LncRNA gas5 regulates granulosa cell apoptosis and viability following radiation by X-ray through sponging miR-205-5p and Wnt/β-catenin signaling pathway ingranulosa cell tumor of ovary

Purpose: The study explored the role of lncRNA gas5 in ovarian granulosa cells exposed to X-ray in granulosa cell tumor of ovary(GCTO). Methods:Exposed the KGN cell line (KALANG, Beijing, China) to X-ray to mimic the radiotherapy for GCSO patients in vitro, cell viability was checked by CCK8 assays. RT-qPCR detected the RNA expression of apoptosis-related genes while Western Blot for biomarkers in wnt/β-catenin signaling. Differential expressions of lncRNA gas5 were examined after cells exposed to X ray for 0,24,48hs. We over expressed gas5 and assessed resultant cell viabilities, apoptosis and signaling. The sponging between gas5 and miR-205-5p was verified through Luciferase Assay. CCK8, RT-qPCR and Western Blot were applied for investigations into the correlation between miR-205-5p and cell viability and apoptosis after miR-205-5p augmentation. Similarly, the interactions between the gas5 and miR-205-5p were assessed after co-transfection of miR-205-5p mimics and oe-gas5. Last, wnt inhibitor was used to study the role of signaling pathway in KGN cells. Results: Exposure of KGN toX-ray reduced cell viabilities and increased apoptosis. Gas5 had reduced expression in cells while miR-205-5p increased. Gas5 upregulation could protect the cells from apoptosis and add to the cell viability and activation of wnt//β-catenin signaling. lncRNA gas5 targeted miR-205-5p and miR-205-5p mimics could counteract functions of up-regulated lncRNA gas5, regulating Wnt/β-catenin signaling pathway. Inactivation in Wnt/β-catenin could suppress cell viability. Conclusions: lncRNA gas5 regulated the cell apoptosis and viability after cellular radiation, which might be a potential therapeutic target to combine into radiotherapy for GCTO patients in clinical stage.


INTRODUCTION
Ovarian follicle is one of the most dynamically evolving structures among female organs which plays an important role in regulating progressions of proliferation, differentiation and apoptosis in granulosa cell tumor of ovary (GCTO). After primordial follicles are activated and grow into primary follicles and secondary follicles, granulosa cells begin to proliferate and form layers [1]. Granular cell layer is a composition of fast-changing ovarian follicles. Granulosa cells are often observed in follicular initial cells undergoing apoptosis in atretic follicles before oocyte and theca cells, which indicate that they could be initiator of follicular atresia [2,3]. In addition, granulosa cells can secrete several factors including gonadal steroids, endocrine hormones and growth factors, which are important for their growth and survival [4].
X-rays are types of electromagnetic radiation which are present in the outer space and are well known for their ability to penetrate human tissues [6]. They are frequently used in the diagnosis and treatment of diseases ,leading to pathological damage to tissues [5]. Among cell organelles, mitochondrion is the most sensitive to ionizing radiation [6]. Impaired mitochondrial function is an important index of oxidative damage [7]. With the advances in technology, damages from radiation, especially their effects on reproductive system, are receiving growing attention. Researches have also proved that oxidative stress is an essential pathological factor in the infertility of both males and females [8].
LncRNAs are non-coding RNAs which have more than 200 nucleotides in length [9]. These RNAs can regulate tumor growth through different mechanisms [10]. Researches have also proven that lncRNAs are important in the control of radio resistance of cancers [11]. When exposed to X-ray, expression of lncRNA-p21 increased, inhibiting β-catenin signaling and inducing apoptosis, leading to increase in sensitivity of CRC to radiation [12]. Up-regulated lncRNA ANRIL could increase resistance of cancers to radiation through suppressing apoptosis and inducing proliferation. Furthermore, its function in regulating tumors is mediated through negative controlling of miR-125a, which is a kind of tumor suppressor [13]. Though, noncoding RNAs played important roles in tumor cell growth and apoptosis as well as resistance to radiation. There are few research related to gas5 in GCTO.
The purpose of this study was to evaluate the role of lncRNA gas5 and miR-205-5p in ovarian granulosa cells exposed to X-ray in granulosa cell tumor of ovary in order to determine the mechanisms in regulating reproduction in female.

Cell culture
Human ovarian granulosa cell line KGN was purchased (KALANG, Beijing, China). The KGN cells were incubated in RPMI-1640 medium contained 20% fetal bovine serum (FBS), 100 umol/ml penicillin and streptomycin. After incubation, cells in log phase were collected and exposed to 10GY X-ray for 0h, 24h and 48h before harvest. The radiation was generated using Philips RT250 (Kimtron, USA).

CCK-8
After 0, 24 and 48h exposure of 10GY X-ray, the cells were seeded into 96 well plate with 1×10 5 cells per well and incubated at 37℃, 5%CO2. CCK-8 (10μl solution) was added into the plate at 24h, 48h and 72h and the optical density (OD) values of cells were determined at 450nm using microplate reader (Thermo Fisher, USA). The cells exposed to the X-ray for 24, 48 h after transfection were selected to undergo cell viability assays in order to determine the correlations between the gene expression and cell viability.

Western blot
Cells exposed to X-ray were washed with PBS twice and then lysed with 250 µl RIPA reagent (Beyotime, Shanghai, China) for 20 min. Then the supernatant liquid was centrifuged at 1000 rpm for 5 min and the total proteins were quantified using BCA assay kit (Beyotime, Shanghai, China) [15]. This was followed by the separation of the total proteins (40μg) using SDS-PAGE and transfer into PVDF membranes. THe the membranes were blocked with 8% nonfat milk powder at room temperature for at least 2 h [16]. This was followed by the addition of primary antibodies and incubation of the membranes at 4℃ overnight. Primary antibodies adopted were as follows: Anti-Wnt3a antibody (1 µg/ml ab28472), Anti-beta Catenin antibody (1:5000, ab32572) and anti-GAPDH (1:1500; ab181602). Then membranes were rinsed and Goat Anti-Mouse IgG H&L (HRP) (1:800; ab205719) and Goat Anti-Rabbit IgG H&L (HRP) (1:800; ab205718) were added and the mixture was incubated for 1h at room temperature. Pierce™ ECL Plus Western Blotting Substrate (Thermo Fisher, USA) was used for development and gray values of proteins were measured with GAPDH as the internal reference.

Statistical analysis
All experiments were carried out three times as appropriate. Data are presented as mean ± SD and analyzed with SPSS 19.0 (IBM, USA) using Student's. t-test. P < 0.05 was considered statistically significant.

X-ray suppressed cell proliferation and promoted apoptosis
Compared to normal cells, cells that were radiated had lower level of cell viabilities ( Figure  1 A) which reduced gradually with time. The xray radiated cells had lower expressions of antiapoptosis genes (Bcl-2 and Bcl-xL) and higher level of caspase-3, suggesting that irradiation promoted apoptosis (Figure 1 B -D).

LncRNA gas5 expressed lower protein levels and promoted proliferation in x-ray exposed cells and inhibited apoptosis
Expression of lncRNA gas5 was detected in cells exposed to x-ray for 0 h, 24 h and 48 h. Compared to normal cells (0 h), the expression of lncRNA gas5 was lower in X-ray treated cells (Figure 2A). The expression of lncRNA gas5 decreased significantly as the exposure time increased (Figure2B, C). When lncRNA gas5 in cells was upregulated with 24h and 48h exposure to X-Ray, CCK8 results showed that increased lncRNA gas5 promoted viability ( Figure 2D). Moreover, cell viability was higher in cells that were pretreated with radiation for 24h compared to those for 48h ( Figure 2E). On the other hand, overexpression of lncRNA gas5 up regulated the expressions of Bcl-2 and Bcl-xL but inhibited expression of caspase-3 ( Figure 2F, G). Expression of wnt3a and beta-catenin was increased by overexpressed lncRNA gas5 ( Figure 2H). Starbase v2.0 predicted that miR-205-5p targeted lncRNA gas5 (Figure 3 A). Therefore, luciferase reporter assay was applied to determine the binding sites between lncRNA gas5 and miR-205-5p. The luciferase activity only decreased significantly in the group that was co-transfected with wild-type of gas5 and miR-205-5p mimics, which indicated that wild type of miR-205-5p can directly bind lncRNA gas5 (Figure 3 B). MiR-205-5p expression was higher in the group with X-ray exposure than untreated cells (Figure 3 C). Thereafter, miR-205-5p was knocked down by transfecting miR-205-5p inhibitor into the cells with pre-exposure to radiation for 24h and 48h. Lower expression of miR-205-5p was detected ( Figure 3 D and E). Correspondingly, cell viability was measured, which disclosed that in cells with pre-exposure to radiation for both 24 and 48 h groups, inhibited miR-205-5p could promote viability of cells ( Figure 3 F and G). As for apoptosis, inhibition of miR-205-5p could activate Bcl-2 and Bcl-xL and silence caspase-3, signifying deterred cellular apoptosis ( Figure 3 H and I).

LncRNA gas5 regulated cell progression by targeting miR-205-5p via Wnt/β-catenin signaling pathway
After assessment of the functions of lncRNA gas5 and miR-205-5p individually, interplays in between were further studied. First, when miR-205-5p was up-regulated, expression of lncRNA gas5 decreased ( Figure 4A). lncRNA gas5 increased viability of cells while overexpressed miR-205-5p partly reversed the promotive effect generated by upregulated gas5 ( Figure 4B). After that, apoptosis was detected through expressions of Bcl-2, Bcl-xL and caspase-3. Upregulated miR-205-5p restored Bcl-2 and Bcl-xL and reversed the suppression of caspase-3 induced by lncRNA gas5 upregulation ( Figure  4C). Moreover, expressions of Wnt 3a and βcatenin were both increased by upregulated lncRNA gas5 while miR-205-5p mimics offset the boost ( Figure 4D). In order to make sure that Wnt/β-catenin signaling pathway played a part in the cellular functions, IWP-4, a Wnt/β-catenin signaling pathway inhibitor was introduced. This could silence Wnt 3a and β-catenin to be able to investigate the changes in cell viabilities brought by the inactivation of Wnt/β-catenin signaling ( Figure 4E, F). The CCK8 results unveiled the reduced cell viabilities after the IWP-4 treatment.

DISCUSSION
The present study mainly explored the role of lncRNA gas5 in granulosa cell tumor of ovary after X-ray treatment in vitro. At first, the GCTOlike cell line, KGN, was acquired and treated with X-ray for 0,24 and 48 hours to mimic the radiotherapy for GCTO patients in vitro. Cell viability decreased and apoptosis increased when the period of treatment increased. Gas5 was silenced when the cells were radiated. The upregulation of gas5 can promote the cell viability and inhibit apoptosis, suggesting that gas5 might deter the pro-apoptosis effect of xray.
In addition, bioinformatics and Luciferase assays confirmed the targeted gene of lncRNA gas5, miR-205-5p. Therefore, the role of miR-205-5p in cellular functions was studied and it was discovered that miR-205-5p expression increased as radiation was pre-treated; miR-205-5p inhibitor promoted the cellular viabilities and deterred apoptosis. On the investigation of interactions between gas5 and miR-205-5p in modulations of the radiation-exposed cell functions, it was observed that miR-205-5p mimics can partially reverse the cellular changes in cell viability and apoptosis induced by gas5 upregulation.
GCTO was a type of gynecological oncology which occurs rarely [17]. The treatments for GCTO include radio-therapy [18]. In recent years, studies indicate that RNAs are involved in GCTO progression. For instance, the presence of TRET C228T mutation tended to appear in recurrent tumors instead of primary ones in GCTO [19]. Silencing FOXO/PTEN in Granulosa cells corelated with the progression of GCTO [20]. There have been some previous studies concerning lncRNAs and GCTO. It was disclosed that lnc LET can induce cell apoptosis and inhibit cell viability in GCTO-like cells; lncRNA SRA might promote cell growth and suppress cell viability in GCTO mice and lncRNA MEG3 promoted the cell viability and inhibit viabilities in GCTO mouse cells via p53/p66 signaling pathway [21-23].
Although LncRNA gas5 was widely reported to participate in the regulation of ovarian disorders [24,25], there is no literature relating to gas5 and GCTO. lncRNAs can be regulated by radiation as it showed radio-resistance in many kinds of cancer cells [26,27]. Researches have also discovered that lncRNAs transcripts were changed because of ultraviolet rays or ionizing radiation in peripheral blood mononuclear cell (PBMC)[28], thymocyte[29] and melanocyte [30]. LncRNA gas5 was discovered in various cancers to play a regulatory role in radiotherapy efficacy [31][32][33][34][35]. This explains why the role of gas5 in GCTO regarding radiation in vitro was investigated.
Wnt signaling pathway was first verified in 1982 and Wnt1 was the first gene of Wnt family [36], which is an important signaling pathway in regulating cell progression. Previous researches have proved that Wnt/β-catenin signaling pathway could activate expression of survivin, an anti-apoptosis gene, to increase radioresistance of progenitor cells of mammary glands [37]. In this study, it was found that wnt/beta-catenin signaling changed as X-ray exposure, gas5 or miR-205-5p adjusted, suggesting that the signaling pathway might be involved with the regulatory mechanism beneath the lncRNA gas5. As a result, the signaling pathway inhibitor, IWP-4, was introduced to silence the signaling. The corresponding changes in cell viability showed that resultant cellular viabilities was inhibited by IWP-4. This is an indication that the lncRNA gas5/miR-205-5p axis might regulate the cell viability and apoptosis via wnt/beta-catenin signaling in GCTO-radiation cellular model.

CONCLUSION
LncRNA gas5 was expressed lower in GCTOradiation cellular model and upregulation inhibited cell apoptosis and promoted cell proliferation by suppressing miR-205-5p and activating Wnt/β-catenin signaling, indicating that it could be a potential target to be considered combining with radiotherapy in GCTO. However, the present study requires more substantial animal and clinical studies for further validation.