was unpromising and the prognosis within five years remained disappointing. 5 Therefore, it is critical to get a deep understanding of latent molecular mechanism underlying OV progression.
Long noncoding RNAs (lncRNAs), over 200 nucleotides in length, are subtype of non-coding RNAs without protein-coding ability. Emerging literatures illustrated that logical functions of MAGI2-AS3 in OV need more detailed explanations. The current study corroborated that overexpression of MAGI2-AS3 executed inhibitory activity in OV via hindering cell proliferation, cell cycle, migration as well as invasion while promoted apoptosis. Moreover MAGI2-AS3 bound with miR-525-5p and negatively regulated the expression of miR-525-5p. Further studies testified that MXD1 was a downstream target of miR-525-5p and the competing relationship between MAGI2-AS3 and MXD1 were confirmed by RNA pull down. Based on the combination between MAX and MYC, we analyzed the effects of MAGI2-AS3 on MXD1 and MYC, unveiling the competing relationship between MXD1 and MYC for binding to MAX. Finally, we constructed rescue assays to certify that MAGI2-AS3 suppressed the course of OV via enhancing MXD1 expression. In summary, MAGI2-AS3 repressed the progression of OV by targeting miR-525-5p/MXD1 axis, offering a novel insight into understanding OV at the molecular level.

K E Y W O R D S
MAGI2-AS3, miR-525-5p, MXD1, ovarian cancer lncRNAs participated in cancer initiation and progression, such as chemoresistance, metastasis and differentiation. 6,7 Importantly, numerous lncRNAs have been reported in OV. For example, lncRNA MALAT1 was highly expressed in OV cells and accelerated the invasion and proliferation in OV. 8 LncRNA SLC27A2 affected chemoresistance of OV to cisplatin via modulating miR-411. 9 LINC00319 boosted OV development via targeting miR-423-5p/NACC1 axis. 10 The recent studies revealed that lncRNA MAGI2-AS3 exerted inhibitory functions in bladder cancer 11 and breast cancer. 12 Nevertheless, the physiological functions of MAGI2-AS3 in OV need more specific explanations.
The purpose of our study was to identify the function and mechanism of MAGI2-AS3 in the process of OV.

'-CCGGGCCTCCATGTTAC C A T A C A A T C T C G A G A T T G T A T G G T A ACATGGAGGCTTTTTG-3') and nonspecific shRNAs as NC (termed shCtrl) (shCtrl: 5'-CCGGCTTACCTGATTCACCA C A G A T C T C G A G A T C T G T G G T G A A T C A G G T A
AGTTTTTG-3') as well as shRNAs specific to MAGI2-AS3 (sh-MAGI2-AS3: 5'-CCGGTTATAGGAAAGCTTTTA

PCR)
Total RNAs extracted from OV cells were achieved by Trizol reagent (Invitrogen) for converting into cDNA using Reverse Transcription Kit (Toyobo). The specific primers were synthesized by Invitrogen (18418012). SYBR Green Super Mix (Bio-Rad) was acquired for qPCR. Results were processed by the comparative 2 −ΔΔCT method. GAPDH and U6 acted as the loading controls to normalize the data. All primers were listed in Table S1.

| Cell proliferation assay
Proliferative ability of OV cells was assayed by EdU incorporation assay kit (Ribobio) after transfection. Cells were fixed and treated with 0.5% Troxin X-100 before staining with DAPI (Beyotime) for nuclear counterstaining. Cells were observed by fluorescent microscope (Leica).

| Flow cytometry
Cell apoptosis was quantitated by flow cytometry after double-staining with Annexin V-labeled with 7AAD and PE (BD Biosciences) as per the standard method. Transfected OV cells in the Binding buffer were stained in the dark for 15 min and then analyzed. To analyze cell cycle, cell samples were treated with 70% ethanol and then stained by propidium iodide (PI) and processed with flow cytometry. The proportions of cells in G0/ G1, S or G2/M phase were separately determined.

| Cell invasion and migration assay
24-well transwell chamber (Corning) with the upper chamber coated with Matrigel (BD Bioscience) or not was used for cell invasion or migration assay. Transfected OV cells in serum-free medium were put into the upper chamber and complete culture medium was placed into the lower chamber. After 24 hours of incubation, invading or migrating OV cells were stained with 0.1% crystal violet in methanol for counting.

| RNA pull down
Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific) was acquired for conducting RNA pull down assay, as instructed by provider. The miR-525-5p-WT and miR-525-5p-Mut were biotin-labeled via Biotin RNA Labeling Mix (Roche), then in vitro transcribed. Thereafter, the Bio-miR-525-5p-WT/Mut probes or control Bio-NC probe were cultured with the protein extracts of OV cells and magnetic beads. Following washing, the pulled-down compounds were detected by qRT-PCR analysis.

| RNA immunoprecipitation
The lysed OV cells in RNA immunoprecipitation (RIP) lysis buffer were incubated with the magnetic beads conjugated to human anti-Ago2 antibody or normal mouse anti-IgG antibody (Millipore). The RNAs in immunoprecipitate were extracted and purified for qRT-PCR.

| Western blot
Total protein samples from OV cells were used for electrophoresis on the 10% SDS-PAGE and transfer onto the PVDF membranes. Following sealing with 5% nonfat milk, membranes were incubated with the specific primary antibodies against MXD1, MYC and control GAPDH all night, and then with appropriate secondary antibodies for 2 hours. All samples were assayed by the enhanced chemiluminescence reagent (Santa Cruz Biotechnology. Antibodies were all from Abcam.

| Co-immunoprecipitation (Co-IP)
Lysates of SUN8 and Caov3 cells were acquired from the IP lysis buffer for cultivation with the antibodies against MAX and negative control IgG overnight at 4°C. After adding beads for 2 hours, the eluted protein samples were measured by western blot.

| Statistical analysis
The experimental data were analyzed statistically by unpaired t tests or one-way ANOVA using Graphpad Prism 6 software and exhibited as the mean values ± standard deviation (SD). Bio-triplicate repeats were included in each assay. The P-value threshold was set as 0.05 to show the statistical significance.

| Overexpression of MAGI2-AS3 obstructed cell growth and motility in OV
Data from GEPIA displayed that MAGI2-AS3 showed a low expression level in OV tissues compared with adjacent normal tissue ( Figure 1A). To assess the physiological function of MAGI2-AS3 in OV, we next detected the expression of MAGI2-AS3 in normal ovarian epithelial cell line (IOSE) and OV cell lines (SKOV3, SNU119, OVCAR-3, SUN8, and Caov3). Consistently, MAGI2-AS3 was lowly expressed among OV cell lines in comparison with IOSE cell line ( Figure 1B). Thereafter, SUN8 and Caov3 cells presenting lower MAGI2-AS3 expression were used for gain-offunction assay. SUN8 and Caov3 cells were transfected with overexpressing plasmids targeting MAGI2-AS3, resulting in conspicuous up-regulation of MAGI2-AS3 expression ( Figure 1C). EdU assays were adopted for evaluating cell proliferation in response to overexpression of MAGI2-AS3. Results revealed that MAGI2-AS3 overexpression caused a noticeable decrease of cell proliferation ( Figure 1D). Besides, we also observed that overexpressed MAGI2-AS3 arrested cell cycle transition from G0/G1 to S or G2/M phase ( Figure 1E). In terms of apoptosis, data of cytometry analysis manifested that up-regulation of MAGI2-AS3 remarkably boosted the apoptosis of SUN8 and Caov3 cells ( Figure 1F). Additionally, the migratory and invasive capacities were both dramatically declined by MAGI2-AS3 overexpression in transwell assays ( Figure 1G,H). Altogether, MAGI2-AS3 was lowly expressed in OV cells and overexpression of MAGI2-AS3 hindered cell growth and motility in OV.

| MAGI2-AS3 regulated MXD1 to suppress MYC
Previous studies confirmed that MXD1 could compete with MYC for the binding of MAX and thereby inhibited the transcriptional function of MYC. 18 Thus, we supposed MAGI2-AS3  Figure 4B). Interestingly, we found that the expressions of MYC downstream genes (CDK4, CCND2 and BMI1) were lessened by overexpression of MAGI2-AS3 ( Figure 4C,D). Besides, the results of luciferase reporter assays displayed that the luciferase activity of these genes promoters was obviously diminished by up-regulation of MAGI2-AS3 ( Figure 4E). Afterwards, Co-IP analysis showed that interaction between MAX and MXD1 was increased while combination of MAX with MYC was dropped by up-regulation of MAGI2-AS3 ( Figure 4F). To summarize, MAGI2-AS3 could repress MYC transcriptional activity by strengthening interaction of MXD1 with MAX.

| DISCUSSION
With the development of medical level, the routine therapies for OV, including surgery, radiotherapy, and chemotherapy, have been improved. However, the overall survival rate was barely improved in the past few decades. 19 Therefore, our study was aimed to investigate the underlying mechanism of OV.
The significant effects of lncRNAs in OV cells have been explored. For example, lncRNA HDAC1 silencing boosted the chemotherapy response in OV. 20 LncRNA HOTAIR facilitated motility and proliferation in OV via modulating PIK3P3. 21 LncRNA CCAT1 accelerated metastasis and predicted poor prognosis in OV. 22 LncRNA MAGI2-AS3 was identified to be a tumor inhibitor in breast cancer 23 and glioma. 24 In our study, we found that MAGI2-AS3 was lowly expressed in OV cells and acted as a powerful factor in repressing OV development through suppressing cell proliferation, cell cycle, migration and invasion and promoting cell apoptosis in OV. Above data suggested that MAGI2-AS3 exerted anti-tumor effect in OV.
In terms of MAGI2-AS3-manipulated ceRNA mechanism in OV, we used dada from starBase and DIANA to search potential miRNAs which could bind with MAGI2-AS3. As expected, miR-525-5p was found through filtration. MiR-525-5p was down-regulated in cervical cancer and cancer cell proliferation and migration as well as exacerbated cell apoptosis. 25 In the present study, miR-525-5p expression was in a high level in OV cells. Luciferase reporter and RNA pull down assays verified the combination between MAGI2-AS3 and miR-525-5p. It was also disclosed that the expression of miR-525-5p was negatively regulated by MAGI2-AS3. These results displayed that MAGI2-AS3 sponged miR-525-5p in OV.
MXD1 was reported to have inhibitory functions in various cancers such as breast cancer 26 and lung cancer. 27 In this study, MXD1 was identified as a downstream target of miR-525-5p. The competition between MXD1 and MAGI2-AS3 for the binding of miR-525-5p was proved by RNA pull down and luciferase reporter assay. Based on that MXD1 could inhibit MYC by competitively interacting with MAX, 18 we assumed that MAGI2-AS3 could directly regulate MYC expression but this hypothesis was further overturned. Then, we found that overexpressed MAGI2-AS3 declined the expressions of CDK4, CCND2 and BMI1 which were all regulated by MYC. [28][29][30] Moreover it was confirmed that MAGI2-AS3 could contribute MXD1 to bind with MAX but inhibit MYC to bind with MAX, which suggested that MAGI2-AS3 suppress MYC by mediating MXD1. Finally, we used rescue assays to certify that MAGI2-AS3/miR-525-5p/MXD1 axis repressed OC cell growth and motility.
To sum up, our study suggested that MAGI2-AS3 acted as a tumor inhibitor in OV through targeting miR-525-5p/ MXD1 axis to suppress MYC signaling. This discovery will provide guidance for exploring the potential OV treatment.