HPV E7 affects the function of cervical cancer cells via the TAL1/lnc‑EBIC/KLHDC7B axis
- Authors:
- Published online on: March 4, 2021 https://doi.org/10.3892/or.2021.8002
- Article Number: 51
Abstract
Introduction
High-risk human papillomaviruses (HPVs), particularly HPV16 and HPV18, are the main cause of cervical cancer, which ranks as the fourth most frequently diagnosed cancer and the fourth leading cause of cancer-associated deaths in women, worldwide (1). Additionally, the incidence and mortality rates of cervical cancer are increasing in several countries (2). HPV E6 and E7 are important viral oncogenes that act in concert to maintain the malignant phenotype of cervical cancer. These genes constitute attractive therapeutic targets, as it has been demonstrated that E6/E7 inhibition rapidly induces senescence in HPV-positive cancer cells (3). However, infection with oncogenic HPV alone is not sufficient for cancer development; genetic variations and epigenetic alterations are required for the development of precancerous lesions and cervical cancer (4,5). Thus, improving the clinical management of patients and developing an effective novel treatment strategy for patients with cervical cancer is a major challenge.
To determine the additional alterations that occur in cervical cancer, previous studies have demonstrated that E6 inactivates p53 by binding to the cellular ubiquitin ligase E6-associated protein, thereby preventing the replicative senescence of cervical cancer cells (6–8). As E6 contains a PDZ binding motif (PBM; including postsynaptic density protein 95/DLG/zona occludins-1) at the extreme C terminus, it can further bind to a number of tumor suppressor protein-containing PDZ domains, including DLG, scribble and membrane associated guanylate kinase, WW and PDZ domain-containing 1 (9). In addition, the transforming activity of E7 is strongly increased when E6 is co-expressed; however, high-risk HPV E7 modulates the expression and degradation of several host proteins, including retinoblastoma (pRB), p107, p130, tyrosine-protein phosphatase non- receptor type14 (PTPN14) and p21, which leads to unscheduled cell cycle progression (10).
Recently, accumulating evidence has further confirmed that the modulation of long non-coding RNA (lncRNA/lnc) expression is an important aspect of oncogenic activity in high-risk HPV E6 and E7 proteins. For example, HPV 16 E6 increased the expression of lnc-cervical carcinoma expressed PCNA regulatory (CCEPR) and lnc-family with sequence similarity 83 member H antisense RNA 1 (FAM83H-AS1) through a mechanism that is not directly dependent on p53 inactivation, which thereby promoted proliferation and migration, and inhibited the apoptosis of cervical cancer cells (11,12). It has been previously demonstrated that HPV16/18 E6 and E7 proteins were associated with increased enhancer of zeste homolog 2 (EZH2)-binding lncRNA in cervical cancer (lnc-EBIC) expression in cervical cancer cells. The study additionally determined that E6 promoted lnc-EBIC expression to sequester certain tumor repressor microRNAs (miRs), including miR-375 and miR-139 that target HPV16/18 E6/E7 mRNA, thus forming a positive feedback loop that mutually derepressed gene expression in cervical cancer cells (13). Lnc-EBIC is a pseudogene that is highly expressed in human cervical cancer tissues and cell lines, which interacts with EZH2 to repress the expression of E-cadherin and thus promote cellular proliferation and invasion (14,15). In addition to E6, a previous study determined that HPV18 E7 also stimulated lnc-EBIC expression in HeLa cells (13). However, the tumorigenic roles and potential molecular mechanism of the E7/lnc-EBIC axis on cervical cancer has not been fully elucidated.
Transcriptome sequencing was performed in a previous study to analyze the effects of lnc-EBIC depletion on the mRNA levels of certain protein-coding genes (13). Oncogenic Kelch domain-containing 7B (KLHDC7B) was identified to be correlated with lnc-EBIC expression, and was determined to interact with Kelch-containing proteins via the C-terminal Kelch domain (16,17). Moreover, KLHDC7B was identified to be upregulated in breast cancer and could promote breast tumorigenesis by modulating genes involved in the interferon signaling pathway (16,18). In addition, recent transcriptome analysis further suggested that KLHDC7B could be used as a biomarker for prognostic prediction and may be involved in the development and progression of cervical cancer (19).
In the present study, the relationship between HPV16/18 E7 and lnc-EBIC in cervical cancer cells was investigated, and the effects of lnc-EBIC on the proliferation (using CCK-8 and EdU/DAPI staining assay), apoptosis [utilizing flow cytometer Annexin V/propidium iodide (PI) assay], cell invasion and migration (using Transwell assays) of HPV+ and HPV− cervical cancer cells were assessed, to provide a novel mechanism and potential therapeutic target for cervical cancer.
Materials and methods
Cell culture and transfection
Human cervical cancer cell lines, including HeLa (HPV18+), CaSki (HPV16+) and C33A (HPV16−/18−), were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in DMEM-low glucose (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Beyotime Institute of Biotechnology), in a humidified atmosphere at 37°C with 5% CO2. Cells in the exponential phase of growth were used in the experiments. The protein-coding plasmids pCMV-Tag2B-HPV18 E7 and pCMV-Tag2B-HPV16 E7 were previously described (13). The nucleotide sequences of lnc-EBIC were synthesized and inserted into pcDNA3.1 vectors (Thermo Fisher Scientific, Inc.) to construct the pcDNA3.1-lnc-EBIC overexpression plasmids. Empty vectors were used as negative controls (NCs). A total of 4 µg of each plasmid vector was added to 100 µl of serum-free medium, and the volume of 100 µl of each mixture was added to 1×105/ml cells in 6-cell culture plates. TAL1 small interfering (si)RNA duplexes were designed and synthesized at concentration of 100 nM (Guangzhou RiboBio Co., Ltd.). The sequences are as follows: siHPV18-E7, 5′-CCTTCTATGTCACGAGCAA-3′; siHPV16-E7, 5′-CACCTACATTGCATGAATA-3′; silnc-EBIC, 5′-GGGAGTAAAGACTCCAGTA-3′; siTAL1, 5′-AACCATGGAATCAACAAGGAT-3′; siKLHDC7B, 5′-CAGTGACAATGACTGGGATAGTGCT-3′; siRNA NC, 5′-GTTCTCCGAACGTGTCACGT-3′. Plasmids were transiently transfected into cells using Lipofectamine® 2000 as instructed by the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. After 48 h transfection, the selective overexpression and silencing of HPV16 E7, HPV18 E7, lnc-EBIC and TAL1 were detected by reverse transcription-quantitative (RT-q) PCR and western blotting.
RT-qPCR analysis
Total RNA (1 µg) was extracted from cells using the TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using an RT-PCR kit (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. qPCR was performed using SYBR Green (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. The following primers were utilized: HPV16E7 forward, 5′-AGCAGAACCGGACAGAGCCCA-3′ and reverse, 5′-TGTACGCACAACCGAAGCGT-3′; HPV18E7 forward, 5′-TGAAATTCCGGTTGACCTTC-3′ and reverse, 5′-TCGGGCTGGTAAATGTTGAT-3′; lnc-EBIC forward, 5′-AAGGGCGTCGTGGTTCCAACTC-3′ and reverse, 5′-AGCATTGCCGTCCTGGGTGTAG-3′; TAL1 forward, 5′-CAACTGGAAAATCCAAAGGCTATGG-3′ and reverse, 5′-GACGCAATTCCTCCACAGTACACAG-3′; KLHDC7B forward, 5′-TGGGAACGAACACTCTTAC-3′ and reverse, 5′-CAGCAACTGAACACTTGAC-3′. A total of 20 µl reaction mixture contained 1.5 µl of cDNA, 10 µl of 2× SYBR Primer Ex TagII (TaKaRa), 7.5 µl of ddH2O and 1 µl of primers (10 µM). The ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to perform the amplification reaction, using the following thermal cycling profile: 94°C for 10 min, followed by 40 cycles of amplification (94°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec), and 72°C for 10 min. Each experiment was performed in triplicate and was analyzed using the 2−ΔΔCq method (20).
Western blot analysis
RIPA buffer (cat. no. P0013C; Beyotime Institute of Biotechnology) was used for the extraction and concentration determination of total protein from cells. Protein concentrations were determined with a BCA Protein Assay kit. Protein samples (30 µg) were separated via SDS-PAGE (8–10%) and transferred onto polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked using 5% non-fat milk for 2 h at room temperature, and then incubated with the following primary antibodies at 4°C overnight: HPV16 E7 (cat. no. sc-6981; 1:1,000; 21 kDa) and HPV18 E7 (cat. no. sc-365035; 1:1,000; 15 kDa) both from Santa Cruz Biotechnology, lnc., p21 (product code ab109520; 1:1,000; 21 kDa), caspase-3 (product code ab32351; 1:5,000; 32 kDa), Bcl-2 (product code ab32124; 1:1,000; 26 kDa), c-JUN (product code ab32137; 1:5,000; 36 kDa), cysteine-rich 61 (Cyr61; product code ab24448; 1:1,000; 42 kDa), myosin regulatory light polypeptide 9 (MYL9; product code ab191393; 1:1,000; 20 kDa) and GAPDH (product code ab9485; 1:2,500; 37 kDa) all from Abcam. The membranes were subsequently incubated with HRP-conjugated IgG secondary antibodies (product code ab7090; 1:4,000; Abcam) at room temperature for 2 h. The chemiluminescence intensity was detected using an ECL kit (EMD Millipore) according to the manufacturer's protocol and ImageJ v1.8.0 software (National Institutes of Health) was used to analyze the gray value of the target band.
Cell Counting Kit-8 (CCK-8) assay
Cells were seeded into 96-well plates (Corning, Inc.) at a density of 1×104/100 µl and incubated at 37°C with 5% CO2 for 24 h. CCK-8 (10 µl/ml; Dojindo Molecular Technologies, Inc.) was subsequently added to each well and incubated at 37°C with 5% CO2 for a further 4 h, after which the absorbance was measured using a microplate reader (Bio-Rad Laboratories, Inc.) at 450 nm.
Flow cytometric assay
The apoptosis of HeLa, CaSki and C33A cells was detected via flow cytometry. After transfection for 48 h, cells were collected and stained using an Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Fluorescence signals were detected using a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo 7.6.5 software (FlowJo LLC).
EDU and DAPI staining assay
Cellular proliferation and apoptosis was assessed using the BeyoClick™ EdU Cell Proliferation kit with Alexa Fluor 488 (Beyotime Institute of Biotechnology) and DAPI dihydrochloride (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. A total of 1×104 cells were seeded in 24-well plates and incubated at 37°C and 5% CO2 with 10% FBS-medium for 12 h. Cells were then fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100 for 20 min at room temperature. Cells were washed thrice with PBS and cultured at room temperature with 100 µl Click Reaction Mixture (50 µM) for 20 min in the dark. Cell nuclei were counterstained with 100 µl DAPI (1 mg/ml) at room temperature for 5 min. A fluorescence microscope of 10×20 (Carl Zeiss AG) was used to count the number of proliferative/apoptotic cells in three random fields of view per slide.
Transwell assay
For the cell invasion assay, the upper chamber (8-µm pore size; Costar; Coring, Inc.) was supplemented with Matrigel, while an upper chamber without Matrigel was used for the migration assay. Cells (5×104) were resuspended in serum-free medium and plated into the upper chamber. Complete medium in the lower chamber was used as a chemical attractant. After incubation at 37°C with 5% CO2 for 48 h, the migrated or invasive cells attached to the lower chamber surface were fixed with 4% formaldehyde at room temperature for 15 min and stained with 0.5% crystal violet at room temperature for 30 min. The invasive or migrated cells in three random fields of view were subsequently imaged under an inverted light microscope. Experiments were performed independently and in triplicate.
Bioinformatics analysis
To determine the potential transcription factors that regulate lnc-EBIC expression, the promoter sequence of lnc-EBIC was extracted from the UCSC Genome Browser bioinformatics program (http://www.genome.ucsc.edu) (21) and analyzed via the Gene Transcription Regulation Database (GTRD; http://gtrd.biouml.org) (22), JASPAR (http://jaspar.genereg.net/) (23) and ChIP-Atlas-Enrichment Analysis program (http://chip-atlas.org) (24).
Statistical analysis
Data are presented as the mean ± SD and analyzed using GraphPad Prism V 6.00 software (GraphPad, Inc.). Statistical significance was determined using a paired Student's t-test or ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
HPV16/18 E7 promotes the expression of lnc-EBIC
To determine whether lnc-EBIC is involved in cervical cancer progression, Tag2B-HPV18-E7 and Tag2B-HPV16-E7 were transfected into HeLa and CasKi cells, respectively. As presented in Fig. 1, the protein expression of E7 was increased in HeLa (Fig. 1A and C) and CasKi cells (Fig. 1E and G). Additionally, the expression of lnc-EBIC was significantly increased (Fig. 1A and E). To further confirm whether HPV16/18 E7 regulated the expression of lnc-EBIC, siRNA specific to HPV18 E7 and HPV16 E7 was transfected into HeLa and CasKi cells to knockdown endogenous E7 expression. The interfering efficiency of HPV16/18 E7 is presented in Fig. 1D and H. The results of RT-qPCR demonstrated that HPV16/18 E7 silencing significantly blocked the expression of lnc-EBIC (Fig. 1B and F). The results indicated that the HPV16/18 E7 protein promoted the excessive expression of lnc-EBIC in cervical cancer cells.
lnc-EBIC overexpression regulates the proliferation, apoptosis, the cell cycle, migration and invasion of HPV− C33A cervical cancer cells
To investigate the role of lnc-EBIC in cervical cancer, the pcDNA3.1-lnc-EBIC overexpression plasmid and corresponding NC were transfected into HPV− C33A cells (Fig. 2A). The results of the CCK-8 assay revealed that cell viability was significantly increased in C33A cells transfected with pcDNA3.1-lnc-EBIC compared with pcDNA3.1-transfected cells after 72 h (Fig. 2B). Additionally, EdU staining further confirmed that the upregulation of lnc-EBIC enhanced the proliferation of C33A cells (Fig. 2C). The effect of this upregulation on the cell cycle was then assessed via flow cytometry. The results demonstrated a markedly increased number of cells in the S and G2 phases (Fig. 2D), indicating that the upregulation of lnc-EBIC enhanced the cell cycle transition of cervical cancer cells. The results of the Transwell assay further demonstrated that cell migration and invasion were markedly increased in C33A cells transfected with pcDNA3.1-lnc-EBIC (Fig. 2E). In addition, an Annexin V-FITC/PI assay was performed to evaluate the apoptosis of C33A cells, the results of which revealed that the apoptotic rate was significantly reduced in lnc-EBIC-overexpression cells compared with the control (Fig. 2F). Moreover, the protein expression of pro-apoptotic p21 and Cleaved caspase-3 were decreased, and anti-apoptotic Bcl-2, c-JUN, Cyr61 and MYL9 were increased in lnc-EBIC-overexpressing C33A cells compared with pcDNA3.1-transfected C33A cells (Fig. 2G). The results indicated that lnc-EBIC promoted cell proliferation, cell cycle progression, migration and invasion, and inhibited the apoptosis of cervical cancer cells in HPV− cervical cancer cells.
lnc-EBIC is required for the HPV16/18 protein, E7, to serve tumorigenic activities in cervical cancer cells
To determine the function of lnc-EBIC in E7-mediated tumorigenesis, siRNA targeting lnc-EBIC was co-transfected with an E7-overexpression plasmid in HeLa and CasKi cells (Fig. S1A and B). Western blot analysis revealed that lnc-EBIC knockdown significantly inhibited the promotive effects of E7 on anti-apoptotic proteins c-JUN, Bcl-2, Cyr61 and MYL9 in HPV+ cervical cancer cells, and increased the expression of pro-apoptotic proteins p21 and Cleaved caspase-3, which were decreased by E7 overexpression (Figs. 3A and S1C). The EdU staining and Annexin V-FITC/PI assay further confirmed that lnc-EBIC knockdown suppressed the tumorigenic effects of E7 on the proliferation and apoptosis of HeLa (Fig. 3B and C) and CasKi (Fig. S1D and E) cells. A series of Transwell assays were performed to evaluate the influence of lnc-EBIC on the migration and invasion of HeLa and CasKi cells. Compared with HPV16/18 E7 overexpression alone, co-transfection with lnc-EBIC siRNA significantly inhibited the migration and invasion of HeLa (Fig. 3D) and CasKi (Fig. S1F) cells. The results indicated that lnc-EBIC may be an important mediator of E7 that enhances tumorigenic activities in cervical cancer cells.
HPV16/18 E7 protein promotes lnc-EBIC expression by inhibiting TAL1 expression
HPV E7 is not a DNA-binding transcription factor (3,25). Thus, the effect of HPV E7 on lnc-EBIC expression may be mediated by cellular transcription factors. TAL1 was identified in the three databases. The present study demonstrated that E7 overexpression in HeLa (Fig. 4A-C) and CasKi cells (Fig. 4D-F) significantly decreased the expression of TAL1, with the opposite effect when E7 knockdown. To further confirm whether E7 depended on TAL1 suppression to enhance lnc-EBIC expression, lnc-EBIC levels were assessed in cervical cancer cells transfected with siRNA against TAL1 alone or in the presence of E7 (Fig. 4G-I). In C33A, HeLa and CasKi cervical cancer cells, TAL1 knockdown significantly increased the expression of lnc-EBIC, an effect that was significantly suppressed following E7 inhibition (Fig. 4J-L). The results suggested that TAL1 inactivation may serve an important role in HPV E7-induced lnc-EBIC upregulation.
HPV16/18 E7 protein depends on lnc-EBIC to suppress KLHDC7B expression
Previous studies have demonstrated that KLHDC7B promotes HPV viral replication and secretion in HPV-infected cervical intraepithelial neoplasia (26). Furthermore, transcriptome sequencing analysis conducted in a previous study revealed that KLHDC7B was decreased in siRNA lnc-EBIC transfected HeLa cells (13). The expression of KLHDC7B in pcDNA3.1 and pcDNA3.1-lnc-EBIC transfected C33A cells was therefore assessed in the present study. The results revealed that the mRNA expression of KLHDC7B was significantly increased in pcDNA3.1-lnc-EBIC C33A cells compared with pcDNA3.1-transfected cells (Fig. 5A). Conversely, lnc-EBIC knockdown significantly decreased the expression of KLHDC7B in C33A cells (Fig. 5B). These results were further confirmed via western blot analysis (Fig. 5C). To determine whether the regulatory role of lnc-EBIC on KLHDC7B expression was associated with HPV16/18 E7, HeLa cells were co-transfected with Tag2B-HPV18-E7 and siRNA lnc-EBIC. Western blot analysis revealed that lnc-EBIC knockdown markedly decreased the expression of KLHDC7B in HPV18 E7-overexpression cells (Fig. 5D and E). Similar results were obtained in HPV16 E7-transfected CasKi cells (Fig. 5F and G). The results indicated that KLHDC7B could be upregulated by lnc-EBIC and that this effect might be further enhanced by HPV16/18 E7 in cervical cancer cells.
KLHDC7B cooperates with lnc-EBIC to promote the tumorigenic activities of cervical cancer cells
To elucidate whether KLHDC7B was involved in lnc-EBIC-mediated tumorigenic activity, C33A cells were transfected with the lnc-EBIC overexpression plasmid alone or in the presence of siRNA KLHDC7B. As presented in Fig. 6A, lnc-EBIC overexpression significantly increased the expression of anti-apoptotic c-JUN, Bcl-2, Cyr61 and MYL9, and reduced the expression of pro-apoptotic p21 and Cleaved caspase-3 in C33A cells. Moreover, as predicted, KLHDC7B knockdown significantly suppressed the effects of lnc-EBIC on the expression of apoptotic proteins (Fig. 6A) and markedly inhibited the tumor-promotive effects of lnc-EBIC, including increased cellular proliferation (Fig. 6B), migration and invasion (Fig. 6D), and decreased apoptosis (Fig. 6C) in C33A cells. The results demonstrated that KLHDC7B served an important role in the lnc-EBIC-mediated tumorigenic activities of cervical cancer cells.
Discussion
lncRNAs serve key regulatory roles in the occurrence and progression of cervical cancer. For example, breast cancer anti-estrogen resistance 4, a lapatinib-responsive lncRNA (an EGFR/HER2 inhibitor) enhanced cell proliferation in estrogen-resistant breast cancer, and serves as a metastasis-promoting lncRNA in cervical cancer (27). A nine-lncRNA signature composed of ATXN8 opposite strand lncRNA, chromosome 5 Open Reading Frame 60, DIO3 opposite strand upstream RNA, EMX2 opposite strand/antisense RNA, inactivation escape 1, KCNQ1 downstream neighbor protein, KCNQ1 overlapping transcript 1, loss of heterozygosity on chromosome 12 region 2 and RFPL1 antisense RNA 1 exhibits great potency for the prediction of cervical cancer recurrence (28). Moreover, recent studies have indicated that lncRNAs including growth arrest-specific 5, H19 imprinted maternally expressed transcript (non-protein coding), FAM83H antisense RNA 1, metastasis-associated lung adenocarcinoma transcript 1 and CCEPR (11,12) can be exploited by HPVs to perform tumorigenic activities. However, these lncRNAs are specifically regulated by E6; less is known about the lncRNAs that are regulated by E7. The present study revealed that oncogenic lnc-EBIC could be exploited by HPV16/18 E7 to accelerate the proliferation, migration and invasion, and inhibit apoptosis in cervical cancer cells. lnc-EBIC also exhibited oncogenic activities even in HPV− cervical cancer cells. Therefore, lnc-EBIC may be a novel therapeutic target for patients with HPV+ and HPV− cervical cancers.
lncRNAs regulate a variety of critical cellular processes by promoting or repressing transcription, serving as epigenetic regulators or as scaffolds to interact with various proteins in cervical cancer (29,30). Lnc-cervical cancer DExH-box helicase 9 (DHX9) suppressive transcript (lnc-CCDST), a recently identified tumor-suppressive lncRNA that can be abolished by E7, has been revealed to promote pro-oncogenic DHX9 degradation by serving as a scaffold to facilitate the formation of mouse double minute 2 (MDM2) and DHX9 complexes, while not influencing the mRNA expression of DHX9 (31). Lnc-EBIC has been confirmed to act as a competing endogenous RNA that sequesters tumor repressors miR-375 and miR-139, which target HPV16/18 E6/E7 mRNA (13), and to serve as a scaffold that interacts with EZH2, thus repressing E-cadherin expression (14,15). The present study further demonstrated that lnc-EBIC promoted cellular proliferation, migration and invasion, and inhibited apoptosis in cervical cancer cells by enhancing KLHDC7B expression.
The transcription factor TAL1 is an essential regulator of hematopoiesis that promotes prostate cancer cell growth via the MAPK/ERK, PI3K/AKT and AMPK signaling pathways (32). However, its role in cervical cancer remains unknown. The present study revealed that TAL1 was significantly downregulated in HPV E7 cervical cells. Furthermore, it was demonstrated that the inactivation of TAL1 may serve an important role in HPV E7-induced lnc-EBIC upregulation.
KLHDC7B is associated with an aggressive subtype of cancer and predicts a poor prognosis in patients with breast (16) and laryngeal (33) cancers. Furthermore, KLHDC7B is upregulated in HPV-induced vulvar intraepithelial neoplasia (26) and can be used as a biomarker for the diagnosis and prognostic prediction of patients with cervical cancer (19). In the present study, interfering KLHDC7B expression was observed to significantly inhibit the oncogenic activities of lnc-EBIC. Thus, KLHDC7B may be a pivotal target of lnc-EBIC in cervical cancer cells. As both mRNA and protein levels of KLHDC7B were enhanced by lnc-EBIC, the exact interaction pathway between lnc-EBIC and KLHDC7B requires further elucidation.
E7 performs oncogenic activities by modulating the expression of several host proteins, including pRB, p107, p130, p21, octamer-binding transcription factor 4 and PTPN14 (34,35). The present study demonstrated that HPV E7 was dependent on the inhibition of TAL1 to promote lnc-EBIC expression. TAL1 is a transcription factor that is aberrantly expressed in 60% of cases of human T-cell acute lymphoblastic leukemia (T-ALL) cases, activating several important oncogenes, including the MYC, MYB, Notch1, cyclin E, and tribbles pseudokinase 2 (36,37). Bioinformatic analysis was performed in the present study to identify the lncRNAs that are regulated by TAL1 in T-ALL cells (38). The results revealed that lnc-EBIC was one such lncRNA. Additionally, a transcriptome profiling study determined that TAL1 was overexpressed in gastric-type cervical cancer that was not associated with HPV infection (39). TAL1 knockdown in HPV+ (HeLa and CasKi) and HPV− (C33A) cells in the present study induced a significant increase in lnc-EBIC expression. TAL1 may therefore represent a novel target for E7 in HPV infection. However, its role in cervical cancer progression requires further clarification.
In conclusion, the present study revealed that oncogenic lnc-EBIC can be exploited by HPV16/18 E7 to increase cellular proliferation, migration and invasion, and decrease apoptosis in cervical cancer cells. Molecular analysis revealed that E7 is dependent on the TAL1/lnc-EBIC/KLHDC7B axis to perform its tumor-promotive activities. Furthermore, lnc-EBIC exhibited oncogenic activity by enhancing KLHDC7B expression in HPV− cervical cancer cells. Thus, the lnc-EBIC/KLHDC7B axis represents a novel molecular mechanism and potential therapeutic target for both HPV+ and HPV− cervical cancer.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
This work was supported by the National Natural Science Foundation of China (grant no. 81201604), The Open Research Fund Program of the State Key Laboratory of Virology of China (grant no. 2015KF010), the Natural Science Foundation of Wuhan Municipal Health Commission (grant no. WX18Q27), The Top Medical Young Talents of Hubei Province (2019) and The Yellow Crane Talents Fund (2016).
Availability of data and materials
All the datasets generated and/or analyzed during the present study are included in this published article.
Authors' contributions
JW, FX and XL performed the experiments, contributed to data analysis and wrote the manuscript. XM, XC, XS and YY analyzed the data. CY, YX and HX conceptualized the study design, contributed to data analysis and experimental materials. All authors have read and approved the final version of this manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References
|
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A and Bray F: Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. Feb 4–2021.(Online ahead of print). View Article : Google Scholar : PubMed/NCBI | |
|
Boda D, Neagu M, Constantin C, Voinescu RN, Caruntu C, Zurac S, Spandidos DA, Drakoulis N, Tsoukalas D and Tsatsakis AM: HPV strain distribution in patients with genital warts in a female population sample. Oncol Lett. 12:1779–1782. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Hoppe-Seyler K, Bossler F, Braun JA, Herrmann AL and Hoppe-Seyler F: The HPV E6/E7 oncogenes: Key factors for viral carcinogenesis and therapeutic targets. Trends Microbiol. 26:158–168. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Dardiotis E, Siokas V, Garas A, Paraskevaidis E, Kyrgiou M, Xiromerisiou G, Deligeoroglou E, Galazios G, Kontomanolis EN, Spandidos DA, et al: Genetic variations in the SULF1 gene alter the risk of cervical cancer and precancerous lesions. Oncol Lett. 16:3833–3841. 2018.PubMed/NCBI | |
|
Boda D, Docea AO, Calina D, Ilie MA, Caruntu C, Zurac S, Neagu M, Constantin C, Branisteanu DE, Voiculescu V, et al: Human papilloma virus: Apprehending the link with carcinogenesis and unveiling new research avenues (Review). Int J Oncol. 52:637–655. 2018.PubMed/NCBI | |
|
Li S, Hong X, Wei Z, Xie M, Li W, Liu G, Guo H, Yang J, Wei W and Zhang S: Ubiquitination of the HPV oncoprotein E6 is critical for E6/E6AP-mediated p53 degradation. Front Microbiol. 10:24832019. View Article : Google Scholar : PubMed/NCBI | |
|
Martinez-Zapien D, Ruiz FX, Poirson J, Mitschler A, Ramirez J, Forster A, Cousido-Siah A, Masson M, Vande Pol S, Podjarny A, et al: Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53. Nature. 529:541–545. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Celegato M, Messa L, Goracci L, Mercorelli B, Bertagnin C, Spyrakis F, Suarez I, Cousido-Siah A, Trave G, Banks L, et al: A novel small-molecule inhibitor of the human papillomavirus E6-p53 interaction that reactivates p53 function and blocks cancer cells growth. Cancer Lett. 470:115–125. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Ganti K, Massimi P, Manzo-Merino J, Tomaic V, Pim D, Playford MP, Lizano M, Roberts S, Kranjec C, Doorbar J and Banks L: Interaction of the human papillomavirus E6 oncoprotein with sorting nexin 27 modulates endocytic cargo transport pathways. PLoS Pathog. 12:e10058542016. View Article : Google Scholar : PubMed/NCBI | |
|
Hatterschide J, Bohidar AE, Grace M, Nulton TJ, Kim HW, Windle B, Morgan IM, Munger K and White EA: PTPN14 degradation by high-risk human papillomavirus E7 limits keratinocyte differentiation and contributes to HPV-mediated oncogenesis. Proc Natl Acad Sci USA. 116:7033–7042. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Barr JA, Hayes KE, Brownmiller T, Harold AD, Jagannathan R, Lockman PR, Khan S and Martinez I: Long non-coding RNA FAM83H-AS1 is regulated by human papillomavirus 16 E6 independently of p53 in cervical cancer cells. Sci Rep. 9:36622019. View Article : Google Scholar : PubMed/NCBI | |
|
Sharma S and Munger K: Expression of the cervical carcinoma expressed PCNA regulatory (CCEPR) long noncoding RNA is driven by the human papillomavirus E6 protein and modulates cell proliferation independent of PCNA. Virology. 518:8–13. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
He H, Liu X, Liu Y, Zhang M, Lai Y, Hao Y, Wang Q, Shi D, Wang N, Luo XG, et al: Human papillomavirus E6/E7 and long noncoding RNA TMPOP2 mutually upregulated gene expression in cervical cancer cells. J Virol. 93:e01808–18. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Dong J, Su M, Chang W, Zhang K, Wu S and Xu T: Long non-coding RNAs on the stage of cervical cancer (Review). Oncol Rep. 38:1923–1931. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Sun NX, Ye C, Zhao Q, Zhang Q, Xu C, Wang SB, Jin ZJ, Sun SH, Wang F and Li W: Long noncoding RNA-EBIC promotes tumor cell invasion by binding to EZH2 and repressing E-cadherin in cervical cancer. PLoS One. 9:e1003402014. View Article : Google Scholar : PubMed/NCBI | |
|
Jeong G, Bae H, Jeong D, Ham J, Park S, Kim HW, Kang HS and Kim SJ: A Kelch domain-containing KLHDC7B and a long non-coding RNA ST8SIA6-AS1 act oppositely on breast cancer cell proliferation via the interferon signaling pathway. Sci Rep. 8:129222018. View Article : Google Scholar : PubMed/NCBI | |
|
Martin-Pardillos A and Cajal SRY: Characterization of Kelch domain-containing protein 7B in breast tumours and breast cancer cell lines. Oncol Lett. 18:2853–2860. 2019.PubMed/NCBI | |
|
Beltrán-Anaya FO, Romero-Córdoba S, Rebollar-Vega R, Arrieta O, Bautista-Piña V, Dominguez-Reyes C, Villegas-Carlos F, Tenorio-Torres A, Alfaro-Riuz L, Jiménez-Morales S, et al: Expression of long non-coding RNA ENSG00000226738 (LncKLHDC7B) is enriched in the immunomodulatory triple-negative breast cancer subtype and its alteration promotes cell migration, invasion, and resistance to cell death. Mol Oncol. 13:909–927. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Guo P, Wang D, Wu J, Yang J, Ren T, Zhu B and Xiang Y: The landscape of alternative splicing in cervical squamous cell carcinoma. Onco Targets Ther. 8:73–79. 2015.PubMed/NCBI | |
|
Wu X, Zheng X, Cheng J, Zhang K and Ma C: LncRNA TUG1 regulates proliferation and apoptosis by regulating miR-148b/IGF2 axis in ox-LDL-stimulated VSMC and HUVEC. Life Sci. 243:1172872020. View Article : Google Scholar : PubMed/NCBI | |
|
Kuhn RM, Haussler D and Kent WJ: The UCSC genome browser and associated tools. Brief Bioinform. 14:144–161. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Mishra GP, Ghosh A, Jha A and Raghav SK: BedSect: An integrated web server application to perform intersection, visualization, and functional annotation of genomic regions from multiple datasets. Front Genet. 11:32020. View Article : Google Scholar : PubMed/NCBI | |
|
Liu Y, Chen X, Cheng R, Yang F, Yu M, Wang C, Cui S, Hong Y, Liang H, Liu M, et al: The Jun/miR-22/HuR regulatory axis contributes to tumourigenesis in colorectal cancer. Mol Cancer. 17:112018. View Article : Google Scholar : PubMed/NCBI | |
|
Li Z, Zhao Z, Cai Z, Sun Y, Li L, Yao F, Yang L, Zhou Y, Zhu H, Fu Y, et al: Runx2 (Runt-related transcription factor 2)-mediated microcalcification is a novel pathological characteristic and potential mediator of abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol. 40:1352–1369. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Tian S, Zhang L, Li Y, Cao D, Quan S, Guo Y, Yang X and Yang T: Human papillomavirus E7 oncoprotein promotes proliferation and migration through the transcription factor E2F1 in cervical cancer cells. Anticancer Agents Med Chem. Nov 5–2020.(Epub ahead of print). View Article : Google Scholar | |
|
Santegoets LA, Seters M, Helmerhorst TJ, Heijmans-Antonissen C, Hanifi-Moghaddam P, Ewing PC, van Ijcken WF, van der Spek PJ, van der Meijden WI and Blok LJ: HPV related VIN: Highly proliferative and diminished responsiveness to extracellular signals. Int J Cancer. 121:759–766. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Integrated genomic and molecular characterization of cervical cancer. Nature. 543:378–384. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Mao Y, Dong L, Zheng Y, Dong J and Li X: Prediction of Recurrence in Cervical Cancer Using a Nine-lncRNA Signature. Front Genet. 10:2842019. View Article : Google Scholar : PubMed/NCBI | |
|
Hosseini ES, Meryet-Figuiere M, Sabzalipoor H, Kashani HH, Nikzad H and Asemi Z: Dysregulated expression of long noncoding RNAs in gynecologic cancers. Mol Cancer. 16:1072017. View Article : Google Scholar : PubMed/NCBI | |
|
Feng S, Liu W, Bai X, Pan W, Jia Z, Zhang S, Zhu Y and Tan W: LncRNA-CTS promotes metastasis and epithelial-to-mesenchymal transition through regulating miR-505/ZEB2 axis in cervical cancer. Cancer Lett. 465:105–117. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Ding X, Jia X, Wang C, Xu J, Gao SJ and Lu C: A DHX9-lncRNA-MDM2 interaction regulates cell invasion and angiogenesis of cervical cancer. Cell Death Differ. 26:1750–1765. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Wu X, Xiao Y, Yan W, Ji Z and Zheng G: The human oncogene SCL/TAL1 interrupting locus (STIL) promotes tumor growth through MAPK/ERK, PI3K/Akt and AMPK pathways in prostate cancer. Gene. 686:220–227. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang G, Fan E, Yue G, Zhong Q, Shuai Y, Wu M, Feng G, Chen Q and Gou X: Five genes as a novel signature for predicting the prognosis of patients with laryngeal cancer. J Cell Biochem. Oct 31–2019.(Epub ahead of print). | |
|
Westrich JA, Warren CJ, Klausner MJ, Guo K, Liu CW, Santiago ML and Pyeon D: Human papillomavirus 16 E7 stabilizes APOBEC3A protein by inhibiting cullin 2-dependent protein degradation. J Virol. 92:e01318–17. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Panayiotou T, Michael S, Zaravinos A, Demirag E, Achilleos C and Strati K: Human papillomavirus E7 binds Oct4 and regulates its activity in HPV-associated cervical cancers. PLoS Pathog. 16:e10084682020. View Article : Google Scholar : PubMed/NCBI | |
|
Sanda T, Lawton LN, Barrasa MI, Fan ZP, Kohlhammer H, Gutierrez A, Ma W, Tatarek J, Ahn Y, Kelliher MA, et al: Core transcriptional regulatory circuit controlled by the TAL1 complex in human T cell acute lymphoblastic leukemia. Cancer Cell. 22:209–221. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Mansour MR, Sanda T, Lawton LN, Li X, Kreslavsky T, Novina CD, Brand M, Gutierrez A, Kelliher MA, Jamieson CH, et al: The TAL1 complex targets the FBXW7 tumor suppressor by activating miR-223 in human T cell acute lymphoblastic leukemia. J Exp Med. 210:1545–1557. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Ngoc PCT, Tan SH, Tan TK, Chan MM, Li Z, Yeoh AEJ, Tenen DG and Sanda T: Identification of novel lncRNAs regulated by the TAL1 complex in T-cell acute lymphoblastic leukemia. Leukemia. 32:2138–2151. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Vasseur D, Lopez J, Croce S, Tondeur G, Bonin L, Descotes F, Golfier F and Devouassoux-Shisheboran M: Transcriptome profiling of gastric-type endocervical adenocarcinomas identifies key signaling pathways for tumor progression. Gynecol Oncol. 157:775–782. 2020. View Article : Google Scholar : PubMed/NCBI |
