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LncRNACASC9 promotes proliferation, metastasis, and cell cycle inovarian carcinoma cells through cyclinG1/TP53/MMP7 signaling

ABSTRACT Ovarian cancer (OC) brings about serious physical and psychological burden for female patients. LncRNA CASC9 has been reported to be intimately linked with the occurrence and development of several tumors. However, the biological role of lncRNA CASC9 in OC still lacks sufficient evidence. The expressions of CASC9 and miR-488-3p in OC cell lines and xenograft mice were detected by qRT-PCR assay. Cell Counting Kit-8 (CCK-8) assay was used to assess cell inhibition rate and cell proliferation in OVCAR-3 and OVCAR-3/DDP cells. Wound healing assay and transwell assay were performed to evaluate the capacity of migration and invasion, respectively. In addition, cell apoptosis was measured by TUNEL assay and cell cycle was assessed by flow cytometric analysis. Moreover, western blotting was carried out to detect the cyclinG1 (CCNG1)/TP53/MMP7 signaling and apoptosis-related proteins. Furthermore, luciferase reporter assay was performed to verify the combination of CASC9 with CCNG1 and miR-488-3p. The results of our study revealed that CASC9 expression was upregulated while miR-488-3p and CCNG1 expression was downregulated in OC cells with significant higher TP53 and MMP7 protein levels compared with normal ovarian surface epithelial cells. Additionally, luciferase reporter assay confirmed CASC9 bond to miR-488-3p/CCNG1. CASC9 silencing inhibited cell proliferation, migration, and invasion whereas promoted cell inhibition rate and apoptosis in vitro and in vivo. However, CASC9 overexpression showed the opposite effects. In summary, LncRNA CASC9 played a regulative role in ovarian carcinoma by cyclinG1/TP53/MMP7 signaling via binding to miR-488-3p in vivo and in vitro.


Introduction
Ovarian cancer (OC) is the commonest gynecological cancer that ranks the eighth-leading cause of female cancer death around the world [1]. The disease usually appears in postmenopausal women, along with 3-4 months of abdominal pain and distension [2]. The symptoms of ovarian carcinoma is unconspicuous and asymptomatic until advanced stages, and the lack of screening methods and early diagnostics of this disease, leading to little chance of a cure [3]. Thus, this cancer often is identified as the 'silent killer' and survival rates for women with advanced stage ovarian carcinoma range from 20% to 30% [4]. So far, advanced equipment and technology in surgery and chemotherapy have been applied for treating this disease, which promote the high incidence of complete remissions, but the recurrence rate is also high [5,6]. Therefore, it is vital to further explore the carcinogenesis and underlying mechanism of OC for female patients.
Long noncoding RNAs (lncRNAs) are a kind of transcripts with over 200 nucleotides in length, and they play crucial roles in a variety of biological activities in organism [7,8]. Numerous studies have shown that lncRNAs take part in the cellular progresses, such as gene transcription, epigenetic regulation, and post-transcription regulation in many cancers [9,10]. LncRNAs can regulate tumor cell proliferation, invasive, migration, cell cycle, and epithelial-mesenchymal transition [11][12][13]. The long noncoding RNA cancer susceptibility candidate 9 (CASC9), located in human chromosome 8q21.11, was first identified in esophageal squamous cell carcinoma. Recent studies have reported that lncRNA CASC9 was aberrantly expressed in several cancers. For example, CASC9 was upregulated in esophageal squamous cell carcinoma and accelerated cell metastasis via regulating LAMC2/CREB-binding protein [14]. Luo et al. reported that CASC9 was overexpressed in colorectal cancer and higher CASC9 level was implicated in poor patient outcomes. CASC9 promoted colorectal cancer cell proliferation in vitro and in vivo by binding to CPSF3 via regulating TGF-β signaling [15]. However, the biological role of CASC9 and potential mechanism in OC is elusive.
In this study, we are aimed to explore the functional role of CASC9 in OC. Given its high expression in OC cells, we hypothesized that CASC9 may play a tumor-promoting role in OC. Then we investigated the expression of CASC9 in OC cell lines and detected the influences of CASC9 overexpression or silencing on cell proliferation, invasion, migration, apoptosis, and cell cycling in OC or DPP-resistant OC cells and in mouse model. In addition, the oncogenic mechanism of CASC9 in OC was also demonstrated.

Cell culture
Human immortalized ovarian surface epithelial cells IOSE-80 and OC cell lines SKOV3, OVCAR-3, TOV-21 G and CoC1 were purchased from China Center for Type Culture Collection. The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C in a humid wet atmosphere containing 5% CO 2 at 37°C.

Establishment of DDP-resistant OC cells
DDP-resistant OVCAR cells (OVCAR-3/DDP) were induced by increasing doses of DDP (Sigma-Aldrich Co., USA). Firstly, OVCAR cells were cultured in the medium containing 0.02 mg/L DDP every day and the DDP concentration was gradually increased until the cells were stable in the medium with 0.2 mg/L DDP. Then the DDPresistant cell culture lasted until the experiments were executed.

Cell Counting Kit-8 (CCK-8) assay
OVCAR-3 and OVCAR-3/DDP cells were seeded into 96-well plates at a density of 8 × 10 3 cells/well until reaching 80-90% confluence. After the cell attachment, 10 µL DDP (25 µg/mL) was added to each well in treated group. The control group was set by adding medium only and the negative control group was set by adding OVCAR-3 or OVCAR-3/DDP cells with medium. Each group was repeated six times. After the exposure period of 72 h, 10 μL of WST-8 was added to each well d for another 3 h incubation. Finally, the absorbance was detected by a microplate reader (BioTek Instruments, VT) at the wavelength of 490 nm. The cell inhibition rate = 1 − (A experimental group −A control group )/(A NC group − A control group ) × 100%. All of the experiments were performed in triplicate.
To assess the cell proliferative ability, OVCAR-3 and OVCAR-3/DDPs were placed in 96-well plates for 48 h and treated with 10 μL CCK-8 reagent for 2 h in the dark. The OD value at 450 nm was measured with the microplate reader [18]. Six parallel wells were set for this assay and all assays were performed in triplicate.

Wound healing assay
OVCAR-3 and transfected OVCAR-3/DDP cells were seeded into a six-well plate and were allowed to grow to 90% confluency. The cell monolayers were then wounded with white pipette tips to make a straight scratch. After washing three times with phosphate-buffered saline (PBS) to remove cell debris, the cells were incubated for 24 h. The number of migrated cells in the scratch was counted with an inverted microscopy [19].

Transwell assay
A transwell assay was employed to measure tumor invasion ability after transfection with shRNA-CASC9 or OverExp-CASC9. OVCAR-3 and transfected OVCAR-3/DDP cells were placed into the upper wells of the transwell chambers with 0.1 mL of matrigel (Becton Dickinson, MA). At the same time the medium with 10% fetal bovine serum was added in the lower chamber. The cells on the upper wells of the transwell chambers were removed with a cotton swab. After 48 h, the invaded cells on the bottom of the chamber were stained with H&E and counted with a microscope. The invasion assay were repeated at least three times [20].

TdT-mediated dUTP nick-end labeling (TUNEL) assay
TUNEL assay was carried out to evaluate effects of CASC9 expression on the apoptosis of OVCAR-3/ DDP cells or animal models [21]. Briefly, the treated cells and the sections from mouse model were fixed with 4% paraformaldehyde and then incubated with proteinase K for 15 min in 37°C. After washing three times, cells and sections were inactivated by 3% H 2 O 2 at room temperature for 15 min. Then, cells and tissues were stained by 3, 3-diaminobenzidine (DAB) and hematoxylin for staining the cell nuclei. TUNEL-positive cells were quantified by counting positively stained cells. Five randomly chosen fields were counted for each group.

Xenograft experiments
All procedures using animals were approved by Animal Care and Use Committee of Tangdu Hospital, Air Force Medical University and performed in accordance with Chinese legislation regarding experiment animals. Four-week-old sixty female nude mice (weight 14-16 g) were raised in pathogen-free rooms at 25 ± 2°C with 12 h light/dark cycles, 22 ± 2°C, and fed with standard laboratory food and water. The mice were injected subcutaneously with 1 × 10 5 OVCAR-3 and OVCAR-3/DDP cells transfected with shRNA-CASC9 or OverExp-CASC9 or empty vectors on flanks (12 mice per group). When the tumors appeared, six mice in each group received intraperitoneal injections of 8 mg/ kg DDP and another six mice were treated with PBS per day for 21 days. The tumor volumes in each group were measured every 3 days. After DDP treatment for 21 days, all mice were sacrificed and the tumors were excised. Tumor volumes and weight were measured, and tumor tissues were stored for further research. Tumor volume was measured with a caliper and calculated using the formula: volume = (length × width 2 )/ 2 [23].

Statistical analysis
SPSS 19.0 statistical software (IBM, Armonk, NY, USA) were performed to analyze the data. All results were presented by the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for multiple comparisons and two group comparisons were performed with a Student's t-test. Statistical significance was defined as P values < 0.05.

Results
In this study, we explored the biological roles and the potential molecular mechanism of LncRNA CASC9 in OC. The data showed the upregulation of CASC9 and downregulation of miR-488-3p in vivo and in vitro. CASC9 silencing inhibited cell proliferation, invasion and migration but promoted apoptosis and blocked cell cycle. CASC9 overexpression revealed the opposite results. CASC9 upregulated CCNG1/TP53/MMP7 expression by targeting miR-488-3p. Overall, our study demonstrated a functional role of the CASC9/miR-488-3p/ CCNG1 axis in OC, providing a potential novel diagnostic and therapeutic target in OC treatment.

Expressions of CCNG1/TP53/MMP7 and LncRNA CASC9/miR-488-3p were aberrant in ovarian carcinoma
To explore the roles of CASC9 and potential molecular mechanism in OC, we first detected the expressions of CASC9 and its downstream gene in several OC cell lines by western blot assay and qRT-PCR. As shown in Figure 1a, the CCNG1 level was decreased while protein levels of TP53 and MMP7 were increased in OC cells compared with human ovarian surface epithelial cell line IOSE-80. In addition, CASC9 expression was significantly elevated and miR-488-3p expression was downregulated in OC cell lines compared with human ovarian surface epithelial cell line IOSE-80 ( Figures 1B and 1 C). Given the results above, OVCAR-3 cell line was used for further experiments.

CASC9 was involved in cell proliferation, migration, and invasion of OVCAR-3 and OVCAR-3/DDP cells
To investigate the biological role of CASC9 in OC cells and DDP-resistant OC cells, DDP-resistant OVCAR-3 cells were cultured and the cell inhibition rate was evaluated (Figure 2a). Then, we transfected shRNA-CASC9-1/2 and OverExp-CASC9-1/2 into OVCAR-3 cells and the transfection efficiency was detected (Figure 2b). According to Figure 2c, the proliferation ability of OVCAR-3/ DDP cells was remarkably inhibited by shRNA-CASC9 whereas CASC9 overexpression enhanced the proliferation compared with the negative control. Additionally, CASC9 silencing improved cell inhibitory rate obviously but CASC9 overexpression repressed the inhibitory rate in OVCAR-3/ DDP cells (Figure 2d). Consistently, cell migration and invasion capacity of OVCAR-3/DDP cells were suppressed by transfection with shRNA-CASC9 while CASC9 overexpression had an opposite effects on migration and invasion ( Figures 2E  and 2 F).

CASC9 inhibited the apoptosis and cell cycle of OVCAR-3 and OVCAR-3/DDP cells
Next, we explored the effects of CASC9 expression on cell apoptosis and cell cycle in OVCAR-3 and OVCAR-3/DDP cells. As presented in Figure 3a, Tunel assay showed that cell apoptosis was extremely induced by shRNA-CASC9 but undermined by OverExp-CASC9 in OVCAR-3/DDP cells when compared with the negative control cells. Furthermore, western blot analysis results showed that Bcl-2 level were markedly inhibited whereas levels of Bax and cleaved caspase 3 was increased in CASC9 silenced cells. However, CASC9 overexpression reduced levels of Bax and cleaved caspase 3 but accelerated Bcl-2 level (Figure 3b). In addition, shRNA-CASC9 induced a G1 cell cycle arrest and reduced the percentage of cells in G2 phase. Nevertheless, CASC9 overexpression decreased the G1 phase population, and the same time increased the G2 phase population in OVCAR-3/DDP cells (Figure 3c).

CCNG1/TP53/MMP7 was regulated by aberrant expression of CASC9
To study the potential mechanism by which CASC9 expression affects OC cells, the expressions of CCNG1/TP53/MMP7 pathway were investigated. As shown in Figure 4, the protein levels of CCNG1, TP53, and MMP7 were noticeably improved in OVCAR-3/DDP cells compared with that in OVCAR-3 cells. Inhibition of CASC9 repressed the contents of CCNG1, TP53, and MMP7 while upregulation of CASC9 promoted the protein expression levels of CCNG1, TP53, and MMP7 in OVCAR-3/DDP cells as compared with the negative control.

Interference of CASC9 inhibited tumor growth and accelerated apoptosis in vivo
To further determine the effects of CASC9 on OC, we structured an OC xenograft mouse model ( Figure 7a). As shown in Figures 7B and 7 C, the weight and volume of tumor in mice were significantly increased after injection with cells transfected with OverExp CASC9 compared with the negative control, but CASC9 silencing suppressed the elevated weight and volume of tumor. In addition, the results from tunel assay and western bolt assay revealed that CASC9 overexpression specifically prevented cell apoptosis in model mice while CASC9 knockdown exhibited the opposite results (Figures 7D and 8B). Moreover, we also measured the expressions of CCNG1/ TP53/MMP7 pathway in tumor tissues. The results showed that CASC9 overexpression increased the levels of CCNG1, TP53 and MMP7, but silencing of CASC9 blocked the expressions of CCNG1/ TP53/MMP7 pathway in tumor tissues of mice (Figure 8a). Also, qRT-PCR results showed that upregulation of CASC9 increased CASC9 expression while reduced miR-488-3p expression in tissues. Nevertheless, CASC9 silencing have the inverse effects on expressions of CASC9 and miR-488-3p (Figures 8C and8 D).

Discussion
Ovarian cancer is the most common gynecological malignancies and there is a lack of effective treatments for ovarian cancer to date [25]. Accumulating studies have shown that special lncRNAs play important roles in Pathology and Pathophysiology of ovarian cancer [26][27][28]. In the present study, we demonstrated that CASC9 was upregulated in several OC cells compared with ovarian surface epithelial cells. In addition, CASC9 silencing inhibited cell proliferation, migration and invasion, facilitated cell apoptosis and blocked cell cycle; CASC9 overexpression showed the opposite functions. Further mechanistic investigations showed that CASC9 regulated OV progress through cyclinG1/TP53/MMP7 pathway via targeting miR-488-3p. On the basis of the previous researches, CASC9 has played oncogenic roles in several types of tumors [29,30]. A previous study revealed that CASC9 was significantly overexpressed in esophageal squamous cell carcinoma and CASC9 silencing markedly suppressed cell migration and invasion [31]. Shao et al. reported that CASC9 contributed to breast cancer cell proliferation and cell cycle progression, and repressed cell apoptosis by sponging the miR-195/497 cluster [32]. Recent studies also showed that dysregulation of CASC9 were involved in lung cancer, bladder cancer, and nasopharyngeal carcinoma [33][34][35]. In addition, Hu et al. also revealed that CASC9 was upregulated in ovarian cancer tissues and cell lines. CASC9 was associated with cell proliferation and metastasis, and promoted tumor growth by facilitating LIN7A expression via targeting miR-758-3p in ovarian cancer [36]. In our study, upregulated CASC9 expression was observed in several OC cells and in vivo. Knockdown of CASC9 notably inhibited OVCAR-3/DDP cell proliferation, migration and invasion, enhanced cell inhibitory rate and apoptosis, and blocked cell cycle progression, as well as inhibition of tumor growth in Xenograft mice. However, the opposite results were observed after transfection with overexpressed CASC9 and in mouse models, which was in line with previous results.
It is widely accepted that lncRNAs can serve as ceRNA for microRNAs to modulate expression of specific mRNA [37]. Based on bioinformatics analysis, miR-488-3p was identified as a potential target of CASC9. miR-488-3p has been reported to express variously in many different tissues and diseases, especially in cancers [38]. For example, miR-488 was downregulated in gastric cancer tissues and cells. miR-488 displayed anti -tumor activity by inhibition of cell proliferation, cell cycle, colony information, and migration in gastric cancer [39]. Moreover, miR-488-3p was found to be implicated in chemoresistance of ovarian cancer [40]. Another study revealed that higher CASC9 expression was related with malignant phenotypes of papillary thyroid cancer (PTC) and CASC9 expression was negatively related to miR-488-3p. lncRNA CASC9 accelerate PTC progress by inhibition of miR-488-3p /ADAM9 pathway [36]. In the study, we verified the combination of CASC9 and miR-488-3p by luciferase reporter assay and revealed that overexpression of miR-488-3p rescues the decreased cell inhibitory rate and apoptosis rate induced by CASC9 overexpression, which indicates CASC9 may play roles in OC by regulating miR-488-3p expression. LncRNA CASC9 is one of long noncoding RNAs and it cannot be transcribed into proteins, thus we can't detect CASC9 expression by immunohistochemistry or western blot. To ensure the consistency of the results, we used the same method (qRT-PCR assay) to analysis the expression of CASC9 and miR-488-3p in OC cell lines and xenograft mice. Other technologies such as immunohistochemistry or western blot to further explore the effects of CASC9 and miR-488-3p in OC cell lines and xenograft mice are needed. In addition, most experiments in this study were done in OC cells and xenograft mice. We will verify our results in clinical and further animal experiments in next study.
Several cyclin proteins were expressed abnormally in various human cancer, such as Cyclin A, B, D1, D3, and E [41]. CCNG1 is a subtype of Cyclin G of the cyclin family and it induces failure of mitosis, polyploid formation, and blockage of M phase arrest [42]. It was found that Cyclin G1 was upregulated in epithelial ovarian cancer tissues was and was associated with poor survival [43]. CCNG1 could facilitate the degradation of TP53 by the MDM2 pathway Figure 8. Effects of CASC9 on protein expressions involved in CCNG1/TP53/MMP7 and apoptosis. A, Protein levels of CCNG1, TP53 and MMP7 in tumor tissues of mice was evaluated by western blot assay transfected with shRNA-CASC9 or OverExp-CASC9. B, Protein levels of Bax, Bcl-2, caspase 3 and cleaved caspase 3 in tumor tissues of mice were identified by western blot analysis after transfection with shRNA-CASC9 or OverExp-CASC9. C-D, Expression of CASC9 and miR-488-3p in tumor tissues of mice was determined with qRT-PCR after transfection with shRNA-CASC9 or OverExp-CASC9. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***P < 0.001 versus OVCAR-3 cells; # P < 0.05, ## P < 0.01, ### P < 0.001 versus OVCAR-3/DDP+vector. and the inactivation of TP53 can induce the high expression of CCNG1 [44,45]. Besides, MMP7 is generally regulated by TP53 and functions as an oncogene for OC [46]. Thus, we hypothesized that cyclinG1/TP53/MMP7 was associated with the regulation of CASC9/miR-488-3p in OC. In our study, we used luciferase reporter assay to confirm the binding of miR-488-3p and CCNG1. The expressions of TP53 and MMP7 were significantly upregulated in OC cell lines. Furthermore, CASC9 silencing or overexpression regulated the protein levels of cyclinG1/TP53/MMP7 in vitro and in vivo. Thus, the data demonstrated that cyclinG1/TP53/MMP7 was mediated by CASC9 in OC cells.

Conclusion
In summary, our findings for the first time showed that CASC9 exerts oncogenic roles in OC cells and OC mice models. Furthermore, a cyclinG1/TP53/ MMP7 pathway is involved in the regulation of OC progression by CCNG1/miR-488-3p, providing novel underlying mechanisms and therapeutic targets for patients with OC.