Long non-coding RNA ADAMTS9-AS1 attenuates ferroptosis by Targeting microRNA-587/solute carrier family 7 member 11 axis in epithelial ovarian cancer

ABSTRACT Epithelial ovarian cancer (EOC) accounts for approximately 90% of all ovarian cancer cases and is the most common cause of gynecological cancer death. Understanding the molecular mechanisms of EOC will help develop better diagnostics and more effective treatments. This study aimed to investigate whether long non-coding RNA ADAMTS9-AS1 (ADAMTS9-AS1) could regulate solute carrier family 7 member 11 (SLC7A11) expression and inhibit ferroptosis by sponging micoRNA-587 in EOC progression. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting results showed that ADAMTS9-AS1 expression was elevated in EOC cells; microRNA-587 expression was up-regulated and SLC7A11 expression was down-regulated after knocking down ADAMTS9-AS1 by transfection with siRNAs; however, microRNA-587 inhibitor reversed SLC7A11 expression in ADAMTS9-AS1 knocking down cells. Ferroptosis related marker detection and cell function assay confirmed that knocking down ADAMTS9-AS1 inhibited EOC cells proliferation and migration by promoting ferroptosis. Overexpression of micoRNA-587 also promoted ferroptosis while inhibited cells proliferation and migration in EOC cells. Additionally, micoRNA-587 inhibitor reversed the effect of ADAMTS9-AS1 silence on the ferroptosis and cell function. Moreover, dual-luciferase reporter gene assay and RNA immunoprecipitation assay confirmed that miR-587 was as a sponge for ADAMTS9-AS1 and SLC7A11. In conclusion, our study found that ADAMTS9-AS1 attenuated ferroptosis by targeting miR-587/SLC7A11 axis in EOC. Our study provides a new therapeutic target for EOC.


Introduction
Ovarian cancer (OC) is a silent cancer, and the deadliest gynecological malignant tumor [1]. Epithelial ovarian cancer (EOC) accounts for about 90% of all OC cases, usually appearing in advanced stages, and is the most common cause of death from gynecological cancer [2]. In which, high grade serous ovarian cancer is the most lethal gynecologic cancer [3,4]. Despite advances in surgical techniques and conventional chemotherapy, EOC patients often relapse due to chemotherapy resistance within a few years of initial treatment, and the long-term survival rate of EOC patients (more than 5 years) is less than 30% [5,6]. EOC pathogenesis is a complex biological process involving gene and epigenetic changes [7,8]. Therefore, more extensive studies are urgently needed to better understand the molecular mechanisms underlying EOC progression.
LncRNA ADAMTS9-AS1 (ADAMTS9-AS1) could play an essential role as a cancer-promoting or cancer-suppressing molecule in various cancers, such as in colorectal cancer, ADAMTS9-AS1 promoted cell proliferation and epithelial-mesenchymal transition [14]. Additionally, previous study has found that ADAMTS9-AS1 expression was elevated in EOC [10]. However, the specific mechanism that plays a role in EOC has not been elucidated. Before conducting experiment, we used miRanda-3.3a software predicted that ADAMTS9-AS1 had binding site with miR-587. miR-587 has been reported to promote or inhibit cancer in different tumors, including prostate cancer [15], non-small-cell lung cancer [16], hepatocellular carcinoma [17], etc.
However, its function in EOC has not been reported.
Before conducting experiment, we also used miRanda-3.3a predicted that downstream targets of miR-587 was SLC7A11. SLC7A11, also called xCT, was the glutamate/cystine antiporter solute carrier family 7 member 11 [18]. Exploring SLC7A11 regulation mechanism has always been a significant research focus, and several studies on SLC7A11 have been involved. SLC7A11 has been reported to be overexpressed in cancer and associated with poor prognosis in patients. These diseases included melanoma [19], glioma [20], nonsmall cell lung cancer [21], etc. In OC, SLC7A11 was reported to be an independent risk prognostic factor for overall survival [22]. Moreover, SLC7A11, as a key suppressor of ferroptosis, has been reported to play a role in inhibiting ferroptosis in glioma and OC [23,24]. But it is unknown whether ADAMTS9-AS1 plays a role in EOC progression by regulating miR-587/SLC7A11 axis and thereby inhibiting ferroptosis.
Based on the above background, we hypothesis that ADAMTS9-AS1 could regulate miR-587/ SLC7A11 axis and inhibit ferroptosis in EOC progression. To verify this hypothesis, we firstly investigate the function of ADAMTS9-AS1, and then confirmed the relationship between ADAMTS9-AS1, miR-587, and SLC7A11. Finally, rescue experiments were performed to verify the potential regulation mechanism. Our study was intended to provide new drug targets for EOC diagnosis and treatment.
To investigate the role of miR-587 in EOC progression, miR-587 mimics negative control (miR-NC) and miR-587 mimics were transfected with OVCAR3 and CAOV-3 cells for 24 h or 48 h. The specific groups were miR-NC and miR-587 mimics. miR-NC and miR-587 mimics purchased from RioBio.

Clone formation assay
OVCAR3 and CAOV-3 cells with the indicated treatment were placed in 6-well plate (1,000 cells per well). The cells were placed in a 37°C 5% CO 2 and saturated humidity incubator for 2 to 3 weeks, during which liquid was appropriately changed. Then phosphate-buffered saline solution (PBS) was used to wash the colonies, stained with 0.1% crystal violet for 15 min. The clone formation ability was observed under a microscope (Carl Zeiss, German) and counted using ImageJ software.

Transwell assay
OVCAR3 and CAOV-3 cells migration ability was examined by Transwell (BD Biosciences) as previous study with a few revision [12]. The upper chambers were filled with 200 μL of serum-free RPMI-1640 medium (for OVCAR3) or DMEM medium (for CAOV-3) containing 5 × 10 4 cells. And 600 μL of culture medium was added to the lower compartments. After 24 h, we discarded the chamber culture medium and washed them twice with PBS. The cells on the upper ventricle were wiped off with a wet cotton swab. Cells were fixed 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 5 min. Stained cells number was counted with a light microscope (magnification, ×200) in 5 random fields of view.

Detection of Fe 2+, iron and reactive oxygen species (ROS) level
Iron assay kit (#MAK025, Sigma) and ROS assay kit (#S0033S, Beyotime) were performed to examine the intracellular Fe 2+ , Iron and ROS levels in OVCAR3 and CAOV-3 cells according to manufacturers' instructions.

Western blotting
As previous study, we used western blotting to evaluate the gene protein expression [12]. RIPA lysis buffer (#P0013B, Beyotime) extracted total protein from cells according to the instructions and quantified according to BCA protein assay Kit (BL521A, Biosharp). The protein supernatant was mixed with sodium dodecyl sulfate, sodium salt polyacrylamide gelelectrophoresis loading buffer and then bathed in boiling water for 5 min.
Proteins were adsorbed on polyvinylidene fluoride membranes by gel electrophoresis and sealed in 5% skim milk solution for 2 h. Then SLC7A11, GPX4, β-actin and GAPDH (Abcam) antibodies were cultured overnight at 4°C, and secondary antibody was incubated for 2 h at 25°C. Using βactin or GAPDH as internal reference, protein level was measured by ECL reagent (#WBULS0100, Sigma, USA).

RNA immunoprecipitation (RIP) assay
As previous study, we used RIP assay to confirm whether Ago2 can enriched ADAMTS9-AS1, miR-587, and SLC7A11 [12]. The EZ-Magna RIP™ kit (#17-701, EMD Millipore) was used in the assay. The cells were lysed in RIP lysis buffer, and treated with magnetic beads coupled to an anti-Ago2 antibody or anti-rabbit IgG at 4°C overnight. Besides, 10 μL whole-cell lysates was used as Input and served as the positive control. IgG was used as negative control. The relative enrichment of ADAMTS9-AS1, miR-587, and SLC7A11 in the immunoprecipitated RNAs was detected via qRT-PCR.

Statistical analysis
Data was quantified by means ± standard deviation (SD) and compared using Student<apos;>s t-test or one-way analysis of variance (ANOVA) (P < 0.05 as statistically significant) based on SPSS 16.0 software.

Results
In our study, we speculated that ADAMTS9-AS1 regulates miR-587/SLC7A11 axis to affect cell proliferation and migration by inhibit ferroptosis in EOC progression. To solve this problem, we firstly investigate the function of ADAMTS9-AS1 on cell proliferation, migration, and ferroptosis in EOC. Then we confirmed the regulation relationship of ADAMTS9-AS1, miR-587, and SLC7A11. Finally, we conducted rescue experiments to verify the potential regulation mechanism that ADAMTS9-AS1 regulated miR-587/ SLC7A11 axis to affect cell function by inhibiting ferroptosis in EOC progression. Our study was intended to provide new drug targets for EOC diagnosis and treatment.

Knocking down ADAMTS9-AS1 could inhibit EOC cells proliferation and migration by promoting ferroptosis
Firstly, OSE and EOC cells (ES-2, OVCAR3, CAOV-3 and SK-OV-3 cells) were cultured and collected, the ADAMTS9-AS1 expression was assessed by qRT-PCR. Results indicated that ADAMTS9-AS1 was highly expressed in EOC cells, and had the highest expression in OVCAR3 and CAOV-3 cells ( Figure 1A). Therefore, OVCAR3 and CAOV-3 cells were selected for further study. Then, OVCAR3 and CAOV-3 cells were transfected with siRNAs target ADAMTS9-AS1 for 24 h to silence the ADAMTS9-AS1 expression, si-NC as the control group. The results showed the synthesized si-ADAMTS9-AS1 (including si-ADAMTS9-AS1-1 and si-ADAMTS9-AS1-2) had interference effect, and si-ADAMTS9-AS1-2 with the best interference effect was used in subsequent experiments ( Figure 1B). To investigate ferroptosis mechanism, we firstly transfected si-ADAMTS9-AS1-2 (or si-NC) into OVCAR3 and CAOV-3 cells for 24 h, then 30 nM ferroptosis inhibitor Fer-1 was used to treat cells for another 48 h. After detection using iron assay kit, ROS assay kit, and western blotting, we found that compared with si-NC group, Fe 2+ , iron, and ROS increased, GPX4 expression decreased after knocking down ADAMTS9-AS1; Fe 2+ , iron, and ROS decreased, GPX4 expression increased in si-ADAMTS9-AS1-2+ Fer-1 group when compared to silence ADAMTS9-AS1 alone group ( Figure 1C-1F). In addition, CCK-8, clone formation assay, and Transwell assay results indicated that compared to si-NC group, knocking down ADAMTS9-AS1 resulted in decreased cell viability and decreased cell proliferation and migration abilities; Fer-1 increased cell viability and increased cell proliferation and migration abilities in si-ADAMTS9-AS1 cells ( Figure 1G-1I). These results suggested knocking down ADAMTS9-AS1 could inhibit EOC cells proliferation and migration by promoting ferroptosis.

ADAMTS9-AS1 negatively regulated miR-587 expression in EOC cells
Next, we used lncLocator to predict ADAMTS9-AS1 subcellular distribution. Among cytoplasm, nucleus, ribosome, cytosol, and exosome, the subcellular localization prediction score of ADAMTS9-AS1 was the highest in cytoplasm (Figure 2A). Cytoplasmic and Nuclear RNA Purification kit separated the cytoplasm and nucleus of OVCAR3 and CAOV-3 cells, then the expression of ADAMTS9-AS1, GAPDH, and U6 in cytoplasm and nucleus was detected using qRT-PCR. GAPDH and U6 was used as control of cytoplasm and nucleus separately. Results showed that GAPDH expression in cytoplasm or U6 expression in nucleus possessed above 80% respectively, confirming that cytoplasm and nucleus separation was successfully ( Figure 2B). And ADAMTS9-AS1 expression in cytoplasm possessed about 60%, confirming that ADAMTS9-AS1 was mainly located in the cytoplasm of OVCAR3 and CAOV-3 cells ( Figure 2B). To investigate the potential mechanism of ADAMTS9-AS1, miRanda-3.3a software was used to predict downstream miRNA. We found that ADAMTS9-AS1 had two binding sites to miR-587 ( Figure 2C). miR-587 have been reported exert promote or inhibit role in different tumors [15,16,28]. Therefore, we detected the miR-587 expression in OVCAR3 and CAOV-3 cells after knockdown ADAMTS9-AS1. Results showed that miR-587 expression was promoted after knocking down ADAMTS9-AS1 ( Figure 2D). In addition, we performed dual-luciferase detection to confirm the binding site between ADAMTS9-AS1 and miR-587. Results showed that ADAMTS9-AS1 regulated miR-587 expression by binding to site 1 and site 2 ( Figure 2E-F). These results showed that ADAMTS9-AS1 negatively regulated miR-587 expression.

Overexpression of miR-587 targeted regulation of SLC7A11 in EOC cells
To investigate the effect of miR-587 on the downstream mRNA, miRanda-3.3a software prediction indicated that there were two binding sites between SLC7A11 and miR-587 ( Figure 3A).

ADAMTS9-AS1 affected ferroptosis, proliferation and migration of EOC cells through miR-587
Finally, we further investigated whether ADAMTS9-AS1 affected ferroptosis and cell function in EOC through miR-587. OVCAR3 and CAOV-3 cells were transfected with si-NC+NC inhibitor, si-ADAMTS9-AS1-2+ NC inhibitor, and si-ADAMTS9-AS1-2+ miR-587 inhibitor for 24 h (for the detection of Fe 2+ levels, Iron expression, ROS levels, cell activity, cell proliferation ability, and cell migration ability) or 48 h (for western blotting). Relative to si-NC+NC inhibitor group, knocking down ADAMTS9-AS1 increased the expression of Fe 2+ and Iron; adding miR-587 inhibitor at the same time reduced the increase of Fe 2+ and Iron induced by knocking down ADAMTS9-AS1 ( Figure 6A-6B). ROS results showed that the ROS increased after ADAMTS9-AS1 was interfered when compared with si-NC +NC inhibitor group; the increase of ROS caused by si-ADAMTS9-AS1-2 was decreased by interfering miR-587 expression simultaneously ( Figure 6C). In addition, western blotting results indicated that the GPX4 expression was decreased after silence of ADAMTS9-AS1 when compared with si-NC+NC inhibitor group; adding miR-587 inhibitor at the same time increased GPX4 expression in ADAMTS9-AS1 knockdown cells ( Figure 6D). Cell function experiments showed that the abilities of cell proliferation (72 h), clone formation, and migration were decreased after knocking down ADAMTS9-AS1; transfected with miR-587 inhibitor at the same time increased the abilities of cell proliferation (72 h), clone formation, and migration in ADAMTS9-AS1 knock down cells ( Figure 6E-6G). All in all, silence of ADAMTS9-AS1 promoted ferroptosis and inhibited the abilities of proliferation and migration in EOC cells; however, these effects were reversed by miR-587 inhibitor, suggesting that ADAMTS9-AS1 affected ferroptosis, proliferation and migration of EOC cells through miR-587.

Discussion
With the further development of the treatment of EOC to personalized medicine, new methods for early diagnosis and prevention using molecular genomics are under development [29]. In this study, we investigated whether ADAMTS9-AS1 could regulate SLC7A11 expression and inhibit ferroptosis by sponging miR-587 in EOC progression through in vitro cell experiments. Our results indicated that ADAMTS9-AS1 attenuated ferroptosis by targeting miR-587/SLC7A11 axis in EOC. At present, there is no reported study on the ADAMTS9-AS1/miR-587/SLC7A11 axis role in regulating ferroptosis of EOC cells, which is our innovation.
lncRNA is considered to be a regulator of gene expression, and lncRNA disorder is involved in many cancers progression, including EOC [30]. It has been reported that ADAMTS9-AS1 overexpression preferentially affected genes related to proliferation and migration [31]. In hepatocellular carcinoma, ADAMTS9-AS1 triggered PI3K/AKT/ mTOR pathway of liver cancer cells, exacerbated cell proliferation and migration [32]. In non-small cell lung cancer, ADAMTS9-AS1 knockdown inhibited cell proliferation and epithelialmesenchymal transformation [33]. Wang H, et al found ADAMTS9-AS1 expression was elevated in EOC [10]. Our study found ADAMTS9-AS1 was highly elevated in EOC cells, and the highest expression was found in OVCAR3 and CAOV-3 cells. This was consistent with the research results of Wang H, et al. In addition, our results revealed knocking down ADAMTS9-AS1 inhibited EOC cells proliferation and migration by promoting ferroptosis. This was consistent with findings in other cancers that ADAMTS9-AS1 affected cancer cells proliferation and migration.
ceRNA is a post-transcriptional transcript that is mutually regulated by competing for shared miRNAs [34]. Interaction between ceRNAs through shared miRNAs represents a new gene regulatory layer, which plays an essential role in physiology and cancer and other diseases development [35]. Fang S, et al reported ADAMTS9-AS1 inhibited invasive phenotype of breast cancer cells by sponging miR-513a-5p and regulating ZFP36 ring finger protein [36]. Zhou Z, et al found ADAMTS9-AS1 inhibited prostate cancer progression via regulating miR-142-5p/cyclin D1 [37]. We found ADAMTS9-AS1 negatively regulated miR-587 expression, and overexpression of miR-587 targeted regulation of SLC7A11. In addition, through miRanda-3.3a prediction and dualluciferase validation, and RIP assay, we confirmed the binding between ADAMTS9-AS1 and miR-587, as well as the binding between miR-587 and SLC7A11, suggesting that ADAMTS9-AS1 regulated SLC7A11 expression through sponging miR-587. Our results revealed ADAMTS9-AS1 /miR-587/SLC7A11 played a vital role in EOC through the ceRNA mechanism.
SLC7A11 is an important oncoprotein, which not only plays a role in defending against oxidative stress and ferroptosis, but also plays a role in influencing malignant tumor behavior and tumor microenvironment [38]. Ferroptosis is a new type of programmed non-apoptotic cell death caused by iron-dependent lipid peroxidation after the inactivation of SLC7A11 andsolute carrier family 3 member 2, which is involved in all kinds of diseases [39]. Ferroptosis, as a promising new antitumor strategy, has shown a vital role in EOC [40]. It has been reported in BRCA-proficient OC, inhibition of SLC7A11 resulted in decreased glutathione biosynthesis and promoted lipid peroxidation and ferroptosis [41]. We verified that overexpression of miR-587 targeted regulation of SLC7A11 promoted ferroptosis and inhibited EOC cells proliferation and migration. In addition, ADAMTS9-AS1 regulated SLC7A11 expression through miR-587, thereby affecting ferroptosis, proliferation and migration of EOC cells. This research provides a possibility for EOC treatment in ferroptosis.
However, we still need to further investigate these effects in animal models. Moreover, samples from EOC patient also need collected to verify our conclusion.

Conclusion
Our study indicated that lncRNA ADAMTS9-AS1 attenuated ferroptosis by targeting miR-587/ SLC7A11 axis in EOC. Our study provides a new target for EOC treatment. It also provides a reference for understanding the molecular mechanism of ferroptosis in EOC.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
Provincial Natural Science Foundation of Jiangxi (20212BAB201398) has provided financial support for this study.