CircAGFG1 promotes cervical cancer progression via miR-370-3p/RAF1 signaling
In past decades, circular RNAs (circRNAs) have achieved increasing attention because of its regulatory role in different kinds of cancers. However, how circAGFG1 regulates cervical cancer (CC) is still largely undiscovered. This study aims to evaluate the role of a novel circRNAs and related molecular mechanism in CC cells.
High or low level of circAGFG1 was detected in CC cells or normal cell line with qRT-PCR. The proliferative and migratory abilities of CC cells were assessed with loss-of function assays. The downstream miRNA and mRNA of circAGFG1 were searched out and proved by using bioinformatics analysis and mechanism experiments. Recue assays were designed to confirm the role of circAGFG1/miR-370-3p/RAF1 axis in CC cell activities.
The levels of circAGFG1 was abundant in CC cells in comparison with normal cervical cell End1/E6E7. The inhibitory effect of decreased circAGFG1 level on the proliferative and migratory abilities of CC cells was assessed. CircAGFG1 and miR-370-3p were localized in the cytoplasm and they can interact with each other. Moreover, miR-370-3p was downregulated in CC cells. We also determined the negative effect of miR-370-3p on RAF1. CircAGFG1 could promote RAF1 expression by absorbing miR-370-3p, thereby activating RAF/MEK/ERK pathway. circAGFG1 promoted proliferation and migration of CC cells via enhancing the activity of RAF/MEK/ERK pathway by sponging miR-370-3p and further regulating RAF1.
The results of this study provided new evidence that circAGFG1 acted as a vital regulator in cervical cancer proliferation and migration, giving great promise to apply it as a potential biomarker for diagnosis and therapy in CC treatment.
KeywordscircAGFG1 miR-370-3p RAF1 Cervical cancer
American Type Culture Collection
competitive endogenous RNA
Dulbecco’s Modified Eagle’s Medium
Fetal bovine serum
Long non-coding RNAs
Raf-1 proto-oncogene, serine/threonine kinase
According to global cancer statistics in 2018, cervical cancer (CC) is acknowledged as the 2nd most commonly-diagnosed tumor, whose fatality rate was the 2nd for female . More than 570,000 patients were diagnosed with CC, and 311,000 death cases were reported in the past year. Considerable advances in treatment have been made over the past decades. However, mortality rate of CC remains high for lagging diagnosis, which was owing to the lack of clear cancer biomarkers . Therefore, discovering molecular mechanisms in CC progression and finding effective therapeutic targets are urgently needed.
Circular RNAs (circRNAs), a newly-discovered non-protein coding RNAs, are featured with a continuous closed loop with no 3′-poly A tail as well as 5′-cap structure . CircRNAs are mostly discovered to mediate gene expression in cancer development through sponging competitive regulators, especially microRNAs (miRNAs) . Recently, mounting evidence has proved that circRNAs can regulate cervical cancer progression via the ceRNA network. For example, circRNA hsa_circRNA_101996 induced the upregulation of TPX2 by restraining miR-8075 to promote CC proliferation and migration . Knockdown of circular RNA hsa_circ_0000263 regulates miR-150-5p/MDM4/p53 pathway and inhibits CC progression . Circ_0067934 modulates miR-545/EIF3C axis to stimulate CC progression . CircRNA8924 serves as an oncogene to facilitate proliferation and migration of CC cells by regulating CBX8 expression via sequestering miR-518d-5p/519-5p family . In our research, CircRNA circAGFG1 with ID of hsa_circ_0058514 (chr2:228356262–228,389,631; circBase: http://www.circbase.org/cgi-bin/simplesearch.cgi) was selected for investigation. CircAGFG1 has been proved to be a facilitator in the progress of triple-negative breast cancer by absorbing miR-195-5p and modulating CCNE1 expression . And circAGFG1 exhausts miR-203 to increase the expression of ZNF281 thereby boosting metastasis of non-small-cell lung cancer . However, its effect on cervical cancer and associated mechanisms in CC have not been totally discovered.
In our research, circAGFG1, miR-370-3p and RAF1 constituted a ceRNA network to regulate cervical cancer cellular processes. MiR-370-3p has been indicated to participate in pancreatic cancer, glioblastoma and bladder cancer and so on [11, 12, 13]. Serine/threonine kinase (RAF1), also names Raf-1 proto-oncogene, is a well-known oncogene in multiple carcinomas, such as lung cancer, glioma and gastric cancer [14, 15, 16]. The correlation among these three genes in cervical cancer is unclear.
In the present study, we first found that circAGFG1 was upregulated in CC and circAGFG1 silencing inhibited the proliferation and migration abilities. We also discovered that circAGFG1 promoted RAF1 expression by sponging miR-370-3p and further activated RAF/MEK/ERK pathway to regulate CC progression. Our study showed that circAGFG1 promoted cervical cancer progression via miR-370-3p/RAF1/MEK/ERK signaling. These data may offer a potent diagnostic biomarker and a novel biological target for CC treatment.
American Type Culture Collection (ATCC; Manassas, VA, USA) was the institute provided cell lines used in these study on 4th, Nov, 2018. Cell lines used in these study including: HeLa (Cat: CCL-2), C-33a (Cat: HTB-31; Cat: CRL-2615), SiHa (Cat: HTB-35), HCC94 and End1/E6E7 (normal cervical cell line). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Carlsbad, CA, USA) added with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin, which were then maintained in a humidified air at 37 °C with 5% CO2. The culture medium was refreshed every 2 days. Cell lines used in this study had been authenticated by STR cell identification on 13th, Nov, 2018. Cell lines were not infected by mycoplasma. All experiments were performed with mycoplasma-free cells. Cells were not contaminated when referring to NCBI.
All plasmids including siRNAs against circAGFG1 containing si/circAGFG1#1 (reduced circAGFG1 expression by 87% in HeLa cells and by 84% in SiHa cells), si/circAGFG1#2 (reduced circAGFG1 expression by 81% in HeLa cells and by 79% in SiHa cells), si/circAGFG1#3 (reduced circAGFG1 expression by 75% in HeLa cells and by 71% in SiHa cells), pcDNA3.1/circAGFG1 (increased circAGFG1 expression to about 114 fold change in HeLa cells and to about 108 fold change in SiHa cells), pcDNA3.1/RAF1 (increased RAF1 expression to about 123 fold change in HeLa cells), miR-370-3p mimic (increased miR-370-3p expression to about 104 fold change in HeLa cells and to about 111 fold change in SiHa cells) and miR-370-3p inhibitor (reduced miR-370-3p expression by 79% in HeLa cells and by 72% in SiHa cells) were constructed. All plasmids and their negative controls were purchased from GenePharma (Shanghai, China) and transfected into CC cells with Lipofectamine® 2000 (Invitrogen) under recommended direction.
Total RNA extraction was conducted with TRIzol reagent (Invitrogen), which were reverse-transcribed into cDNA by utilizing PrimeScript RT Reagent Kit (Takara, Dalian, China) routinely. qRT-PCR were carried out by using TB Green Premix Ex Taq (Takara) on the Bio-Rad CFX96 system (Bio-Rad, CA, USA). Quantification of circRNA and mRNA and miRNA was made by referring to GAPDH or U6. Relative gene expression was determined with the use of 2–ΔΔCT method. Special primers are listed as follows:
F: 5′- CTTCAGGAACGAGGTGGCTGTT-3′.
R: 5′- TGCTGCCTTCACACCACTGAGT-3′.
Cell viability and proliferation assays
1 × 103 CC cells were maintained in 96-pore plates for 1 d and grown for extra 0, 24, 48, 72 and 96 h. Cell Counting Kit-8 (Bosterbio, Wuhan, China) was utilized to evaluate cell viability following the supplier’s protocol at each time point. After 4 h of culture, a spectrophotometric plate reader (BioTek, VT, USA) was applied to measure the optical density at 450 nm for detection of cell viability.
CC cells (200 μL, 2 × 104/mL) were immobilized by 70% alcohol and subsequently cultured with 50 μM EdU (5-Ethynyl-2′-Deoxyuridine) labeling solution (Invitrogen) for 2 h at 37 °C. The fluorescent intensity of EdU was examined at 550 nm through applying Cell Light EdU DNA imaging kit (Invitrogen). Cells were cultivated using 5 μg/mL Hoechst 33342 for 0.5 h for DNA staining. The visualization and photograph of immunostainings were implemented with a fluorescent microscope (Olympus inverted microscope IX71).
Flow cytometry analysis
Apoptosis of indicated cells was measured using flow cytometry analysis in accordance with a previous study .
Transwell migration assay
Cells with a density of 1 × 105 cells/hore were seeded in 6-well plates, suspended in 200 μL serum-free medium and planted into the upper chambers without Matrigel mixture. 500 μL medium with 10% FBS was added into the lower chambers (BD BioCoat, MA, USA) as attractants. After 24 h, cells in the upper chambers were removed. Migrated cells in the lower chamber were fixed by ethanol and stained with crystal violet, which were subsequently photographed and calculated by a light microscope (Olympus Corporation, Tokyo, Japan).
Fluorescence in situ hybridization (FISH)
Specific Cy3-labeled circAGFG1 probe and FITC-labeled miR-370-3p probe were designed and synthesized via RiboBio (Gangzhou, China) at 37 °C for whole night, and dyed utilizing DAPI obeying the guidebooks of the supplier. Slides were photographed with a fluorescence microscope (Leica, Wetzlar, Germany).
Luciferase reporter assay
Binding sequences of circAGFG1 and RAF1 3′UTR for miR-370-3p as well as mutant versions (circAGFG1-WT, RAF1-WT; circAGFG1-MUT, RAF1-MUT) were synthesized and subcloned into luciferase reporter vector pGL3 (Promega, Madison, WI, USA). Then these vectors were co-transfected with NC/mimic or miR-370-3p mimic into HeLa and SiHa cells. For affirming the specificity of miR-370-3p and circAGFG1, we constructed the full length of circABCC2, circLRP6, circSCAF11 and circAGFG1 into luciferase reporter vector pGL3 to observe the changes of these luciferase activities by miR-370-3p mimics. As for the regulation of miR-370-3p and circAGFG1 on the luciferase activity of RAF1, RAF1-WT or RAF1-MUT was co-transfected with NC/mimic, miR-370-3p mimic, miR-370-3p mimic + pcDNA3.1 or miR-370-3p mimic + pcDNA3.1/circAGFG1 into CC cells. The luciferase activities were measured by Dual Luciferase Assay Kit (Promega) according to the manufacturer’s directions.
RNA immunoprecipitation (RIP)
Under the manufacturer’s protocols, RIP was performed with Magna RIP kit (Millipore). Transfected cells were lysed in RNA lysis buffer and cell lysates were incubated with magnetic beads with anti-Argonaute2 (Ago2) or anti-IgG (both from Millipore) at 4 °C for 4 h. After the beads were washed, the immuno-precipitated RNAs were purified and detected by qRT-PCR.
RNA pull-down assay
G-50 Sephadex RNA Columns (Roche) was employed to purify biotinylated RNA synthesized in vitro with the help of T7 RNA polymerase. Biotinylated miR-370-3p sense was named as bio-miR-370-3p-WT probe, and biotinylated miR-370-3p antisense was named as bio-miR-370-3p-MUT probe. Transfected cells were lysed with lysis buffer and incubated with streptavidin-coated magnetic beads to pull down the biotin-labeled RNA complex. Human Ago2 or normal mouse IgG antibody (Millipore) were used. The beads were washed and the precipitates were purified with TRIzol (Takara). The abundance of circAGFG1 was determined with qRT-PCR.
Western blot analysis
Extracted proteins were separated by 10% SDS-PAGE, and then transferred onto PVDF membrane (Bio-Rad). After being blocked with 5% skimmed milk, the membranes were incubated with primary antibodies against RAF1, p-RAF1, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2 and GAPDH at 4 °C. After overnight incubation and then incubated with secondary antibodies at room temperature for 2 h. All antibodies were purchased from Abcam (Burlingame, CA, USA). In the end, bands were measured with Immobilob™ Western Chemiluminescent HRP Substrate (Millipore).
All independent experiments were conducted for three times. SPSS 19.0 software (IBM Corporation, Armonk, NY, USA) was responsible for data analyses. Data of three experimental results were exhibited as the mean ± standard deviation (SD). Student’s t-test and one-way ANOVA were two statistical methods for comparison of difference between two or more groups. P<0.05 is a symbol that indicates statistical significance.
Downregulation of circAGFG1 restrained cell viability, proliferation and migration, and promoted cell apoptosis in cervical cancer
MiR-370-3p was downregulated in CC and negatively modulated by circAGFG1
CircAGFG1 activated RAF/MEK/ERK pathway by sponging miR-370-3p
CircAGFG1 affected CC proliferation and migration via miR-370-3p/RAF1 axis
Competitive endogenous RNA (ceRNA) ceRNAs can act like a molecular sponge of miRNAs, hence reducing miRNAs-regulated inhibition of the target genes . In 2013, circRNAs were reported to be a key subtype of ceRNAs that functioned to absorb miRNAs and abrogated its impact on downstream gene expression [22, 23]. It is evident that circRNAs affect not only occurrence and development of cervical cancers, but also those of other cancers. In lung cancer, circular RNA hsa_circRNA_103809 acts as an oncogene through miR-4302/ZNF121/MYC axis . CircRNA_102171 drives papillary thyroid cancer cell proliferation, migration and invasion via activating CTNNBIP1-dependent β-catenin pathway . CircRNA_100782 promotes pancreatic cancer proliferation through the IL6-STAT3 pathway via sponging miR-124 . In breast cancer, hsa_circRNA_0006528 activates the MAPK/ERK signaling pathway by absorbing miR-7-5p .
Non-coding RNAs are RNAs that have no capability of encoding proteins. Except for long non-coding RNAs (lncRNAs), circRNAs could also compete with protein-coding mRNAs for binding to microRNAs (miRNAs) in ceRNA networks . Herein, we found circAGFG1 (ID: hsa_circ_0058514) that develops carcinogenic impacts on cellular function of triple-negative breast cancer and non-small cell lung cancer [9, 10]. Our study firstly disclosed that circAGFG1 was heightened in CC and circAGFG1 silencing impaired the proliferation and migration properties of CC.
Among assumed targets of circAGFG1, we chose miR-370-3p for further exploration. The well-known tumor inhibitor role of miR-370-3p has been revealed in numerous tumors. For instance, CXCL12-regulated miR-370–3p acts as a tumor suppressor of nonfunctional pituitary adenomas via targeting HMGA2 . LncRNA H19 enhances TGF-β-activated epithelial-mesenchymal transition (EMT) in ovarian cancer through serving as a ceRNA of miR-370-3p . Overexpression of miR-370-3p impairs glioblastoma multiforme resistance to temozolomide via affecting MGMT expression . Then it was observed that circAGFG1 and miR-370-3p were situated in the cytoplasm, and miR-370-3p was negatively modulated by circAGFG1. Further, through qRT-PCR screening, we found the target gene: Raf-1 proto-oncogene, serine/threonine kinase (RAF1), a member in RAF/MEK/ERK pathway. RAF/MEK/ERK pathway is a noted oncogenic pathway functioned in a big amount of cancers [32, 33, 34]. Besides, RAF1 has been indicated to exert carcinogenic property in various carcinomas [14, 15, 16]. Consistently, mechanism experiments affirmed that circAGFG1 exhausted miR-370-3p to regulate RAF1 expression and further activate RAF/MEK/ERK pathway. The relationship among circAGFG1, miR-370-3p and RAF1 was firstly displayed by our research.
In the end, rescue assays certified that circAGFG1 promoted CC proliferation and migration via miR-370-3p/RAF1 axis. These findings provided a potential biomarker and an admissible therapeutic target for CC patients. Nevertheless, other modulation mechanisms of circAGFG1 in cervical cancer remain to be discovered.
In summary, this study revealed that circAGFG1 exerted oncogenic properties in cervical cancer by sponging miR-370-3p to upregulate RAF1. Our research findings potentially help to provide an admissible therapeutic target for CC patients.
We sincerely thank all participants in this investigation.
In our study, FW designed and performed experiment. In addition, experimental materials and methods were provided by FW. JZ was responsible for analyzing experimental data and writing. Both authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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