lncRNA SNHG15 Induced by SOX12 Promotes the Tumorigenic Properties and Chemoresistance in Cervical Cancer via the miR-4735-3p/HIF1a Pathway

Cervical cancer (CC) is one of the most common malignancies in females, with high prevalence and mortality globally. Despite advances in diagnosis and therapeutic strategies developed in recent years, CC is still a major health burden worldwide. The molecular mechanisms underlying the development of CC need to be understood. In this study, we aimed to demonstrate the role of lncRNA SNHG15 in CC progression. Using qRT-PCR, we determined that lncRNA SNHG15 is highly expressed in CC tumor tissues and cells. lncRNA SNHG15 knockdown also reduces the tumorigenic properties of CC in vitro, as determined using the MTT, EdU, flow cytometry, and transwell assays. Using bioinformatics analysis, RNA pull-down, ChIP, and luciferase reporter assays, we verified the molecular mechanisms of lncRNA SNHG15 in CC progression and found that lncRNA SNHG15 expression in CC cells is transcriptionally regulated by SOX12; moreover, lncRNA SNHG15 promotes CC progression via the miR-4735-3p/HIF1a axis. This study can provide a potential target for CC diagnosis or therapeutic strategies in the future.


Introduction
Cervical cancer (CC) ranks next to breast cancer as the most common malignancy and is the third leading cause of cancer-related deaths in females globally [1]. The health burden associated with CC is more severe in developing countries [2], particularly in low-income countries, than in developed countries [2]. Multiple risk factors contribute to the initiation and progression of CC, such as viral infection, genetic influences, and human papillomavirus (HR-HPV) genotype infection [3,4]. Despite improvement in clinical intervention strategies, such as the global application of standard vaccination, surgical resection innovation, and periodic cancer screening in recent decades, the outcomes of patients with CC remain poor. Therefore, the molecular mechanisms underlying CC development need to be understood, and novel therapeutic targets for the prevention and treatment of CC have to be urgently developed.
In this study, we aimed to demonstrate the role of lncRNA SNHG15 in CC progression. We found that lncRNA SNHG15 was abundantly expressed in CC tumors and cells. lncRNA SNHG15 knockdown significantly attenuated tumorigenesis in CC in vitro and in vivo, as determined using the MTT, EdU, flow cytometry, and transwell assays, in addition to animal experiments. Further, our study revealed that lncRNA SNHG15 influenced the chemoresistance of CC cells to cisplatin. We then investigated the molecular mechanisms of lncRNA SNHG15 in CC cells by conducting bioinformatics analysis, RNA pull-down, ChIP, and luciferase reporter assay. It was found that lncRNA SNHG15 expression in CC cells was transcriptionally regulated by SRY-Box Transcription Factor 12 (SOX12). Further, we found that lncRNA SNHG15 upregulated HIF1a expression to regulate tumorigenesis in CC by sponging miR-4735-3p. Thus, our study elucidated the effect of SOX12/lncRNA SNHG15/miR-4735-3p/HIF1a axis on the biological features of CC.

Clinical Samples.
A total of twenty-eight pairs of CC tumor tissues and comparative normal tissues were collected from Xiangyang Central Hospital within the period 2019/06 to 2020/06. All CC tissues were immediately stored in −80°C liquid nitrogen until the experiment was conducted. Informed consent was obtained from each patient. All CC tissues were confirmed by two pathologists, independently. This study was performed following the principles of the Declaration of Helsinki and was approved by the ethical committee of Xiangyang Central Hospital.
2.2. Cell Culture and Transfection. All cervical cancer cell lines (SiHa, HeLa, Caski, C-33A, and MS751) and normal cell line HEKn were commercially obtained from the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured using Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, USA) supplied with 10% fetal bovine serum (FBS) at 37°C in a humidified 5% CO 2 environment.
5-Ethynyl-2 ′ -deoxyuridine (EdU) experiment. The experimental cells were first cultured in a 96-well plate after transfection. The EdU kit (KeyGEN BioTECH) was used to stain cells in the well in accordance with instructions provided by the manufacturer. The results (EdU-positive cells) were recorded by a fluorescence microscopy.
2.6. Cell Apoptosis Detection. The apoptosis rate of infected CC cells was detected by flow cytometry analysis. The infected CC cells were stained using an Annexin Vfluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD Biosciences, USA), and the results were calculated with a flow cytometer using the CellQuest software version 0.9.3.1 (BD Biosciences, USA).

2
Oxidative Medicine and Cellular Longevity 2.7. Cell Migration Detection. The migratory ability of infected CC cells was detected using the transwell assay. In summary, the infected CC cells were seeded in the upper chambers (Costar, USA) supplied with the serum-free RPMI-1640 medium, and the lower chambers without cells were supplied with full medium. After incubation for one day, the migrated cells were fixed with 4% paraformaldehyde and then stained with crystal violet. The results were recorded by a microscopy.
2.8. Murine Xenograft Assay. SiHa and HeLa cells stably transfected with si-SHNG15#1, si-SHNG15#2, or si-NC were injected into mice aged 4 weeks at a density of 1 × 10 6 cells. The dimensions of the tumors were determined, and their weights were recorded after injection for 28 d.
The animal procedures conducted in this study complied with the guidelines set by the ethical committee of Xiangyang Central Hospital.
2.9. RNA Pull-down Assay. SiHa and HeLa cells were infected using 50 nM biotinylated probes, which were synthesized and procured from GenePharma (Shanghai, China). After 2 d, the M-280 streptavidin magnetic beads (Sigma-Aldrich, USA) were used to incubate with the cell lysate at 4°C for 3 h. The beads were then washed, and the bound RNAs were isolated using the TRIzol Reagent. The results were analyzed by qRT-PCR. The experiment was performed 3 times.
2.10. Luciferase Assays. Cells were first cultured in a 24-well plate (1 × 10 4 cells per well) for 2 d. They were then transfected with the indicated vectors using a Lipofectamine 3000 kit (Invitrogen) in accordance with the manufacturer protocols. After 2 d, the luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). The experiment was performed 3 times.

Chromatin Immunoprecipitation (ChIP) Assay.
Infected CC cells were stained with 1% formaldehyde and lysed at room temperature for 10 min. The genome was frag-mented to~500 bp upon sonication. Antibodies were used to incubate the cell lysate for 12 hours. The lysate was subsequently incubated with Protein A Agarose/Salmon Sperm DNA (50% Slurry) beads for 6 hours. The results were analyzed by qRT-PCR assay.
2.12. Statistical Analysis. The GraphPad Prism software (GraphPad, USA) was used to calculate all data in this study. Statistical analysis was performed using two-tailed Student's t-test. p < 0:05 indicated a significant difference. Data in this study are presented using the mean ± square deviation ðSDÞ.

Expression Characteristics of lncRNA SNHG15 in
Cervical Cancer. To identify the possible function of lncRNA SNHG15 in CC progression, we first investigated the expression profile of lncRNA SNHG15 in CC. Twenty-eight pairs of CC tumor tissues and its comparative normal tissues were examined. lncRNA SNHG15 was noticeably upregulated in CC tumor tissues relative to that in normal tissues ( Figure 1(a)). We further found that lncRNA SNHG15 was abundantly expressed in CC cell lines (Figure 1(b)) and mainly located in the cell cytoplasm (Figures 1(c) and 1(d)). On the basis of these combined findings, we hypothesized that lncRNA SNHG15 is involved in CC progression.

lncRNA SNHG15 Knockdown Attenuates CC
Tumorigenesis and Chemoresistance In Vitro. Given the abundant expression of lncRNA SNHG15 in CC tumor tissues and cells, we generated knockdown cell models of lncRNA SNHG15 to explore its cellular function (Figure 2(a)). We first examined the proliferation level of lncRNA SNHG15 knockdown cells by using the MTT assay (Figures 2(b) and 2(c)) and the EdU assay (Figures 2(d) and 2(e)) in SiHa and HeLa cells and found that lncRNA SNHG15 downregulation markedly suppressed CC cell proliferation. lncRNA SNHG15 downregulation significantly promoted the cell apoptosis rate (Figures 2(f) and 2(g)), as    Figure S1); lncRNA SNHG15 overexpression promoted cell proliferation, migration, and chemoresistance to cisplatin but repressed cell apoptosis.

lncRNA SNHG15 Expression in CC Is Transcriptionally
Regulated by SOX12. We investigated the upstream regulator of lncRNA SNHG15 in CC cells. Bioinformatics analysis was conducted, and 6 potential transcriptional regulators were predicted. We found that lncRNA SNHG15 expression was markedly decreased in CC cells under SRY-Box Transcription Factor 12 (SOX12) knockdown (Figures 3(a) and 3(b)) and increased in CC cells upon SOX12 overexpression (Figure 3(c)). The binding motif in SOX12 and the binding sites of the lncRNA SNHG15 promoter were acquired from the JASPAR database (Figures 3(e) and 3(e)). Chromatin immunoprecipitation (ChIP) assay results showed that the lncRNA SNHG15 promoter was pulled down by the SOX12 antibody in the SiHa and HeLa cells (Figure 3(f)).
Moreover, miR-4735-3p expression in CC cells was upregulated under lncRNA SNHG15 knockdown (Figure 4(h)) and upregulated in CC tumors relative to that in normal samples (Figure 4(i)). These data revealed the miR-4735-3p as a downstream target for lncRNA SNHG15 in CC.

miR-4735-3p
Modulates Tumorigenesis and Chemoresistance in Cervical Cancer. We revealed miR-4735-3p as a target for lncRNA SNHG15 in CC cells; however, the function of miR-4735-3p remained unverified. Thus, we conducted a gain-or loss-of-function experiment on miR-4735-3p in CC cells. Cell models were generated as indicated ( Figure 5(a)). CC cell proliferation was markedly suppressed by the miR-4735-3p mimic but promoted by the miR-4735-3p inhibitor, as determined using the MTT assay ( Figures 5(b)  3.6. miR-4735-3p Targets to HIF1a. A total of 13 mRNA targets of miR-4735-3p were found by bioinformatics analysis (Figure 6(a)). We then found that HIF1a expression was decreased in miR-4735-3p overexpressed CC cells (Figures 6(b) and 6(c)) and increased in miR-4735-3p knockdown CC cells (Figure 6(d)), indicating that miR-4735-3p could be a potential target to HIF1a. The binding sites between miR-4735-3p and HIF1a were then obtained as indicated (Figure 6(e)). Data from the RNA pull-down assay showed that HIF1a was noticeably enriched in biotinylated miR-4735-3p probes in CC cells (Figures 6(f) and 6(g)). Meanwhile, luciferase reporter gene assay results confirmed that miR-4735-3p directly sponged to HIF1a in CC cells (Figures 6(h) and 6(i)). The interaction between miR-4735-3p and HIF1a was verified. HIF1a expression in CC  Oxidative Medicine and Cellular Longevity tissues was measured; HIF1a expression in CC tumors was markedly higher than that in comparative normal tissues (Figures 6(j) and 6(k)).   Figure S3A). The cell apoptosis rate increased subsequent to lncRNA SNHG15 knockdown attenuated by HIF1a in CC cells (Figure 7(f)). Moreover, lncRNA SNHG15 knockdown suppressed cell migration level, and those occurrences were rescued by HIF1a in CC cells (Figure 7(g), Supplementary Materials Figure S3B). With regard to the chemoresistance of CC cells to cisplatin, lncRNA SNHG15 knockdown inhibited the IC50 value of SiHa and HeLa cells, but this occurrence was reversed by HIF1a (Figures 7(h) and 7(i)). Our results showed that lncRNA SNHG15 modulated HIF1a expression to regulate CC progression.

lncRNA SNHG15 Knockdown Suppresses CC Tumor
Growth In Vivo. The aforementioned experiments demonstrated the function of lncRNA SNHG15 in vitro. We generated mouse models to examine the role of lncRNA SNHG15 in vivo. lncRNA SNHG15 knockdown cells (10 6 per murine) were subcutaneously injected into the mice, and after 4 weeks, tumors were collected. The representative image of xenograft tumors is presented in Figure 8(a). The tumor volume and the end weight were significantly inhibited by lncRNA NHG15 knockdown (Figures 8(b) and 8(c)). Further, the expression levels of SNHG15, miR-4735-3p, and HIF1a in lncRNA SNHG15 knockdown tumor tissues were measured (Figures 8(d)-8(g)), and t and the trends were consistent with our in vitro experiments. The results obtained suggest that lncRNA SNHG15 regulated tumor growth in murine models via the miR-4735-3p/HIF1a axis.

Discussion
The role of the lncRNA/miRNA/mRNA network in various biological progression has been elucidated in recent decades. As a major cause of cancer initiation and progression, multiple gene mutations have drawn wide scientific interests, and considerable effort has been exerted to elucidate the molecular mechanisms that contribute to tumorigenesis via the lncRNA/miRNA/mRNA network, such as bladder cancer, hepatocellular carcinoma, osteosarcoma, gastric cancer, oral cancer, and CC [21][22][23][24][25][26]. lncRNA-CTS regulates CC progression via the miR-505/ZEB2 axis [27], lncRNA TP73-AS1 modulates CC development via the miR-329-3p/ARF1 axis [28], lncRNA CAR10 promotes CC progression via the miR-125b-5p/PDPK1 pathway [29], and lncRNA DANCR aggravates CC development via the miR-335-5p/ROCK1 axis [30]. Despite the accumulating reports on the lncRNA/miRNA/mRNA network globally, the main mechanism underlying gene dysregulation in cancer development has yet to be clarified. In this study, we clarified elucidated that lncRNA SNHG15 was highly expressed in CC tissues and cells. Downregulated lncRNA SNHG15 attenuated tumorigenic properties in vitro and CC tumor growth in vivo. lncRNA SNHG15 dysregulation also influenced the chemoresistance of CC cells to cisplatin. Cisplatin belongs to the alkylating antineoplastic agent and is used in the clinical management of several malignancies, including CC [31][32][33][34][35][36]. With the wide clinical application of cisplatin, its side effects are been revealed, such as nausea, vomiting, kidney damage, hearing loss, and cell chemoresistance [37]. Understanding the molecular mechanisms underlying chemoresistance, among its side effects, is crucial to improving the therapy efficiency of cisplatin. The results of this study indicate that lncRNA SNHG15 downregulation inhibited the IC50 value of CC cells upon cisplatin treatment.
Further, for the upstream regulator of lncRNA SNHG15, we found that lncRNA SNHG15 expression in CC cells was

Conclusion
In summary, our study elucidates the expression and biological function of lncRNA SNHG15 in CC. This study also indicates that SOX12 could transcriptionally regulate lncRNA SNHG15 expression in CC cells. lncRNA SNHG15 promotes CC progression by regulating the miR-4735-3p/ HIF1a axis, which could be a potential target for the diagnosis of CC or clinical intervention for the disease in the future.

Data Availability
The research data used to support the findings of this study are included within the article.

Ethical Approval
The studies involving human participants were reviewed and approved by the Medical Research Ethics Committee of Xiangyang Central Hospital.