{'fraction_non_alphanumeric': 0.04159592529711375, 'fraction_numerical': 0.031712345379577976, 'mean_word_length': 4.8673070081821415, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
lncRNA SNHG3 acts as oncogene in ovarian cancer through miR-139-5p and Notch1

Ovarian cancer (OC) is a common malignant tumor of the female reproductive system. Long non-coding RNAs (lncRNAs) play an important role in OC occurrence and development. Thus, the function and potential mechanism of lncRNA small nucleolar RNA host gene 3 (SNHG3) was explored in the development of OC. The expression of SNHG3, microRNA (miR)-139-5p and Notch homolog 1, translocation-associated (Drosophila) (Notch1) in OC were detected by RT-qPCR or western blot assay. In addition, CCK-8 and wound-healing assays were used to detect OVCAR3 proliferation and migration ability. The targeting relationship of miR-139-5p with SNHG3 or Notch1 was verified through luciferase reporter assay. Rescue experiments were performed to confirm whether SNHG3 could mediate OVCAR3 proliferation and migration through miR-139-5p and Notch1. In OC tissues and cell lines, the expression of SNHG3 and Notch1 were significantly increased, and the expression of miR-139-5p was significantly decreased. SNHG3 inhibition suppressed the proliferation and migration of OVCAR3 cells. Luciferase reporter experiment confirmed that miR-139-5p could target SNHG3 and Notch1. Transfection of miR-139-5p inhibitor significantly reversed the inhibitory effect of SNHG3 knockdown on OVCAR3 proliferation and migration. Moreover, SNHG3 inhibition or miR-139-5p mimic abolished the promotion of Notch1 overexpression on OVCAR3 proliferation and migration. In conclusion, SNHG3 could accelerate the proliferation and migration of OC cells by regulating miR-139-5p and Notch1.


Introduction
Ovarian cancer (OC) is one of the most lethal gynecological malignancies in the world (1). Early detection of OC is difficult for lack of specific diagnostic markers. Therefore, most patients are in advanced stage when they are diagnosed (2). Although adjuvant chemotherapy has improved the prognosis of patients, the survival rate of patients with OC remains low due to late diagnosis and resistance to chemotherapy (2,3). Therefore, reasonable and effective treatment is the key to improve the survival rate and prolong the survival time of patients with OC. Its occurrence and development involve a variety of genetics and environmental factors (4). As important biomolecules, lncRNAs play an important role in the communication and exchange between cells, and the occurrence, development, metastasis and resistance of tumors (5). In recent years, some progress has been made in the mechanism and treatment of lncRNAs in OC. As research continues, the function of lncRNAs will further be clarified, and it has broad application prospects in the early diagnosis and treatment of OC.
lncRNAs are non-coding RNA molecules, that do not have protein-coding functions, and participate in biological processes in the form of RNAs (6). With the development of technology, an increasing number of studies have revealed that lncRNAs can not only maintain the normal physiological functions of cells, but also play an important role in tumor progression (5). For example, PVT1 could promote gallbladder cancer progression by mediating the miR-143/HK2 axis (7). Luo et al (8) have suggested that NEAT1 could sponge miR-34a to regulate SIRT1 expression, and activate Wnt/β-catenin signaling, thus accelerating colorectal cancer progression. Sun et al (9) have reported XIST could suppress renal cell carcinoma progression by regulating the miR-106b-5p/p21 axis. It has also been reported that various lncRNAs are dysregulated in OC, and their expression levels are related to OC progression, prognosis, patient survival, and response to treatment, such as H19, MALAT1, HOTAIR, NEAT1, MEG3, lncRNA SNHG3 acts as oncogene in ovarian cancer through miR-139-5p and Notch1 XIST, and MALAT1 (10,11). However, there are few studies on small nucleolar RNA host gene 3 (SNHG3) in OC. SNHG3 which is located on 1q36.1 and is also named host gene of U17 (U17HG), is a member of SNHG family (12). In OC, SNHG3 expression has been revealed to be markedly increased, and significantly related to the malignant degree and poor prognosis of OC (13). It has been revealed that SNHG3 participated in energy metabolism in OC by regulating miRNAs and EIF4AⅢ (14). However, its potential mechanisms in OC remain to be explored. Thus, the role of SNHG3 in OC was explored, and the signaling pathways that SNHG3 regulated or participated in OC were identified, in order to clarify the regulation mode of SNHG3 in OC.

Materials and methods
Tissue samples. In the present study, 40 patients with OC and 19 patients with benign OC admitted to Jinan Maternal and Child Health Care Hospital (Jinan, China) from July, 2018 to September, 2019. OC patients were between the ages of 45 and 65 years, with an average age of 51.8±7.6 years. The age of benign OC patients were ranged from 40 to 55 years, with an average age of 48.8±8.6 years. Inclusion criteria included: i) Not receiving any treatment before surgery. ii) Confirmed by pathological examination. iii) The patient underwent follow-up observations. Exclusion criteria included: i) Patients with other malignant tumors. ii) Severe abnormal liver function. iii) Serious lung diseases. iv) Cannot cooperate with the treatment. v) Patients with mental illness or immune disease. This study was approved by the Medical Ethics Committee of Jinan Maternal and Child Health Care Hospital and all subjects signed written informed consent.
Cell culture. Human OC cell lines A2780, SKOV3, OVCAR3 and OV90, and human ovarian epithelial cell line HOSE which were purchased from the American Type Culture Collection (ATCC) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin in a cell incubator at 37˚C and 5% CO 2 . Cell passage was performed when the degree of cell fusion reached 80%. Dual luciferase reporter assay. The binding sites between SNHG3 and miR-139-5p, and the target genes of miR-139-5p were predicted using starBase 3.0 (http://starbase.sysu.edu.cn/ index.php) (15) and TargetScan 7.2 database (www.targetscan. org) (16). The pmirGLO vector (v) was used to construct vectors [wild-type (SNHG3-wt and Notch1-wt) and mutant type (SNHG3-mut and Notch1-mut)] for luciferase reporter assays. Cells were seeded into a 24-well plate. After the cell density in the well reached ~80%, miR-139-5p mimic or miR-NC (GenePharma Co., Ltd, Shanghai, China) was transfected into OC cells with lucif erase reporter vectors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48-h transfection, fluorescence activity was assessed according to the instructions of the dual luciferase detection system (Promega Corp.). The luciferase activity was normalized to that of Renilla luciferase activity. The experiment was repeated three times independently.
Cell Counting Kit-8 (CCK-8) assay. A CCK-8 assay (Dojindo Molecular Technologies, Inc.) was used to detect cell proliferation according to the manufacturer's instructions. The transfected cells were inoculated into 96 wells with 2x10 4 cells/well. After cell attachment, they were cultured for 1, 2, 3 and 4 days, and 10 µl of CCK-8 solution was added. Finally, the absorbance of each well at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Inc.). The experiment was conducted independently in triplicate.
Wound-healing assay. The transfected OC cells were placed into a 6-well plate in a CO 2 incubator for 24 h. After the cells were completely attached to the wall, a straight line perpendicular to the cell surface was gently drawn in the petri dish with the tip of a 10-µl pipette. The non-adherent cells were washed off. Image-Pro Plus software (Media Cybernetics, Inc.) was used to measure the scratch width. Cell scratch width was analyzed and the scratch healing rate was calculated. Scratch healing rate = (scratch width at 0 h -scratch width

SNHG3 expression in OC.
The expression level of SNHG3 in OC tissues was significantly higher than normal tissues (Fig. 1A). Further examination of SNHG3 expression in OC cells revealed a significant increase of SNHG3 expression in different OC cell lines vs. HOSE cells (Fig. 1B). SNHG3 expression was higher in the OVCAR3 cell line than in the other cell lines (Fig. 1B). Based on the aforementioned experimental results, the OVCAR3 cell line was selected for further biological functional experiments.
SNHG3 regulates OC cell proliferation and migration. OC cells were collected after 48-h transfection. Compared with the si-NC group, si-SNHG3 could effectively decrease the expression of SNHG3 in OVCAR3 cells ( Fig. 2A). Subsequently, CCK-8 and wound-healing assays were performed to detect the effects of SNHG3 on cell proliferation and migration. The CCK-8 assay revealed that cell proliferation ability was significantly inhibited after downregulation of the expression level of SNHG3 in OVCAR3 cells (Fig. 2B). Similarly, the scratch healing rate of the si-SNHG3 group was significantly lower than in the si-NC group (Fig. 2C), indicating that OC cells exhibited a decreased migration ability after SNHG3 knockdown.
miR-139-5p regulates the suppressive effects of SNHG3 on OC cells. Considering the relationship of SNHG3 and miR-139-5p, SNHG3 and miR-139-5p expression were inhibited to detect OVCAR3 cell proliferation and invasion capabilities (Fig. 4A). The proliferation ability of cells in the si-SNHG3 group was significantly reduced compared with the si-SNHG3+inhibitor group (Fig. 4B). The wound-healing assay revealed that compared with the si-SNHG3 group, the scratch healing rate of si-SNHG3+inhibitor group was significantly increased at 48 h (Fig. 4C). The results indicated that the suppression of si-SNHG3 on OVCAR3 cell proliferation and migration ability could be reversed by inhibiting miR-139-5p.
Notch1 is a target of miR-139-5p. Then, bioinformatic online analysis (TargetScanHuman 7.2) predicted targets of miR-139-5p. The results revealed that binding sites exist between Notch1 and miR-139-5p (Fig. 5A). A luciferase reporter experiment revealed that compared with the Notch1-wt+miR-NC group, the luciferase activity of the Notch1-wt+mimic group was significantly reduced (Fig. 5B). However, in the Notch1-mut group, no effect on luciferase activity was observed (Fig. 5B). It was thus revealed that miR-139-5p could bind to Notch1.
In order to further verify the regulatory relationship between SNHG3 and miR-139-5p on Notch1, SNHG3 and miR-139-5p expression were interfered in OVCAR3 cells to analyze their effects on Notch1. Notch1 mRNA and protein levels of the si-SNHG3 group were significantly reduced vs. the si-NC group, and miR-139-5p inhibitor reversed this effect (Fig. 5C). Moreover, an evident upregulation of Notch1 (Fig. 5D) as well as a significant positive correlation with SNHG3 expression in OC tissues were observed (Fig. 5E). Collectively, it was theorized that SNHG3 may regulate Notch1 expression through miR-139-5p in OC.

Discussion
OC is a malignant tumor of the reproductive system that affects the health and life of women (3). It includes numerous histological subtypes according to risk factors, origination, Figure 5. miR-139-5p directly targets Notch1 in OVCAR3 cells. (A) Putative target sites between miR-139-5p and Notch1 were obtained by bioinformatics analysis. (B) A luciferase reporter assay was used to confirm the binding sites of SNHG3 and miR-139-5p. (C) RT-qPCR and western blot assays were conducted to detect Notch1 expression in OVCAR3 cells after SNHG3 and miR-139-5p inhibition. (D) RT-qPCR was applied to analyze Notch1 mRNA expression in OC tissues. (E) Spearman's correlation analysis revealed the correlation between SNHG3 and Notch1 in OC tissues. ** P<0.01 and *** P<0.001, compared with miR-NC, si-NC, or the normal group; # P<0.05.compared with si-SNHG3. miR, microRNA; Notch 1, Notch homolog 1, translocation-associated (Drosophila); SNHG3, small nucleolar RNA host gene 3; si-SNHG3, small interfering RNA targeting SNHG3; OC, ovarian cancer; RT-qPCR, reverse transciption-quantitative PCR; NC, negative control. molecular compositions, pathogenesis and treatments, of which epithelial OC accounts for ~85-90%; the most common is HGSOC which accounts for ~70% of ovarian malignancies (1,4). Although there has been great progress in OC prevention and treatment in recent years, most patients with advanced OC will relapse even if the treatment reaches complete remission (18). Therefore, it is particularly important to investigate the pathogenesis of OC.
The progression of OC is a complex process, which is regulated by various cytokines and signaling pathways (19). Previous studies have generally revealed that numerous lncRNAs with dysregulated expression in OC are regarded as potential therapeutic targets (11), including PVT1 (7), NEAT1 (8), and XIST (9). SNHG3 has been reported as an oncogene in OC and its overexpression can promote cell proliferation and invasion while knockdown of SNHG3 has the opposite effect (13). In the present study it was also detected that SNHG3 was upregulated in OC and its knockdown suppressed cell proliferation and migration. This was consistent with previous research and suggested that SNHG3 plays a role as a carcinogen (11). Notably, it was also confirmed that SNHG3 could bind to miR-139-5p.
As a promising cancer biomarker, miR-139-5p has various roles in different types of tumors (20). Yang et al (21) indicated that miR-139-5p expression was decreased in prostate cancer, and was proposed to play an anticancerous function by mediating SOX5. A previous study also revealed that the expression of miR-139-5p was decreased in oral squamous carcinoma cells, and had an inhibitory role in the progression of tumorigenesis through the regulation of HOXA9 (22). However, miR-139-5p was demonstrated to be upregulated in adrenocortical cancer and play a promoting role in cell migration and invasion by mediating NDRG4 expression (23). In OC, low expression of miR-139-5p has been confirmed in previous studies (24,25). In the present study it was observed that miR-139-5p expression was significantly decreased in OC. Additionally, SNHG3 could bind to miR-139-5p and a negative regulatory relationship was revealed of their expression. Furthermore, miR-139-5p inhibition abolished the suppressive effects of SNHG3 on OC cell proliferation and migration. The results indicated that SNHG3 could promote OC progression by inhibiting miR-139-5p.
Studies have revealed that lncRNAs, as carcinogenic or tumor suppressor genes, mainly regulate the expression of miRNA target genes by competitively binding miRNAs, and participate in the occurrence and development of malignant tumors (26). In the present study it was confirmed that Notch1 was a target gene of miR-139-5p. Notch1, as an important part of Notch signaling, plays an oncogenic role in multiple cancers (27). Its high expression has been revealed to lead to poor overall survival of OC patients (28). It was observed that Notch1 was upregulated and facilitated cell proliferation and migration of OC progression. SNHG3 inhibition or miR-139-5p overexpression suppressed Notch1 and its promoting function on OC cell proliferation and migration. The aforementioned results proposed that SNHG3 regulated Notch1 and miR-139-5p in OC progression.
There are some shortcomings in our research. For example, additional OC tissue samples should be collected to further explore the correlation between SNHG3 and clinicopathological features in OC. Moreover, two or more OC cell lines, other experiments (such as cell cloning, Transwell assay and RNA immunoprecipitation) and animal experiments should be introduced to investigate the function of SNHG3 in OC. Furthermore, it is possible there are other miRNAs or target genes/signaling pathways that are regulated by SNHG3. Hence, more research is warranted to fully understand the potential molecular mechanism of SNHG3 in OC.
In summary, SNHG3 expression significantly increased and its knockdown reduced cell proliferation and migration abilities in OC. Concurrently, the mechanism of SNHG3 was explored and preliminarily researched. The results demonstrated that SNHG3 can regulate the expression of Notch1 and miR-139-5p, and thus play a role in the development of OC. This provides a scientific basis for further exploration of the mechanism of OC progression and finding new targets for OC treatment.