circSLC30A7 Inhibits Hepatocellular Carcinoma Cell Proliferation via the miR-767-5p/FBXW7/NOTCH1 Axis

Circular RNAs, noncoding RNAs, have attracted much attention in various human tumor research fields. They regulate the development of various human cancers via microRNA sponges. This study aimed to assess the molecular mechanism of circSLC30A7 in hepatocellular carcinoma (HCC). In our study, we identified that circSLC30A7 was significantly downregulated in HCC cell lines and tissues. Furthermore, gain and loss function experiments were conducted to elucidate the biological functions of circSLC30A7 in HCC cell lines. Mechanistically, circSLC30A7 sponged miR-767-5p, inhibiting the expression of its downstream protein, FBXW7. In summary, this study revealed that circSLC30A7 is an essential tumor suppressor that inhibits HCC tumorigenesis through the miR-767-5p/FBXW7/NOTCH1 axis. Taken together, circSLC30A7 reduces HCC malignancy and can be a biomarker for HCC management.


Introduction
Hepatocellular carcinoma (HCC) is a common human malignancy with high prevalence and mortality rates worldwide [1]. Early-stage HCC patients can be treated using surgical treatments, such as liver resection and transplantation. However, the treatment efficacy and 5-year survival rate for HCC patients are poor due to the lack of highly specific and sensitive early diagnostic biomarkers [2,3]. erefore, it is necessary to elucidate the molecular mechanisms of HCC and identify the molecular biomarkers for HCC prevention and treatment.
In this study, a novel HCC-related tumor suppressor, circRNA, was identified via data mining in three GEO databases (GSE78520, GSE94508, and GSE97332). Hsa_-circRNA_100291 was identified at chr1:101372407-101387397 with a spliced sequence length of 660 nt and named hsa_circ_0000098 at Circbase database, which derived from SLC30A7. Besides, the genomic structure analysis demonstrated that hsa_circRNA_100291 was mapped at the 3, 4, 5, 6, 7, and 8 exons of the SLC30A7 gene, thus naming it as circSLC30A7. is study showed that circSLC30A7 expression level was decreased in HCC cell lines and tissues. Furthermore, circSLC30A7 inhibited the proliferation of HCC cells by binding to miR-767-5p as a miRNA sponge to regulate FBXW7 expression. excised from confirmed HCC patients and received surgery at the Department of Surgery of e First Affiliated Hospital of Nanjing Medical University between March 2018 and October 2019. All fresh tissues were stored in liquid nitrogen at −80°C. No patient received radiotherapy or chemotherapy treatment before surgery. e clinicopathological features of PTC patients are shown in Table 1. All participants signed written informed consent before tissue sample analysis. e study followed the provisions of the Declaration of Helsinki and was approved by the Ethics Committee of e First Affiliated Hospital of Nanjing Medical University.

RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) Assay.
e TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from cell and tissue samples. e Cytoplasmic and Nuclear RNA Purification Kit (Invitrogen, Carlsbad, CA, USA) was used to extract the nuclear and cytoplasmic fractions. e SYBR Premix Ex Taq kit (TaKaRa, Japan) was used to detect the mRNA level of target genes via the StepOnePlus ™ Real-Time PCR system (Applied Biosystems, USA). e relative expression levels were calculated using the 2 −ΔΔCt method. All reactions were conducted in triplicate. e primers are as follows: F: GGTGTAATTGCTTCTGCCATC; R: ATAACAGAAGCT GCCAGTCCA.

RNase R Digestion and Actinomycin D Treatment.
HepG2 and Huh-7 cells were extracted from total RNA. Total RNA (3 μg) was incubated with or without 3 U/mg RNase R (Epicentre Biotechnologies, USA) at 37°C for 15 min for qRT-PCR analysis. e culture medium was added to actinomycin D (100 ng/ml) or DMSO (Amresco, USA) to block new RNA synthesis in HepG2 and Huh-7 cells for stability analysis of circSLC30A7 and its linear isoform. e qRT-PCR was used to examine the relative RNA levels in HepG2 and Huh-7 cells at the indicated time points.

Fluorescent In Situ Hybridization (FISH).
Cy3-labeled probes were specific to circSLC30A7 and were obtained from RiboBio (Shanghai, China). A FISH Kit (RiboBio, Shanghai, China) was used to detect the probe signals following the manufacturer's instructions. Nikon AISi Laser Scanning Confocal Microscope (Nikon, Japan) was used for visualization. e probe is as follows: 5′ ATCAAGCCTAAGCT-TACAAC 3′.
2.6. Cell Proliferation Assays. CCK-8 assay kits, ethynyldeoxyuridine (Edu) assays, and colony formation assays were used for cell proliferation analysis. For the CCK-8 assay, treated cells were cultured in 96-well plates, and CCK-8 reagent (Takara, Nanjing, China) was added at 1, 2, 3, 4, and 5 days. Absorbance quantification was then conducted at 490 nm. Similarly, the cells were incubated in a 96-well plate after transfection for Edu assays. A 20 μM Edu labeling media (KeyGENBioTECH, Nanjing, China) was added to a 96-well plate and then incubated for 2 hours. e cells were treated with 0.5% Triton X-100 and 4% paraformaldehyde, and then an anti-Edu working buffer was used for staining. Fluorescence microscopy was used to visualize the cells, detecting the Edupositive cells. For colony formation assay, the treated cells were placed into 6-well plates and incubated for 14 days. e cells were then fixed in methanol for 30 min and stained using crystal violet solution.   Journal of Oncology then treated with trypsin (without EDTA). e cells were reconstituted in a binding solution, stained with 5 μl of annexin V-FITC (Beyotime, Nanjing, China) reagent, and then incubated in a dark place at room temperature (RT) for 15 minutes. 1 μl of propidium iodide (PI, 50 μg/ml) (Beyotime, China) was added, gently mixed, and then incubated in darkness at RT for 5 min for detection. Flow cytometry (FACScan, BD Biosciences) with the Cell-Quest software was used to analyze the apoptotic cells.

Western Blot Assay.
Protein extracts were separated through a 10% SDS-PAGE gel and then transferred to a 0.4 mm PVDF membrane. e membranes were treated with 5% nonfat milk at RT for 2 h and then incubated with primary antibodies at 4°C overnight. e membrane was then treated with a secondary antibody. e BeyoECL Plus Kit (Beyotime, Nanjing, China) was then used to visualize the protein band. GAPDH was used as an internal control. e primary antibody used included: rabbit anti-GAPDH, anti-FBXW7, anti-Notch1, anti-CyclinD1, anti-PCNA, anti-Bcl-2, and anti-Bax at 1 : 1000 (Cell Signaling Technology, USA).

Tumor Growth in Nude
Mice. HCC cell lines, Huh7 and HepG2, were transfected with vector or circSLC30A7-ov and sh-NC or sh-circSLC30A7, respectively; then subcutaneously injected into the flanks of female athymic specificpathogen-free (SPF) BALB/C nude mice (aged 4-5 weeks). Mice were sacrificed after 28 days, and then tumor weights were evaluated. e Animal Care and Use Committee of e First Affiliated Hospital of Nanjing Medical University approved the study protocols.

Data Analysis.
e SPSS 22.0 software and GraphPad Prism 7 were used for data analyses. Student's t-test and chisquared test were used to assess differences among groups. Pearson correlation analysis was used to determine the relationships among circSLC30A, miR-767-5p, and FBXW7 expressions. p < 0.05 represented statistically significant differences.

circSLC30A7 Is Downregulated in HCC Tissues and Cells.
e analysis of the three GEO databases (GSE78520, GSE94508, and GSE97332) showed that hsa_circRNA_ 100291 (has_circ_0000098 in circbase) was dow-regulated in HCC (hsa_circRNA_100291 was named as circSLC30A7) (Figure 1(a)). Sanger sequencing was further used for headto-tail splicing of circSLC30A7. qRT-PCR was used in forcircSL30A7 expression analysis to explore the role of circSLC30A7 in HCC. circSLC30A7 expression was significantly downregulated in 50 pairs of HCC tissues compared to the matched normal samples (Figure 1(b)). circSLC30A7 level in HCC cells was further assessed. circSLC30A7 was substantially decreased in Huh7, HepG2, SMMC-7221, and Bel-7404 than in HL-7702 (Figure 1(c)). Huh7 and HepG2 were selected to examine the stability of circSLC30A7 via RNase R or Actinomycin D stimulation. e Actinomycin D assay showed that circSLC30A7 had a significantly higher half-life than SLC30A7 (Figure 1(d)). RNase R digestion assay indicated that circSLC30A7 was more resistant to RNase R than the linear isoform SLC30A7 (Figure 1(e)). qRT-PCR analysis ( Figure 1(f )) and fluorescence in situ hybridization (FISH) (Figure 1(g)) confirmed that circSLC30A7 was located in the cytoplasm. Taken together, these results show that circSLC30A7 is downregulated and stable in HCC.

CircSLC30A7 Inhibits Proliferation and Induces Apoptosis in HCC Cells.
e tumor was more than 5 cm large, and the circSLC30A7 expression was substantially downregulated (Table 1). e circSLC30A7-ov and circSLC30A7-sh were transfected in Huh7 and HepG2, respectively, to investigate the effect of circSLC30A on HCC proliferation using the qRT-PCR assay (Figure 2(a)). CCK8 assay showed that circSLC30A7 overexpression significantly reduced the viability of Huh7 cells while circSLC30A7 downregulation increased the viability of HepG2 (Figure 2(b)). Colony formation assay suggested that circSLC30A7 overexpression significantly decreased the colony numbers of Huh7 cells, while circSLC30A7 knockdown showed increased colony numbers in HepG2 cells (Figure 2(c)).
e EdU assays elucidated that the Huh7 cells significantly increased in the circSLC30A7-ov group than the HepG2 cells ( Figure 2(d)). Flow cytometry assay was used to evaluate the influence of circSLC30A7 on the apoptosis of HCC cells. e apoptotic rate of HepG2 cells decreased after circSLC30A7-sh transfection while that of Huh7 cells increased (Figure 2(e)). Also, circSLC30A7 overexpression decreased the protein level of Bcl-2 and elevated that of Bax in Huh7 cells (Figure 2(f )), while it showed opposite effects in HepG2 cells (Figure 2(g)).
Overall, circSLC30A7 overexpression inhibits proliferation and induces apoptosis in HCC cells.

CircSLC30A7 Promotes HCC via NOTCH1 Pathway regulation.
e Gene Set Enrichment Analysis (GSEA) was used further to determine the underlying mechanism of circSLC30A7 on HCC proliferation. FBXW7 expression was positively correlated with GO_REGULATION_NOTCH_ SIGNALING_PATHWAY (Figure 7(a)). erefore, a western blotting assay was used to assess the protein expression of NOTCH1-associated proteins after cotransfection of circSLC30A7 and miR-767-5p. e circSLC30A7 expression increased the protein level of FBXW7 and decreased that of NOTCH1, Cyclin D1, and PCNA. However, miR-767-5p-mimic alleviated the effect in the Huh7 cell ( Figure 7(b)). Similarly, circSLC30A7 silencing reduced the protein level of FBXW7 and amplified that of NOTCH1, Cyclin D1, and PCNA, and the effects were alleviated by anti-miR-767-5p (Figure 7(c)). erefore, circSLC30A7 inhibits HCC proliferation via the miR-767-5p/FBXW7/ NOTCH1 axis.

CircSLC30A7 Inhibits HCC Progression In Vivo.
A xenograft mice model was used to confirm the effect of circSLC30A7 on tumor progression in vivo. Tumor volume and weight were substantially lower in the oe-circSLC30A7 group than in the vector group (Figures 8(a)-8(c)). Furthermore, tumor volume and weight were significantly elevated in the sh-circSLC30A7 group compared with the shcontrol group (Figure 8(d)-8(f )). erefore, circSLC30A7 overexpression inhibits HCC progression in vivo.

Discussion
Several studies have reported that circRNAs are essential in the pathogenesis and progression of various cancers. However, fewer studies have investigated the role of Journal of Oncology 13 circRNAs in HCC. It is necessary to explore the molecular mechanisms of HCC since it is a multistep and multifactorial cancer. Recent reports have shown that several circRNAs are dysregulated in various malignancies, which regulate the initiation and progression of tumors. [16][17][18] For instance, hsa_circ_0000337, expressed in esophageal squamous cell carcinoma tissues, promotes cell invasion, migration, and proliferation. [19] Circ_0067934 also regulates thyroid carcinoma progression. erefore, circRNA can be a prognostic biomarker for various malignancies [20].
In this study, circSLC30A7 (circRNA) was used to investigate its role in HCC progression. Herein, circSLC30A7 expression was significantly downregulated in HCC tissues and cells. Low circSLC30A7 expression was associated with the stage and size of the tumor, implying that circSLC30A7 can be a novel biomarker for HCC diagnosis. circSLC30A7 upregulation decreased tumor growth in vivo, induced apoptosis, and suppressed colony formation, proliferation, invasion, and migration of HCC cells in vitro.
Several studies have also indicated that circRNA containing miRNA binding sites can act as competitive endogenous RNA (ceRNA). Furthermore, some studies have demonstrated that lncRNA and miRNA are associated [21,22]. Circular RNA AKT3 acts as the sponge of miR-198 and promotes gastric cancer cell proliferation [23]. Circular RNA circTADA2A promotes osteosarcoma cell metastasis and proliferation by sponging miR-203a-3p and modulating CREB3 expression [24]. Similarly, circRNA_100269 is significantly decreased in gastric cancer, inhibiting tumor cell growth [25]. circSLC30A7 can function as a miRNA sponge to decrease HCC malignant phenotypes since it is located in the cytoplasm. In this study, the regulatory mechanism of circSLC30A7 in HCC was assessed. Bioinformatics analysis indicated that CircSLC30A7 was negatively correlated with miR-767-5p in HCC tissues. Luciferase reporter assay indicated that miR-767-5p was a direct target of circSLC30A7 and miR-767-5p knockdown counteracted the tumor-inhibiting activity of circSLC30A7, showing that miR-767-5p is associated with circSLC30A7-mediated cellular senescence in HCC.
Four bioinformatics algorithms were used to predict four potential downstream target genes of the circSLC30A7/miR-767-5p axis in HCC. Further experiments revealed that FBXW7 was the downstream effector of the circSLC30A7/ miR-767-5p axis and mediated cellular senescence in HCC. FBXW7 is a tumor suppressor [26]. However, FBXW7 and miR-767-5p were negatively correlated in HCC tissues. e protein level of FBXW7 mRNA decreased in HCC tissues. qRT-PCR and western blot showed that circSLC30A7 negatively regulated FBXW7 via the miR-767-5p. Besides, the GSEA assay and western blotting assay indicated that the NOTCH1 signaling pathway was associated with circSLC30A7.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
All tissues used in this study were acquired with written informed consent. e study followed the provisions of the Declaration of Helsinki and was approved by the Ethics Committee of e First Affiliated Hospital of Nanjing Medical University. e number of approval from the Animal Care and Use Committee of e First Affiliated Hospital of Nanjing Medical University is IACUC2008022.

Conflicts of Interest
e authors declare that there are no conflicts of interest.