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High-risk human papillomavirus (HPV)16 and 18 are the primary cause of cervical cancer (CC) and long non-coding RNAs (lncRNAs/lncs) are often abnormally expressed in patients with CC. The authors' previous study indicated that oncogenic enhancer of zeste homolog 2 (EZH2)-binding lncRNA in cervical cancer (lnc-EBIC) serves a role in the tumorigenic activity of the HPV E6 protein in patients with CC. However, whether HPV E7 affects the development of CC through lnc-EBIC, and the potential mechanisms underlying this remains unclear. Therefore, the present study investigated the effects of lnc-EBIC and HPV E7 in cervical cancer cell lines HeLa, CaSki and C33A
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 (
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 (
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 (
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 (
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.
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 (
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 (
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.
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.
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).
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.
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.
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 (
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.
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
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 (
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 (
HPV E7 is not a DNA-binding transcription factor (
Previous studies have demonstrated that KLHDC7B promotes HPV viral replication and secretion in HPV-infected cervical intraepithelial neoplasia (
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
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 (
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 (
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 (
KLHDC7B is associated with an aggressive subtype of cancer and predicts a poor prognosis in patients with breast (
E7 performs oncogenic activities by modulating the expression of several host proteins, including pRB, p107, p130, p21, octamer-binding transcription factor 4 and PTPN14 (
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.
Not applicable.
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).
All the datasets generated and/or analyzed during the present study are included in this published article.
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.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
HPV16/18 E7 promotes the expression of lnc-EBIC. (A) Overexpression of HPV18 E7 enhanced the expression of lnc-EBIC in HeLa cells. (B) Depletion of HPV18 E7 reduced the expression of lnc-EBIC in HeLa cells. mRNA levels of HPV18 E7 and lnc-EBIC were detected by RT-qPCR. (C and D) The protein levels of HPV18 E7 were detected in HeLa cells; western blotting was performed with whole cell extracts of HeLa cells transfected with Tag2B-HPV18-E7 and siHPV18-E7. (E) Expression of lnc-EBIC was upregulated when the HPV16 E7 was overexpressed in CasKi cells. (F) Depletion of HPV16 E7 reduced the expression of lnc-EBIC in HeLa cells. mRNA levels of HPV16 E7 and lnc-EBIC were detected by RT-qPCR. (G and H) The protein levels of HPV16 E7 were detected in CasKi cells. Western blotting was performed with whole cell extracts of CasKi cells transfected with Tag2B-HPV16-E7 and siHPV16-E7. GAPDH was the loading control. Data are presented as the means ± SD; n=3. *P<0.05. HPV, human papillomavirus; lnc, long non-coding RNA; lnc-EBIC, enhancer of zeste homolog 2-binding lncRNA in cervical cancer; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; NC, negative control.
lnc-EBIC affects the function of cervical cancer cells. (A) The expression of lnc-EBIC in C33A cells was detected by RT-qPCR. C33A cells were transfected with pcDNA3.1 and pcDNA3.1-lnc-EBIC, respectively. (B) The proliferation of C33A cells was increased following lnc-EBIC overexpression. CCK-8 assays were performed at 24 h intervals as indicated. (C) The fractions of S-phase C33A cells were increased upon lnc-EBIC-overexpression. EdU assays were applied to visualize cells in the S-phase of the cell cycle. (D) Cell cycle analysis of the lnc-EBIC-overexpressed C33A cells. PI-stained C33A cells were subjected to FACS. (E) Transwell assays indicate that lnc-EBIC-overexpression increased C33A cell migration and invasion. (F) Overexpression of lnc-EBIC decreased the percentage of apoptotic cells. The percentage of cells in each quadrant is indicated. (G) The western blot analysis of cellular functional proteins on cell cycle, apoptosis, migration lysates from lnc-EBIC-overexpressed in C33A cells. The blots revealed lnc-EBIC increased the expression of c-JUN, Bcl-2, Cyr61, MYL9 and reduced the expression of p21 and Cleaved caspase-3. Data are presented as the means ± SD. n=3. *P<0.05 and **P<0.01. lnc, long non-coding RNA; lnc-EBIC, enhancer of zeste homolog 2-binding lncRNA in cervical cancer; RT-qPCR, reverse transcription-quantitative PCR; CCK-8, Cell Counting Kit-8.
HPV E7 affects the function of Hela cells by regulating lnc-EBIC. (A) The western blot analysis of cellular functional proteins on cell cycle, apoptosis, migration lysates from HeLa cells. (B) The fraction of S-phase HPV18 E7 overexpressed HeLa cells was increased with co-transfection of HPV18 E7 and silnc-EBIC. (C) Interference of lnc-EBIC attenuated the effect of HPV E7 on apoptosis in HeLa cells. The percentage of cells in each quadrant is indicated. (D) Transwell assays indicated that interference of lnc-EBIC attenuated the effect of HPV E7 on migration and invasion in HeLa cells. Data are presented as the means ± SD, n=3. *P<0.05 and **P<0.01. HPV, human papillomavirus; lnc, long non-coding RNA; lnc-EBIC, enhancer of zeste homolog 2-binding lncRNA in cervical cancer; si, small interfering; NC, negative control.
HPV E7 promotes the expression lnc-EBIC by inhibiting TAL1. (A and B) The overexpression and silencing of HPV18 E7 altered the expression of TAL1 in HeLa cells. (C) The protein levels of TAL1 were decreased following the overexpression of HPV18 E7 in HeLa cells and increased following the silencing of HPV18 E7 in HeLa cells. (D and E) The overexpression and silencing of HPV16 E7 altered the expression of TAL1 in CasKi cells. (F) The protein levels of TAL1 were decreased following the overexpression and increased following the silencing of HPV16 E7 in CasKi cells. (G) The protein levels of TAL1 were downregulated following the knockdown of TAL1 in C33A cells. (H and I) The protein levels of TAL1 were detected in HeLa and CasKi cells. (J) The mRNA levels of lnc-EBIC were upregulated following the knockdown of TAL1 in C33A cells. (K and L) TAL1 altered the effect of HPV E7 on lnc-EBIC in HeLa and CasKi cells. mRNA levels of TAL1 were detected by RT-qPCR. The protein level of TAL1 was detected by western blotting. GAPDH was used as the protein loading control. Data are presented as the means ± SD, n=3. *P<0.05 and **P<0.01. HPV, human papillomavirus; lnc, long non-coding RNA; lnc-EBIC, enhancer of zeste homolog 2-binding lncRNA in cervical cancer; TAL1, TAL BHLH transcription factor 1, erythroid differentiation factor; si, small interfering; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR.
lnc-EBIC regulates the expression of KLHDC7B in C33A cells. (A and B) lnc-EBIC could positively regulate the mRNA levels of KLHDC7B in C33A cells. (C) Western blot analysis indicated that overexpression of lnc-EBIC boosted the protein levels of KLHDC7B and knockdown of lnc-EBIC suppressed the protein levels of KLHDC7B in C33A cells. GAPDH was used as the protein loading control. (D and E) HPV18 E7 influenced the mRNA and protein levels of KLHDC7B by regulating lnc-EBIC in C33A cells. (F and G) HPV16 E7 influenced the mRNA and protein levels of KLHDC7B by regulating lnc-EBIC in C33A cells. The mRNA levels of lnc-EBIC and KLHDC7B were detected by RT-qPCR. The protein level of KLHDC7B was detected by western blotting. GAPDH was used as the protein loading control. Data are presented as the means ± SD, n=3. *P<0.05 and **P<0.01. lnc, long non-coding RNA; lnc-EBIC, enhancer of zeste homolog 2-binding lncRNA in cervical cancer; KLHDC7B, Kelch domain-containing 7B; HPV, human papillomavirus; si, small interfering; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR.
lnc-EBIC affects the function of cervical cancer cells by regulating KLHDC7B. (A) The western blot analysis of cellular functional proteins on cell cycle, apoptosis, migration lysates from C33A cells. (B) The fraction of S-phase lnc-EBIC-overexpressed C33A cells was increased with co-transfection of pcDNA3.1-lnc-EBIC and siKLHDC7B. (C) Interference of KLHDC7B attenuated the effect of lnc-EBIC on apoptosis in C33A cells. The percentage of cells in each quadrant is indicated. (D) Transwell assays suggested that interference of KLHDC7B attenuated the effect of lnc-EBIC on migration and invasion in C33A cells. Differences between NC, pcDNA3.1-lnc-EBIC alone and co-transfection of pcDNA3.1-lnc-EBIC and siKLHDC7B were compared. Data are presented as the means ± SD, n=3. *P<0.05 and **P<0.01. lnc, long non-coding RNA; lnc-EBIC, enhancer of zeste homolog 2-binding lncRNA in cervical cancer; KLHDC7B, Kelch domain-containing 7B; NC, negative control; si, small interfering.