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Histone Demethylase KDM3A Promotes Cervical Cancer Malignancy Through the ETS1/KIF14/Hedgehog Axis

Authors Liu J, Li D, Zhang X, Li Y, Ou J

Received 12 August 2020

Accepted for publication 22 October 2020

Published 19 November 2020 Volume 2020:13 Pages 11957—11973

DOI https://doi.org/10.2147/OTT.S276559

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr XuYu Yang

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Jinyu Liu,1,* Dongqing Li,2,* Xin Zhang,3 Yanyan Li,1 Jian Ou4

1Frist Department of Gynecologic Oncology, Jilin Cancer Hospital, Changchun 130012, Jilin, People’s Republic of China; 2Second Department of Gynecologic Oncology, Jilin Cancer Hospital, Changchun 130012, Jilin, People’s Republic of China; 3Department of Rheumatology and Immunology, China-Japan Union Hospital of Jilin University, Changchun 130033, Jilin, People’s Republic of China; 4Department of Gynecological Oncology Radiotherapy, Jilin Cancer Hospital, Changchun 130012, Jilin, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Jian Ou
Department of Gynecological Oncology Radiotherapy, Jilin Cancer Hospital, No. 1018, Huguang Road, Changchun 130012, Jilin, People’s Republic of China
Tel/Fax +86-18686500760
Email [email protected]

Background: Lysine demethylase 3A (KDM3A) has been increasingly recognized as an important epigenetic regulator involved in cancer development. This study aims to explore the relevance of KDM3A to cervical cancer (CC) progression and the molecules involved.
Materials and Methods: Tumor and the adjacent tissues from CC patients were collected. KDM3A expression in tissues and CC cell lines and its correlation with the survival and prognosis of patients were determined. Malignant potentials of CC cells and the angiogenesis ability of HUVECs were measured to evaluate the function of KDM3A on CC progression. The interactions among KDM3A, H3K9me2 and ETS1, and the binding between ETS1 and KIF14 were validated through ChIP and luciferase assays. Altered expression of ETS1 and KIF14 was introduced to explore their roles in CC development.
Results: KDM3A was abundantly expressed in CC tissues and cells and linked to dismal prognosis of CC patients. Knockdown of KDM3A suppressed malignant behaviors of CC cells. KDM3A was found to increase ETS1 expression through the demethylation of H3K9me2. Overexpression of ETS1 blocked the inhibiting roles of sh-KDM3A. ETS1 could bind to the promoter region of KIF14 to trigger its transcription. Overexpression ofKIF14aggravated the malignant behaviors of CC cells and the angiogenesis ability of HUVECs, and it activated the Hedgehog signaling pathway. Artificial activation of Hedgehog by Sag1.5 diminished the effects of sh-KDM3A. These changes were reproduced in vivo.
Conclusion: This study evidenced that KDM3A promotes ETS1-mediated KIF14 transcription to promote CC progression with the involvement of the Hedgehog activation.

Keywords: lysine demethylase 3A, ETS proto-oncogene 1, kinesin family member 14, Hedgehog signaling pathway, cervical cancer

Introduction

Cervical cancer (CC) represents the fourth gynecological malignancy both in terms of incidence and mortality among females, with an estimated 570,000 diagnoses and 311,000 deaths in 2018 across the globe.1 Specifically, the morbidity rate varies worldwide, with the highest rate in Eastern Africa and the lowest incidence in Western Asia; and it is the second most prevailing cancer in women in South East Asia.2 Chemo- and radiotherapies are well recognized as the primary interventions for CC control, but the overall survival rate of the advanced patients who underwent these conventional therapies are still low, at about 40%.3 Besides, the radiation exposure itself may bring side-effects and a risk fact for cancer development.4 Gene-based therapy has been accepted as a less-invasive promising approach that brings the development of many treating options focusing on different aspects,5 which requires an increased understanding in the molecules of action.

Though initially less studied, the epigenetic mechanisms which mediate transcriptional regulation and dysregulation in cancer have aroused increasing focus among researchers in this field, mainly including the acetylation and methylation modifications.6 Lysine demethylase 3A (KDM3A), also termed Jumonji domain-containing 1A (JMJD1A) and Jumonji C (domain-containing histone demethylase 2A) JHDM2A, is a histone H3 lysine 9 (H3K9) dimethyl and monomethyl (me2/1) demethylase has been implicated in many cellular processes from proliferation to progression, and is reported as an advanced target for cancer treatment.7,8 But the role of KDM3A in CC development remains less concerned. Interestingly, a previous report suggested that KDM3A could interactwith the ETS proto-oncogene transcription factor 1 (ETS1) flanking its start site through a H3K9me2 demethylation manner, which leads to the further metastasis of Ewing sarcoma cells.9 ETS1 is a key member of the ETS domain family and is implicated in many essential processes in cancer development, such as invasion, epithelial-to-mesenchymal transition (EMT), drug-resistance and angiogenesis.10 Similarly, it was demonstrated as a promising therapeutic target for CC as well.11 We therefore speculated that there is a similar interaction between KDM3A and ETS1 in CC development. Moreover, A previous study noted that ETS1 can promote the transcription activity of kinesin family member 14 (KIF14),12 which has been documented to be abundantly expressed in CC and associated with chemo-resistance and unfavorable prognosis.13 Therefore, we hypothesized that ETS1 possibly regulates KIF14 transcription to govern CC development. Hence, this study was performed in both cell and animal models to validate the functions of the above factors in CC progression and their interactions, as well as the potential pathway implicated.

Materials and Methods

Ethics Statement

The study was performed as per the Declaration of Helsinki and ratified by the Ethics Committee of Jilin Cancer Hospital. All patients signed an informed consent form before enrollment. The animal experimental protocol was approved by the Committee on the Ethics of Animal Experiments of Jilin Cancer Hospital. All animal procedures were performed in line with the Guide for the Care and Use of Laboratory Animals published by National Institutes of Health (NIH Publication No.85–23, revised 1996). Great efforts were made to reduce the usage and pain in animals.

Sample Collection

Tumor tissues and the adjacent tissues were collected from 50 CC patients who were treated in Jilin Cancer Hospital from January 2013 to April 2014. The first-time diagnosed patients with complete clinical information were free of any other malignancies or a history of preoperative radiotherapy and chemotherapy. The tissues were collected during surgery and instantly preserved at −80°C. A 5-year follow-up study was performed.

Cell Transfection

An immortalized cervical epithelial cell line (H8) was from Cell Resource Center, the Chinese Academy of Sciences (Shanghai, China), and CC cell lines (HT-8, CaSki, HeLa, and SiHa) were from American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultivated in 10% fetal bovine serum (FBS)-supplemented DMEM containing 1% penicillin/streptomycin (all from Sigma-Aldrich Chemical Company, St Louis, MO, USA) at constant 37°C with 5% CO2. The medium was refreshed every 3 days.

HeLa and SiHa cells were used for the following transfection. The short hairpin (sh) RNAs targeting KDM3A (sh-KDM3A1, 2#), overexpressing vector of ETS-1 (oe-ETS1) and KIF14 (oe-KIF14), and the corresponding control vectors were acquired from GenePharma Co., Ltd. (Shanghai, China). All transfection was performed in line with the protocols of a Lipofectamine 2000 kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). A Hedgehog pathway-specific agonist Sag1.5 (HY-124,899) acquired from MedChemExpress (Monmouth Junction, NJ, USA) was used to activate the Hedgehog pathway in CC cells, and dimethyl sulphoxide (DMSO, Sigma-Aldrich) served as a control.

Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)

An RNeasy Mini Kit (Qiagen Sciences, Valencia, CA, USA) was utilized to extract total RNA from cells or tissues. Then, 1.5 μg RNA was reversely transcribed to cDNA using the TaqMan Reverse Transcription Reagent (Applied Biosystems Inc., Carlsbad, CA, USA). Then, real-time qPCR was performed on a Prism 7900HT System (Applied Biosystems) and a SYBR Green PCR Master Mix (Applied Biosystems). The 2−ΔΔCt method was used to determine gene expression. The primers are exhibited in Table 1, and GAPDH served as an internal reference.

Table 1 Primer Sequences for RT-qPCR

Immunohistochemical (IHC) Staining

The tumor samples were fixed in paraformaldehyde, embedded in paraffin, cut into sections (4 μm), dewaxed, and rehydrated. Then, the sections were soaked in boiled 10 mM citrate buffer (pH=6.0) for 2 minutes of antigen retrieval, and then blocked with 100 μL H2O2 for 10 minutes. Then, the sections were co-cultured with the primary antibodies against KDM3A (1:50, ab91252, Abcam Inc., Cambridge, MA, USA), ETS1 (1:500, ab220361, Abcam), KIF14 (1:100, #PA5-87,769, Thermo Fisher) and Gli1 (1:200, #MA5-32,553, Thermo Fisher) at 4°C overnight, and then with the secondary antibodies anti-mouse IgG H&L (HRP) (ab205719, Abcam) and goat anti-rabbit IgG H&L (HRP) (ab6721, Abcam) at 20°C for 1 hour, followed by a 10-minute incubation with horseradish peroxidase (HRP)-labeled streptomycin (KIT-9710, MXB Biotechnologies, China). The staining was developed by 3.3’-diaminobenzidine, and the tissues were observed under a microscope (Nikon Eclipse NI, Japan).

Western Blot Analysis

Tissues and cells were collected by proteinase inhibitor-contained radio-immunoprecipitation assay (RIPA) cell lysis buffer on ice. After protein quantification by a bicinchoninic acid (BCA) kit (Pierce Waltham, MA, USA), an equal volume of protein lysates was separated by 8–12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Next, the membranes were blocked with 5% skimmed milk for 15 minutes and cultured with the primary antibodies against KDM3A (1:1,000, ab91252, Abcam), ETS1 (1:1,000, ab220361, Abcam), H3K9me2 (1:1,000, ab1220, Abcam), GAPDH (1:10,000, ab181602, Abcam), Gli1 (1:1,000, #MA5-32,553, Thermo Fisher), and Gli3 (1:1,500, ab181130, Abcam) at 4°C overnight, followed by further incubation with the secondary antibodies anti-mouse IgG H&L (HRP) (ab205719, Abcam) and goat anti-rabbit IgG H&L (HRP) (ab6721, Abcam) at 20°C for 1 hour. Then, the blot bands were developed by enhanced chemiluminescence (ECL) reagent (Millipore) and scanned using an immune blotting system (Tanon 5200, China).

Cell Counting Kit-8 (CCK-8) Method

A CCK-8 kit was used to measure cell proliferation. Briefly, transfected cells were sorted into 96-well plates at 2,000 cells per well. Then, each well was loaded with 10 μL CCK-8 solution at different time points for another 2 hours of incubation at 37°C. The optical density (OD) at 450 nm was determined utilizing a spectrophotometer (Synergy H1, Bio-Tek, USA).

Colony Formation Assay

After transfection, cells were sorted in 6-well plates at 1×105 cells per well for 2 weeks of incubation. Then, the cells were immobilized in 4% paraformaldehyde for 30 minutes and stained with crystal violet (Sigma-Aldrich) for 15 minutes, and the number of colonies (over 50 cells) were observed under an inverted microscope (Olympus Optical Co., Ltd, Tokyo, Japan) with five random fields observed.

5-Ethynyl-2’-Deoxyuridine (EdU) Labeling Assay

A Cell-Light EdU DNA cell proliferation kit (RiboBio, Guangzhou, China) was used for DNA replication and proliferation measurement. Cells were sorted in 96-well plates at 2×104 cells per well. After 36 hours, each well was filled with 50 μM EdU solution for another 3 hours of incubation. Then, the cells were fixed and stained by Apollo solution. Nuclei were stained by 4’, 6-diamidino-2-phenylindole (DAPI), and the staining images were captured under the inverted microscope. Five random fields were included, and the red staining (EdU-positive cells) and blue staining (DAPI-stained cells) were calculated.

Terminal Deoxynucleotidyl Transferase (TdT)-Mediated dUTP Nick End Labeling (TUNEL)

Apoptosis of cells was determined using a TUNEL kit from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Briefly, cells were immobilized for 30 minutes and cultured in phosphate buffer saline (PBS) containing 0.3% Triton X-100 for 5 minutes of incubation. Then, the cells were additionally incubated with 50 μL fresh TUNEL reagent at 37°C avoiding light exposure for 60 minutes. Thereafter, the slides were anti-quenched by fluorescent mounting media, and the relative fluorescence intensity was determined by an EVOS FL microscope (Invitrogen, Thermo Fisher).

Flow Cytometry

An Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Thermo Fisher) was used for apoptosis detection. Briefly, cells after transfection were centrifuged at 1,000 g for 5 minutes with the supernatant discarded. Next, the cells were resuspended in PBS, and the suspension containing 5×104 cells was centrifuged again to discard the supernatant, Then, the cells were resuspended in the Annexin V-FITC mixture and then stained with propidium iodide (PI) solution for 10–20 minutes of incubation at 20–25°C in dark condition. Cell apoptosis was evaluated using a flow cytometer (FACS Calibur, BD Biosciences, San Jose, CA, USA), and the data were analyzed by Flow J.

Wound-Healing Assay

Cells were sorted in 6-well plates. When the cell confluence reached 90–100%, a scratch was produced on the cells using a 200 µL pipette tip. Thereafter, the cells were cultured in FBS-free medium containing berberine chloride hydrate, an inhibitor for cancer cell growth (Gibco Company, Grand Island, NY, USA). The scratch width was measured at 0 and 48 hours using an inverted microscope (Olympus), and the migration rate of cells was evaluated by Image J.

Transwell Assay

Cell invasion was measured by a Transwell assay. The apical chambers (Millipore) were pre-coated with Matrigel (BD Biosciences). Then, each apical chamber was filled with 200 μL serum-free cell suspension containing 1×105 transfected cells, while each basolateral chamber was added with 600 μL 10%FBS-DMEM. After 24 hours of incubation at 37°C, the non-invaded cells on the upper membrane were wiped out using cotton swabs, while the invaded cells were fixed for 30 minutes and stained by crystal violet for 20 minutes. The cells were observed under the inverted microscope, and the average value of five included fields of views was calculated.

Tube Formation Assay

A human umbilical vein endothelial cell (HUVEC) line purchased from ATCC was used for angiogenesis measurement. The cells were sorted in Matrigel-coated (90 μL) 24-well plates at 37°C for 30 minutes for polymerization, and then seeded into wells filled with different HeLa and SiHa cell-conditioned media. After a further 6 hours of incubation, the tube formation by HUVECs was imaged under the inverted microscope.

Immunofluorescence Staining

CC cells were fixed and permeated in 0.1%Triton X-100 for 30 minutes. After blockade by 5% skimmed milk, the cells were cultivated with anti-KDM3A (ab243641) and anti-ETS1 (ab186844) at 4°C overnight, and then with goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 20°C for 1 hour. Then, the cells were rinsed and counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), and observed by a fluorescence microscope (Olympus). All antibodies were from Abcam.

Luciferase Reporter Assay

The putative binding sites between ETS1 and the promoter region of KIF14 were obtained from a bioinformatics system Jasper (http://jaspar.genereg.net/). The binding sites were PCR amplified and inserted to pGL3 vectors (Promega, Corp., Madison, WI, USA) to construct KIF14 promoter luciferase vectors (named Promoter). Then, the promoter was co-transfected with either oe-ETS1 or oe-NC into 293T cells (ATCC). Forty-eight hours later, the luciferase activity was determined using a Luciferase Reporter Assay System (Promega).

Chromatin Immunoprecipitation (ChIP) Assay

An EZ-Magna ChIP kit (EMD Millipore) was used to verify the interactions among KDM3A, and the promoter region of ETS1 and KIF14. Briefly, CC cell lines were incubated in 1% formaldehyde solution for 15 minutes of crosslink and then quenched by glycine. Following ultrasonication, the DNA fragments were obtained. Specifically, for the binding relationship between KDM3A and ETS1, the promoter of ETS1 was enriched by the magnet beads conjugated with anti-KDM3A in HeLa cells, while that was enriched by the magnet beads conjugated with anti-H3K9me2 in SiHa cells. For the binding relationship between ETS1 and KIF14, the promoter of KIF14 was enriched by the magnet-beads conjugated with anti-ETS1 in both HeLa and SiHa cells. Magnet beads conjugated with IgG served as a control. The precipitated chromatin DNA was analyzed by RT-qPCR.

Xenograft Tumor in Nude Mice

Twenty NU-Foxn1nu mice (4–6 weeks old) were from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). HeLa and SiHa cells with stable sh-KDM3A or sh-NC transfection were implanted into the mice through a subcutaneous injection. From the 10th day after cell implantation, the tumor size was measured using a vernier caliper every 5 days. The tumor volume (V) was determined as follows: V=length×width2×0.52. The mice were euthanized through an overdose of pentobarbital sodium (150 mg/kg, intraperitoneal injection) on the 30th day. Then, the tumors were collected and weighed for histological examination.

Statistical Analysis

Prism 8.0 (GraphPad, La Jolla, CA, USA) was used for data analysis. Data were obtained from no less than three independent experiments and presented as mean±standard deviation (SD). Differences were analyzed by paired t-test (two groups) and one-way or two-way analysis of variance (ANOVA), followed by Tukey’s multiple test (over two groups). The Log-rank (Mantel-Cox) test was used to record the survival rate of patients. Enumeration data were analyzed by Fisher’s exact test. The correlations between variables were evaluated by Spearman’s rank correlation coefficient. *P<0.05 represents statistical significance.

Results

KDM3A is Abundantly Expressed in CC Tissues and Indicates a Dismal Prognosis in Patients

Though the oncogenic roles of KDM3A have been mentioned, its function in CC remains largely unknown. Here, we first examined mRNA expression of KDM3A in the tumor tissues and the paired adjacent tissues from 50 CC patients, which suggested that the KDM3A expression in CC tissues was increased (Figure 1A). Based on this, the following IHC staining results showed that the staining intensity of KDM3A was higher in tumor tissues than in the adjacent tissues (Figure 1B). According to the average value (2.9) of KDM3A expression in tumor tissues, the included patients were allocated into the high KDM3A expression group (n=25) and poor KDM3A expression group (n=25). It was found that CC patients with higher KDM3A expression had a significantly worse 5-year survival rate (Figure 1C). The clinical characteristics of patients are exhibited in Table 2, which suggested that a high KDM3A expression profile in patients was correlated with worse clinical presentations such as increased lymphatic metastasis, while there was limited tumor differentiation.

Table 2 Correlations Between the Clinicopathologic Characteristics and KDM3A Expression in Patients with Cervical Cancer (n=50)

Figure 1 KDM3A is abundantly expressed in CC and indicates a dismal prognosis in patients. (A) mRNA expression of KDM3A in CC and adjacent tissues determined by RT-qPCR (n=50); (B) staining intensity of KDM3A in CC tumor and the adjacent tissues by IHC staining (n=50); (C) correlation between KDM3A expression and the 5-year survival rate in CC patients. In panels (A) and (B), data were analyzed by paired t test, **P<0.01; in panel (C), correlation was analyzed by the Log-rank (Mantel-Cox) test, #P<0.05.

Knockdown of KDM3A Suppresses CC Cell Growth

Following the findings above, we then measured expression of KDM3A in H8 and CC cell lines (HT-8, CaSki, HeLa, and SiHa). It was found that the mRNA and protein expression of KDM3A was notably increased in CC cell lines compared to that in H8 cells (Figure 2A and B). The HeLa and SiHa cells, owning the highest KDM3A expression, were chosen for the following experiments.

Figure 2 Knockdown of KDM3A suppresses CC cell growth. mRNA (A) and protein (B) expression of KDM3A in H8 and CC cell lines (HT-8, CaSki, HeLa, and SiHa) determined by RT-qPCR and Western blot analysis, respectively; mRNA (C) and protein (D) expression of KDM3A in HeLa and SiHa cells after sh-KDM3A 1# and sh-KDM3A 2# transfection measured by T-qPCR and Western blot analysis, respectively; (E) proliferation of CC cells determined by the CCK-8 method; (F) colony formation ability of CC cells; (G) expression of apoptosis-related factors Bax and Bcl-2 in CC cells determined by RT-qPCR; (H) apoptosis rate of CC cells determined by TUNEL. In panels (AD, FH), data were analyzed by one-way ANOVA, while data in panel (E) were analyzed by two-way ANOVA, followed by Tukey’s multiple comparison test. *P<0.05, **P<0.01 compared to H8; #P<0.05 compared to sh-NC.

Next, sh-KDM3A 1# and sh-KDM3A 2# were transfected into HeLa and SiHa cells, and they both showed effective interference on KDM3A expression (Figure 2C and D). The sh-KDM3A 1# with a better interfering efficacy was chosen for further experiments. After KDM3A knockdown, the proliferation ability according to the CCK-8 assay, and the colony formation ability of cells were notably reduced (Figure 2E and F). As for cell apoptosis, the RT-qPCR results identified an increase in Bax (pro-apoptotic) expression, with a decline in Bcl-2 (anti-apoptotic) expression upon KDM3A knockdown (Figure 2G). More obviously, the TUNEL staining results showed that the apoptosis rate of cells was notably enhanced after sh-KDM3A administration (Figure 2H).

Knockdown of KDM3A Inhibits Metastasis of CC Cells and Angiogenesis by HUVECs

We further explored the relevance of KDM3A with the metastasis of CC cells. First, expression of the EMT-related factors was measured by RT-qPCR, which identified an increase in E-cadherin while a decline in the expression of N-cadherin and Vimentin upon KDM3A knockdown (Figure 3A). In addition, the wound-healing assay results suggested that the migration of cells was notably decreased when KDM3A was suppressed (Figure 3B). Similarly, the invasive potential of CC cells was reduced following KDM3A downregulation (Figure 3C). These collectively showed that knockdown of KDM3A suppressed metastasis of CC cells. In addition, a tube formation assay was performed by seeding HUVECs in different HeLa and SiHa cell-conditioned medium, which showed that the number of formed tubes by HUVECs was declined after KDM3A knockdown (Figure 3D).

Figure 3 Knockdown of KDM3A inhibits metastasis of CC cells and angiogenesis by HUVECs. (A) Expression of EMT-related factors in HeLa and SiHa cells determined by RT-qPCR; (B) migration ability of CC cells evaluated by a wound-healing assay; (C) invasion ability of CC cells measured by a Transwell assay; (D) angiogenesis ability of HUVECs in different conditioned medium determined by a tube formation assay. In all panels, data were compared by one-way ANOVA. #P<0.05 compared to sh-NC.

KDM3A Activates ETS1 Through Demethylation of H3K9me2

As discussed above, we hypothesized that KDM3A possibly mediated ETS1 activity through the demethylation and histone modification of H3K9me2. Thereafter, we first explored ETS1 expression in the collected CC tissues. The RT-qPCR results suggested that the mRNA expression of ETS1 was notably increased in tumor tissues relative to the paired adjacent tissues (Figure 4A) and shared a positive correlation with KDM3A expression (Figure 4B). Again, the IHC staining showed an increased positive expression of KDM3A in tumor tissues (Figure 4C).

Figure 4 KDM3A activates ETS1 through demethylation of H3K9me2. (A) ETS1 expression in tumor tissues and the paired adjacent tissues determined by RT-qPCR; (B) a positive correlation between KDM3A and ETS1 expression; (C) ETS1 expression in tumor and adjacent tissues evaluated by IHC staining; (D) subcellular localization of ETS1 and KDM3A in cells determined by immunofluorescence staining; (E) mRNA expression of ETS1 in cells determined by RT-qPCR; (F) protein expression of ETS1 and H3K9me2 in cells after sh-KDM3A administration determined by Western blot analysis; (G) interactions among KDM3A, H3k9me2, and ETS1 promoter determined by ChIP assays. In panels (A and C), data were compared by the paired t-test, **P<0.05 compared to Adjacent; in panel (B), correlation was evaluated by Spearman’s rank correlation coefficient, r=0.6545, P<0.0001; data in panels (E and F) were compared by one-way ANOVA, while in panel (G) by two-way ANOVA, followed by Tukey’s multiple comparison test, #P<0.05 compared to sh-KDM3A; @@P<0.01, @@@P<0.001 compared to anti-IgG.

In HeLa and SiHa cells, we identified that both KDM3A and ETS1 were sub-localized in nuclear according to the immunofluorescence staining (Figure 4D). Then, ETS1 expression in CC cells with stable sh-KDM3A transfection was determined by RT-qPCR, which showed that sh-KDM3A notably inhibited ETS1 expression in cells (Figure 4E). Likewise, the protein expression of ETS1 in cells was decreased as well after sh-KDM3A administration (Figure 4F), and, interestingly, the protein level of H3K9me2 in cells was notably increased (Figure 4F). Thereafter, a ChIP assay was performed to validate the regulatory network between KDM3A and ETS1 (Figure 4G). In Hela cells, compared to anti-IgG, an enrichment of ETS1 promoter sequence was found in the immunoprecipitates combined by anti-KDM3A. Following sh-KDM3A administration, the enrichment of ETS1 promoter sequence in the precipitates by anti-KDM3A was notably reduced. In SiHa cells, we found that, compared to anti-IgG, anti-H3KPme2 enriched the ETS1 promoter sequence in the precipitates, while this enrichment was further strengthened upon KDM3A knockdown. These results showed that KDM3A promotes ETS1 transcription through the demethylation and histone modification of H3K9me2.

Overexpression of ETS1 Blocks the Inhibition of Sh-KDM3A on CC Cells

To validate the implication of ETS1 in the KDM3A mediation, overexpression of ETS1 was further introduced into CC cells in the presence of KDM3A knockdown, and the transfection efficacy was, again, confirmed by RT-qPCR and Western blot assays (Figure 5A and B). Then, the CCK-8 method showed that the proliferation of cells suppressed by sh-KDM3A was recovered upon ETS1 overexpression (Figure 5C). Next, the flow cytometry suggested that the apoptosis rate of cells was reduced by the further upregulation of ETS1 (Figure 5D). In addition, the migration and invasion abilities of cells reduced by sh-KDM3A were recovered upon ETS1 overexpression (Figure 5E and F). Moreover, HUVECs were sorted in the conditioned medium of CC cells with stable transfection of oe-ETS1, after which the angiogenesis ability of cells suppressed by sh-KDM3A was notably recovered (Figure 5G).

Figure 5 Overexpression of ETS1 blocks the inhibition of sh-KDM3A on CC cells. (A and B) Transfection efficacy of oe-ETS1 in cells determined by RT-qPCR and Western blot analysis, respectively; (C) proliferation ability of cells measured by colony formation assay; (D) apoptosis rate of cells measured after ETS1 overexpression determined by flow cytometry; migration (E) and invasion (F) abilities of cells determined by wound-healing and Transwell assays, respectively; (G), angiogenesis ability of cells determined by tube formation assay. Data were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison. *P<0.05 compared to sh-KDM3A+oe-NC.

ETS1 Transcriptionally Activates KIF14

As mentioned above, ETS1 has been reported to transcriptionally activate KIF14,12 an oncogene in CC.13 First, mRNA expression of KIF14 in the collected tissues from CC patients was determined, which exhibited that the KIF14 expression was notably increased in the tumor tissues compared to the matched adjacent tissues (Figure 6A) and positively associated with ETS1 expression (Figure 6B). Again, the IHC staining intensity of KIF14 was notably enhanced in tumor tissues (Figure 6C).

Figure 6 ETS1transcriptionally activates KIF14. (A) KIF14 expression in tumor and adjacent tissues determined by RT-qPCR; (B) a positive correlation between ETS1 and KIF14 expression; (C) KIF14 expression in tumor and adjacent tissues measured by IHC staining; (D) KIF14 expression in HeLa and SiHa cells after sh-KDM3A or oe-ETS1 administration determined by RT-qPCR; (E) putative binding sites between ETS1 and KIF14 predicted on Jaspar; (F), the binding site with highest score used for luciferase assay; (G and H), binding relationship between ETS1 and KIF14 Promoter validated by (G) and ChIP (H) assay. In panels (A and C), data were analyzed by paired t-test while data in panel (H) by unpaired t-test, **P<0.01 compared to Adjacent, $$P<0.01 compared to anti-IgG; in panel (B), correlation was evaluated by Spearman’s rank correlation coefficient, r=0.6484, P<0.0001; data in panels (D and G) were compared by one-way ANOVA, #P<0.05 compared to sh-NC, &P<0.05 compared to sh-KDM3A+oe-NC, @P<0.05 compared to Promoter+oe-NC.

We then measured mRNA expression of KIF14 in cells after different transfection. It was found that the KIF14 expression was decreased by sh-KDM3A but then recovered by the further administration of oe-ETS1 (Figure 6D). Then, the putative binding sites between ETS1 and the promoter region of KIF14 were predicted on Jaspar (http://jaspar.genereg.net/) (Figure 6E). Then, the binding site with the highest score (Figure 6F) was selected for luciferase assay to validate the binding relationship. The Promoter was co-transfected with oe-NC or oe-ETS1 in 293T cells. After 48 hours, the luciferase activity of Promoter was notably increased by oe-ETS1 (Figure 6G). In addition, a ChIP assay was performed, which found an enrichment of KIF14 promoter was found in the precipitates by anti-ETS1 (Figure 6H), indicating a direct binding relationship between ETS1 and the promoter of KIF14.

KIF14 Activates the Hedgehog Signaling Pathway and Promotes CC Progression

KIF14has been documented as a positive regulator of the Hedgehog signaling pathway,14 whose activation may lead to further development of CC.15,16 To explore whether KIF14 mediates this signaling pathway in CC, oe-KIF14 was administrated into CC cells, and the successful transfection was validated by RT-qPCR (Figure 7A). Then, the expression of Hedgehog-signaling marker proteins Gli1 and Gli3 was measured by Western blot analysis, which suggested that KIF14 led to a notable increase in the expression of the two proteins (Figure 7B), namely, activating the Hedgehog signaling pathway.

Figure 7 KIF14 activates the Hedgehog signaling pathway and promotes CC progression. (A) Transfection efficacy of oe-KIF14 determined by RT-qPCR; (B) protein levels of Gli1 and Gli3 in cells determined by Western blot analysis; (C) DNA replication in cells determined by EdU staining; (D) apoptosis rate in cells determined flow cytometry; migration (E) and invasion (F) abilities of cells determined by wound-healing and Transwell assays; (G) angiogenesis ability of HUVECs determined by tube formation assay. Data were analyzed by one-way ANOVA, *P<0.05 compared to oe-NC.

Further, the EdU labeling assay results showed that the EdU-positive rate in CC cells was increased by oe-KIF14 (Figure 7C), while the apoptosis rate of cells was reduced (Figure 7D). In addition, the migratory and invasive potentials of CC cells were promoted upon KIF14 upregulation (Figure 7E and F). Moreover, the angiogenesis ability of HUVECs cultivated in conditioned medium with stable transfection of oe-KIF14 was increased (Figure 7G).

Activation of Hedgehog Blocks the Inhibiting Role of Sh-KDM3A in CC

To further validate the involvement of Hedgehog in the above events, a rescue experiment was performed through additional administration of a Hedgehog-specific agonist Sag1.5 in cells pre-treated with sh-KDM3A. Then, it was found that this signaling pathway was notably activated (Figure 8A). Thereafter, the colony formation assay results showed that the proliferation of HeLa and SiHa cells inhibited by sh-KDM3A was recovered by Sag1.5 (Figure 8B). In addition, the promotion on cell apoptosis, and the suppression of sh-KDM3A on cell migration and invasion as well as angiogenesis ability of HUVECs were all blocked by the further usage of Sag1.5 (Figure 8CF). These results indicated that Hedgehog is a important downstream effector of KDM3A in CC progression.

Figure 8 Activation of Hedgehog blocks the inhibiting role of sh-KDM3A in CC. (A) Protein levels of Gli1 and Gli3 in cells determined by Western blot analysis; (B) proliferation of CC cells determined by colony formation assay; (C) apoptosis rate of cells measured by TUNEL assay; migration (D) and invasion (E) abilities of cells determined by wound-healing and Transwell assays; (F) angiogenesis ability of HUVECs determined by tube formation assay. Data were analyzed by one-way ANOVA, *P<0.05 compared to sh-KDM3A+DMSO.

Sh-KDM3A Suppresses Tumor Growth in vivo

HeLa and SiHa cells with stable sh-KDM3A transfection were implanted into the ventral side of mice through a subcutaneous injection. The tumor volume was recorded since the 10th day at a 5-day interval (Figure 9A), which showed that knockdown of KDM3A suppressed growth of xenograft tumors in mice. On the 30th day, the mice were euthanized and the tumor tissues were collected and weighed. Again, the tumor weight was reduced upon KDM3A knockdown (Figure 9B). Furthermore, the IHC staining results showed that the staining intensity of KDM3A, ETS1, KIF14, Gli1, and Gli3 was notably reduced in the tumor tissues from mice implanted with cells with stable sh-KDM3A transfection (Figure 9C). These results showed that sh-KDM3A suppresses tumor growth in vivo.

Figure 9 Sh-KDM3A suppresses tumor growth in vivo. (A) Tumor volume growth in mice by time (n=5); (B) weight of tumors in mice on the 30th day; (C) expression of KDM3A, ETS1, KIF14, Gli1, and Gli3 in tissues of xenograft tumors determined by IHC staining. Data were analyzed by one-way (panel C) or two-way (panels A and B) ANOVA. *P<0.05, **P<0.01 compared to sh-NC.

Discussion

As a leading cause of cancer related death among females, CC remains a huge healthy concern in the modern society, especially in those low-income countries,2 owing to the current limitation in effective treatments. Researchers have made great efforts to explore novel molecular mechanisms and to develop promising factors with prognostic or therapeutic values to overcome the typical malignant behaviors from proliferation to metastasis.17–19 Methylation of histone lysine is crucial for epigenetic correlated gene expression profiles in cancer.20 In this study, we validated an interaction network involving KDM3A/ETS1/KIF14, in which KDM3A governs ETS1-mediated KIF14 transcription to promote CC growth in either cell and animal models with the further involvement of the Hedgehog signaling pathway.

The KDM3A has been reviewed as a promising target for human cancer treatment.8 Its oncogenic role has been well demonstrated.21–23 Here, the initial finding of the current study was that KDM3A was highly expressed in the collected tumor tissues from CC patients, and it led to a dismal 5-year survival rate, increased lymphatic metastasis, and decreased tumor differentiation rate in patients. Similarly, abundant expression of KDM3A has been noted as an independent unfavorable factor in patients with pancreatic tumor20 and colorectal cancer24 and epithelial ovarian cancer.25 A high-expression profile of KDM3A was also found in CC cell lines. In the following loss-of-function studies, we observed that sh-KDM3A suppressed growth and metastatic potential of Hela and SiHa cells. In addition, an increase in the pro-apoptotic factor Bax and the epithelial marker E-cadherin, while a decline in the anti-apoptotic factor Bcl-2 and mesenchymal markers N-cadherin and Vimentin suggested that sh-KDM3A promoted apoptosis while suppressing EMT in CC cells in a cellular perspective. In addition, angiogenesis, a hallmark of cancers that is crucial for tumor growth and metastasis,26 was found to be suppressed as well upon KDM3A downregulation.

KDM3A catalyzes the demethylation of H3K9me1/me2 in vivo and in vitro with a preference for dimethylated residues, therefore governing transcriptional activation.8,20,27,28 Intriguingly, studies have noted that KDM3A could control the transcriptional activation of ETS1 in a similar manner.9,29 ETS1 has been noted as an important oncogene and a treating target in multiple malignancies including breast cancer,30,31 ovarian cancer,32,33 and CC as well.11,34 To validate this potential interaction between KDM3A and ETS1, we first identified nuclear-localization of KDM3A and ETS1 in CC cells, and then a positive correlation between KDM3A and ETS1 was identified. Further, the ChIP assays were performed, which suggested that ETS1 promoter sequences were enriched by either anti-KDM3A or anti-H3K9me2. On this basis, we found that artificial overexpression of ETS1 blocked the inhibitory roles of sh-KDM3A in CC cell growth, suggesting that ETS1 is possibly responsible for the pro-oncogenic role of KDM3A in CC cells.

We then observed a positive relevance between ETS1 and KIF14 expression in the collected tissues and HeLa and SiHa cells. Then, the ChIP and luciferase assays suggested that as a transcription factor, ETS1 had a direct binding relationship with the promoter sequence with KIF14 and activated its transcription, which was partially in line with the previous report.12 KIF4 is a potent oncogene participating in the development of multiple cancers. For instance, it was associated with the metastasis and tumor progression as well as poor treating outcome in gastric cancer35 and prostate cancer.36 As for in CC, it was reported to predict dismal prognosis and chemoresistance.13 Here, after identification of the high-expression profile of KIF14 in collected tissues, we noticed that overexpression of KIF14 promoted the malignant behaviors of CC cells, which validated its oncogenic function in CC. Intriguingly, KIF14 was documented as a positive regulator of the Hedgehog signaling pathway. Here, we found oe-KIF14 promoted the expression of Gli1 and Gli3 expression. This signaling pathway, first identified in fruit fly, is a highly conserved pathway responsible for signal transduction from cell membrane to nucleus, which plays crucial functions for embryonic development and is also implicated in cancer progression.37–39 This is also applied in CC, where the Hedgehog was found to be correlated with radioresistance,40 proliferation and survival,41 and EMT and metastasis16 of CC cells. Importantly, our study noticed that upregulation of the Hedgehog blocked the inhibitory functions of sh-KDM3A in CC cells, implicating its potential participation in KDM3A-mediated events. Consistently, similar results were reproduced in our in vivo experiments, where sh-KDM3A suppressed the growth of xenograft tumors in nude mice and the IHC staining intensity of ETS1, KIF14, Gil1, and Gil3 in the collected tumor tissues.

To conclude, our experimental results provided evidence that KDM3A promotes CC growth in either cell and animal models through triggering ETS1-mediated KIF14 transcription and the further upregulation the Hedgehog signaling pathway (Figure 10). This study may offer new understandings in CC progression and provide novel thoughts into CC treatment.

Figure 10 Diagram presentation of the molecular mechanism. KDM3A encourages the transcription of ETS1 through the demethylation and histone modification of H3K9me2, while ETS1 further binds to the promoter region of KIF14 to promote its transcription activity, which activate the Hedgehog signaling pathway and promote CC progression.

Data Sharing Statement

All the data generated or analyzed during this study are included in this published article.

Funding

There is no funding to report.

Disclosure

The authors declare no conflicts of interest in this work.

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