Long Non-Coding RNA SNHG12 Contributes to Cisplatin Resistance by Mediating WEE1 via miR-503-5p in Cervical Cancer

Background: Emerging evidences have indicated that the aberrant expression of long noncoding RNAs (lncRNAs) was responsible for drug resistance, which represents a major obstacle for chemotherapy failure. Our previous study has showed that small nuclear RNA host gene 12 (SNHG12) was increased and contributed to cell growth and invasion in cervical cancer. In the present study, we aimed to investigate the role of the lncRNA SNHG12 in cisplatin (DDP) resistance and elucidate its underlying mechanisms in cervical cancer. Methods: The expression and prognosis of SNHG12 in cervical cancer tissues were evaluated based on bioinformatics. MTT, colony formation assay and ow cytometer were performed to detect cell viability. Further, Molecular relationships among CTD-3252C9.4, IRF1 and IFI6 were investigated via luciferase reporter assay, western blot, and qRT-PCR. Finally, subcutaneous xenograft model was established to verify our ndings. Results: In the present study, we evaluated the cell apoptosis and half maximal inhibitory concentration (IC50) of cervical cancer upon DDP treatment. Mechanically, we found that SNHG12 upregulated WEE1 expression to regulate cell and DDP resistance via sponging miR-503-5p. Moreover, SNHG12 silencing inhibited the growth of DDP-resistant cervical cancer tumors in vivo. Conclusions: Taken together, our ndings suggested that a SNHG12/miR-503-5p/ WEE1 axis which modulated the chemoresistance of cervical cancer cell to DDP, and provided promising targets for dealing with the chemoresistance of cervical cancer.


Introduction
Cervical cancer is the second most common malignancy amongst women worldwide [1]. Though great efforts have been made to improve the diagnosis and treatment of cancer in recent years; however, resistance to chemotherapy usually exists in the process of treating cancers [2,3]. At present, cisplatin (DDP) was considered to be one of the most effective drugs for the clinical treatment of multiple cancers, including cervical carcinoma [3][4][5]. However, DDP resistance has become a major challenge for cervical carcinoma treatment and the molecular basis for resistance remains unclear.
Increasing evidence indicates that lncRNAs contribute to cancer initiation, progression, as well as chemotherapy resistance [8][9][10]. Previous studies have suggested that SNHG12 was signi cantly evaluated in a variety of cancers, including bladder cancer, osteosarcoma, prostate cancer and cervical cancer [11][12][13][14]. For example, SNHG12 was signi cantly increased in bladder cancer tissues compared to adjacent normal tissues and high expression of SNHG12 was associated with shorter recurrence-free survival time [11]. SNHG12 was reported to promote tumorigenesis and metastasis via modulating STAT3 by sponging miR-125b in cervical cancer [15]. SNHG12 was rstly reported to reverse the resistance to cisplatin by SNHG12-miR-181a-MAPK/Slug axis in NSCLC [16]. Subsequent experiments showed that SNHG12 mediates doxorubicin resistance via miR-320a/MCL1 axis in osteosarcoma [17].
Based on these literatures, we speculated that SNHG12 might play a crucial role in chemoresistance in cervical cancer.
Wee1 is a tyrosine kinase that participates in multiple aspects of tumor biology, including proliferation, migration, invasion, and survival [18,19]. Wee1 is known to be overexpressed in HPV-positive head and neck squamous cell carcinoma (HNSCC) and inhibition of Wee1 has been shown to sensitize tumor cells to DDP [20]. In the current study, we designed experiments to investigate the role of SNHG12 in cervical cancer resistance to DDP. We proposed that SNHG12 might contribute to CDDP resistance via regulating the miR-503-5p/WEE1 axis.

Materials And Methods
Cell culture and construction of the DDP-resistant cervical cancer cell line The HEK 293T cells and human cervical cancer cell lines (HeLa and CaSki) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured as our previously described [14].
Cell viability and colony formation assays Different treatment cells in logarithmic growth phase were seeded into 96-well plates at 8 × 10 3 cells per well with 100 µL culture medium. The medium in the administration group was replaced with culture medium containing different DDP concentrations 24 h later. Cells in each well were added with 10 µL CCK-8 (Dojindo, Japan) 48 h later. After 2 h incubation, the optical density (OD) value was measured at a wavelength of 450 nm using a microplate ELISA reader (Bio-Rad, Hercules, CA). The half inhibitory concentration (IC50) was calculated according to the OD value.
For colony formation assay, 8 × 10 2 cells per well HeLa/DDP and CaSki/DDP cells transduced with or without shSNHG12 were seeded into 6-well plates, the medium was replaced with culture medium containing 8µM DDP 24 h later. 5 × 10 3 cells per well HeLa and CaSki cells transfected with or without SNHG12 were seeded into 6-well plates, the medium was replaced with culture medium containing 200 nM DDP 24 h later. Colonies containing at least 50 cells on the plates were xed with 4% polyoxymethylene and stained with 1% crystal violet for 15 min 2 weeks later.

Cell apoptosis analysis
Cells were treated as above mention. Then cells were harvested and stained with Annexin V-FITC and propidium iodide (BD Biosciences, NJ, USA) according to the manufacturer's instructions. The percentage of apoptotic cells was determined on a FACScalibur ow cytometer and analyzed using CellQuestPro software (Becton Dickinson, USA).

Dual-luciferase reporter gene assay
To construct dual luciferase reporter plasmids, the potential binding sequence of miR-503-5p in SNHG12 or WEE1 and their mutated sequence were separately cloned into pMir-Reporter plasmid (Life Technologies). HEK-293T cells were co-transfected with miR-503-5p mimics or miR-NC, and luciferase reporter constructs using HiPerFect Transfection reagent according to the manufacturer's protocol. The luciferase activity was then detected using a Dual-Luciferase® Reporter Assay System (Promega, USA) at 24 h post-transfection.

Animal experiments
Female athymic BALB/c nude mice (4 weeks old) were obtained from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). All of the animal experiments were performed in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals. This study was approved by the Institutional Animal Care and Use Committee of Obstetrics and Gynecology Hospital of Fudan University.
Approximately 1 × 10 7 HeLa/DDP cells transduced with shSNHG12 in 0.2 ml serum-free medium were subcutaneously injected in the axilla of mice. After the tumors grew to approximately 1000 mm 3 in size, (5 mg/Kg) DDP or saline were administered every 5 days. Mice were sacri ced and the xenograft tissues were harvested and then frozen or para n embedded for immunohistochemical detection.

Statistical analysis
Data were presented as mean ± standard deviation (SD). Group comparison was performed by Student's t-test or One-Way Analysis of Variance followed by Newman-Keuls post hoc test. The Pearson's correlation analysis was performed to assess the correlation between SNHG12 and miR-03-5p or WEE1 expression. A P value < 0.05 was considered as signi cant difference.

Results
LncRNA SNHG12 is up-regulated in DDP-resistant cervical cancer cell lines The differential expression of SNHG12 between cervical cancer tissues and normal tissues using GEPIA2 (http://gepia2.cancer-pku.cn/#index) and UALCAN (http://ualcan.path.uab.edu/) based on TCGA database. The results showed that SNHG12 was dramatically higher in cervical cancer patients than in healthy people (Figs. 1A). The upregulation of SNHG12 wasn't found to be associated with the stage (Figs. 1B) and subtype (Figs. 1C) of cervical cancer. Furthermore, it could be observed that the overall survival rate in the high expression group (n = 77) was lower than that in the low expression group (n = 227) from The Cancer Genome Atlas (TCGA) database (http://kmplot.com/analysis/index.php) (Fig. 1D). Our previous studies have shown that SNHG12 was up-regulated in cervical cancer tissues and cell lines [19], then we further explored whether SNHG12 was involved in the DDP resistance of cervical cancer cells. Interestingly. Then DDP-resistant HeLa/DDP and CaSki/DDP cell line were established with gradually increasing DDP concentration. SNHG12 expression was also signi cantly increased in both HeLa/DDP and CaSki/DDP cells compared with their parental cells by qRT-PCR, respectively (Fig. 1E). Therefore, these prompted us to suspect that SNHG12 might participate in DDP-resistance of cervical cancer.

Inhibition of SNHG12 suppressed the drug resistance of cervical cancer cells to DDP, whereas
To evaluate the role of SNHG12 in the regulation of cervical cancer chemoresistance to DDP, the expression of SNHG12 was knocked down by shRNA in both HeLa/DDP and CaSki/DDP cells. SNHG12 was dramatically decreased after SNHG12 silencing in both HeLa/DDP and CaSki/DDP cells ( Fig. 2A).
After transduction, the effects of SNHG12 on the proliferation and apoptosis of DDP-resistant cells were assessed. Upon 8 µg/ml DDP treatment, the IC50 value of SNHG12 knockdown was markedly lower than that of SCR group (Fig. 2B). Furthermore, apoptosis assay showed that SNHG12 silencing induced DDPresistant cells apoptosis (Fig. 2C). Moreover, knockdown of SNHG12 led to a signi cant repressed anchorage-independent growth upon DDP treatment (Fig. 2D). In contrast, SNHG12 was overexpressed in HeLa and CaSki cells by transfecting SNHG12 and con rmed by qRT-PCR (Fig. 2E). As expected, overexpression of SNHG12 could enhanced the IC50 value (Fig. 2F) and colony formation (Fig. 2G), and inhibited apoptosis (Fig. 2H) of cervical cancer cells. In summary, these results revealed that SNHG12 played an important role in the chemoresistance of cervical cancer cell to DDP.

SNHG12 served as a molecule sponge for miR-503-5p
It is widely acknowledged that lncRNAs can act as competitive RNA (ceRNA) through sponging with miRNAs in various cancers [7]. To determine the potential mechanism of SNHG12, online software program starbase v2.0 (http://starbase.sysu.edu.cn/mirLncRNA.php) was used to predict the possible miRNAs. To better understand the role of SNHG12 in the development of cervical cancer, LinkedOmics was used to analyze its related genes. The top 50 genes that were signi cantly positively or negatively correlated with SNHG12 are indicated with a heat map (Fig. 3A). Further the enrichment functions of GO annotations (Fig. 3B) and KEGG pathways (Fig. 3C) analyzed using GSEA showed that ribosome played a crucial role in biological process of SNHG12. By comparing all of the candidate genes predicted by starbase v2.0, miR-503-5p was selected due to the tumor suppressive roles of miR-503-5p in several cancers (Fig. 3D) [21][22][23]. To further investigate whether SNHG12 was a functional target of miR-503-5p, luciferase reporter was performed and our results showed that overexpressed miR-503-5p could downregulate the wild-type luciferase activity of SNHG12 but not the mutant of SNHG12 (Fig. 3E).
MiR-503-5p was required for SNHG12-mediated drug sensitivity of DDP-tolerated cervical cancer cells To determine the functional signi cance of miR-503-5p in the SNHG12-induced phenotype, a series of restoration assays were performed in DDP resistance of cervical cancer cells. Anti-miR-503-5p was used to inhibit miR-503-5p expression in SNHG12-depleted DDP-tolerant cervical cancer cells and miR-503-5p was signi cantly decreased after miR-503-5p inhibitor transfection (Fig. 4A). As illustrated in Fig. 4B and 4C, decreased IC50 value of SNHG12-depleted DDP-tolerant cervical cancer cells was increased by miR-503-5p inhibitor transfection. Furthermore, inhibition of miR-503-5p could signi cantly abrogate the effect of SNHG12 on DDP-tolerant cervical cancer cells. Colony formation assay could not be performed because of the few numbers of colonies after SNHG12 silencing. Taken together, these results implied that miR-503-5p was required for the biological functions of SNHG12 in cervical cancer cells. WEE1 was directly targeted by miR-503-5p and partly controlled by SNHG12 Using online software program, TargetScan, starbase v2.0and miRDB, a total of 145 genes were identi ed to potentially regulate by miR-503-5p (Fig. 5A). Investigating genetic model of drug response (iGMDR) (https://igmdr.modellab.cn) was used to assess the drug sensitivity of WEE1 in cervical cancer [24]. The results showed the related genes (Fig. 5B) and pathways (Fig. 5C) involved in drug response of WEE1. By comparing all candidate genes predicted by the programs, WEE1 was selected for further experiments due to the ontogenetic role of WEE1 in cervical cancer (Fig. 5D). Luciferase reporter assays were performed and the results revealed that miR-503-5p could dramatically suppress the luciferase activity of the reporter gene in recombinant plasmids containing the wild-type 3′UTRs of WEE1 in HEK-293T cells but did not affect the activities of mutant (Fig. 5E). In concordance with these results, the mRNA and protein expression of WEE1 was signi cantly decreased in miR-503-5p-overexpressed cervical cancer cells (Fig. 5F). Similar results were observed in SNHG12-depleted DDP-tolerant cervical cancer cells (Fig. 5G). Moreover, introduction of anti-miR-503-5p partly abrogated the downregulation of WEE1 induced by SNHG12 silencing in both HeLa/DDP and CaSki/DDP cells (Fig. 5H). In addition, the mRNA level of WEE1 was upregulated in cervical cancer tissues (Fig. 5I). A dramatically negative correlation was noted between miR-503-5p and WEE1 expression (Fig. 5J) and a positive correlation was also noted between SNHG12 and WEE1 expression in cervical cancer tissues (Fig. 5K). Taken together, these data indicated that the miR-503-5p/WEE1 axis mediated the effect of SNHG12 on cervical cancer cells DDP resistance.

SNHG12 silencing decreases cervical cancer cell resistance to DDP therapy in vivo
To investigate the in vivo effect of SNHG12 on cervical cancer DDP resistance, we generated xenografts using SNHG12 stale knockdown HeLa/DDP cells. Once the tumors reached a diameter of 1.0 cm, mice was divided into two groups and treated with either DDP or saline solution, respectively (Fig. 6A). DDP treatment led to a signi cant reduction of tumor volume compared to that of the group without DDP treatment (Fig. 6B). Furthermore, the growth curse and weight of the tumors with DDP treatment were decreased compared with those in the group without DDP treatment ( Fig. 6C and D). In addition, the expression of c-Myc was remarkably decreased in SNHG12 silencing xenografts tissues compared to the PBS-treated xenografts sections (Fig. 6E), which further con rmed that SNHG12 has a positive effect on both the growth and drug resistance in vitro and in vivo.

Discussion
Chemotherapy is regarded as a standard strategy for patients with advanced or recurrent cervical cancer and DDP was the rst-line treatment in chemotherapy of cervical cancer [25,26]. The anti-tumor effects of DDP has shown to promote DNA damage by binding to DNA and crosslinking the DNA strands, which resulted in cell death [27][28][29]. However, DDP resistance became a major obstacle for cancer therapy and compromised the e cacy of DDP to treat advanced or recurrent cervical cancer. Our previous study has shown that SNHG12 is signi cantly upregulated in cervical tissues and knockdown of SNHG12 expression suppressed cell proliferation and invasion by modulating miR-424-5p expression [14].
Recently, a growing body of reports has demonstrated that lncRNAs played functional roles in DDP resistance of cancer cells [30,31]. SNHG12 was been reported to increase in Temozolomide(TMZ)resistant glioblastoma cells and enforcing SNHG12 expression led to the development of acquired TMZ resistance [32]. This study focuses on the importance of further understanding the role of SNHG12 in drug resistance in cervical cancer. In this study, we con rmed the expression of SNHG12 upregulated in DDP-resistant cervical cancer tissues and DDP-resistant cells. Further investigation demonstrated that SNHG12 knockdown decreased the IC50 value of DDP-resistant cervical cancer cells and clone formation, and promoted cell apoptosis, whereas overexpression of SNHG12 had an opposite effects in the presence of DDP. These results indicated that SNHG12 may be a promising new treatment intervention for the patients with cervical cancer.
Numerous reports have suggested that lncRNAs harbored the recognition sequence of many miRNAs and SNHG12 has been predicted to function as ceRNA of several miRNAs, including miR-320, miR-125b and miR-181, miR-195-5p, miR-129-5p [7,12,13,[15][16][17]. In our research, we found that SNHG12 functions as a ceRNA to repress miR-503-5p expression. MiR-503-5p was reported to be downregulated in several cancers, including lung cancer, gastric cancer, ovarian cancer, and correlates with tumor progression and clinical prognosis [22,33,34]. In addition, downregulation of miR-503-5p contribute to cell survival and chemoresistance in ovarian cancer and colorectal carcinoma [22,23]. Consisted with these data, the inhibitory phenomenon of SNHG12 silencing could partially was blocked by miR-503-5p inhibitor in DDP resistance cervical cancer cells. Subsequent study con rmed that WEE1 was a direct target of miR-503-5p and indirectly regulated by SNHG12. WEE1, a known oncogene, has been reported to increase in many cancers and involved in DNA damage and drug resistance [35,36].

Conclusion
In conclusion, our data proved that SNHG12 was upregulated in cervical cancer tissues from patients who did not respond to DDP treatment than those from patients who experienced response to chemotherapy. In vitro experiments demonstrated that overexpression or inhibition of SNHG12 would alter DDP resistance through the miR-503-5p/WEE1 axis. There are some limits in our present study: 1, the effects of SNHG12 on DNA damage; 2, WEE1 inhibitors could effectively overcome the DDP resistance patients, especially high SNHG12 expression; 3, more patients were needed to con rm our data.