Archives of Biochemistry and Biophysics 755 (2024) 109980

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Archives of Biochemistry and Biophysics

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Promotive actions of lncRNA EBLN3P involved in cervical cancer

progression via interacting with miR-29c-3p and TAF15 to modify RCC2
Ting Zou a, b, Yan Gao b, Mingrong Qie a, *
a
Department of Gynecology and Obstetrics, West China Second University Hospital, Sichuan University, Chengdu, 610041, Sichuan Province, PR China
b
Department of Gynecology, Guizhou Provincial People’s Hospital, Guiyang, 550002, Guizhou Province, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Cervical cancer is a common cancer that seriously affects women’s health globally. The key roles of
Cervical cancer long non-coding RNAs (lncRNAs) in the onset and development of cervical cancer have attracted much attention.
EBLN3P Our study aims to uncover the roles of lncRNA EBLN3P and miR-29c-3p and the mechanisms by which EBLN3P
miR-29c-3p
and miR-29c-3p regulate malignancy in cervical cancer.
TAF15
RCC2
Methods: Tumor and adjacent normal tissues were collected from cervical cancer patients, and the expression of
EBLN3P and miR-29c-3p were analyzed via RT-qPCR. The capacities of proliferation, migration, and invasion
were assessed using CCK-8, wound healing and transwell assays. The interaction among EBLN3P, miR-29c-3p
and TAF15 was determined by luciferase, RNA immunoprecipitation and RNA pull-down assays, respectively.
A subcutaneous tumor xenograft mouse model was established to evaluate the functional role of EBLN3P in vivo.
Results: The interaction and reciprocal negative regulation between EBLN3P and miR-29c-3p were uncovered in
cervical cancer cells. Likewise, EBLN3P and miR-29c-3p expression patterns in tumor tissues presented a negative
association. EBLN3P knockdown weakened cell proliferation, migration and invasion, but these effects were
abrogated by miR-29c-3p depletion. Mechanistically, ALKBH5 might impaired EBLN3P stability to reduce its
expression. EBLN3P functioned as a competing endogenous RNA (ceRNA) for miR-29c-3p to relieve its sup­
pression of RCC2. Besides, EBLN3P enhanced RCC2 mRNA stability via interacting with TAF15. Furthermore,
silencing of EBLN3P repressed the tumor growth in mice.
Conclusion: Altogether, lncRNA EBLN3P positively regulates RCC2 expression via competitively binding to miR-
29c-3p and interacting with TAF15, thereby boosting proliferation, migration, and invasion of cervical cancer
cells.

1. Introduction cervical cancer [5]. Therefore, uncovering the mechanisms underlying

the progression of cervical cancer is crucial for a better understanding of
Cervical cancer with an estimated incidence of over 500,000 causes cancer pathogenesis and development of novel therapeutic approaches.
more than 300,000 deaths each year, making it as the fourth common Non-coding RNAs (ncRNAs) are novel classes of regulatory RNAs
malignancy in women globally [1]. Human papillomavirus (HPV) which lack the protein-coding capacity and play vital roles in controlling
infection is considered to be responsible for most cervical cancer cases, the onset and progression of diverse human cancers including cervical
and HPV vaccination and early screening prevent the development of cancer [6,7]. Numerous studies have revealed that microRNAs (miR­
cervical cancer [2,3]. The incidence and mortality have significantly NAs) are dysregulated in cervical cancer and act as important regulators
declined in developed countries, but cervical cancer is still a leading in the tumorigenesis, invasion and metastasis [8]. Zhang et al. reported
cause of cancer death in women due to a lack of HPV vaccination and that miR-218 modulated apoptosis and proliferation in cervical cancer
efficient early screening in low- and middle-income countries [4]. Great through Gli3 [9]. MiR-204 exerted its anti-tumor activity in cervical
advances have been accomplished in the diagnosis and treatment, but cancer via suppressing migration and invasion [10]. In addition, our
the survival rate is still unsatisfactory for patients with advanced previous study has demonstrated that miR-29c-3p, a tumor suppressor in

* Corresponding author. Department of Gynecology and Obstetrics, West China Second University Hospital, Sichuan University, No.20 Renmin South Road,
Chengdu 610041, Sichuan Province, PR China
E-mail address: [email protected] (M. Qie).

https://doi.org/10.1016/j.abb.2024.109980
Received 3 January 2024; Received in revised form 11 March 2024; Accepted 27 March 2024
Available online 29 March 2024
0003-9861/© 2024 Elsevier Inc. All rights reserved.
T. Zou et al. Archives of Biochemistry and Biophysics 755 (2024) 109980

various cancers, represses proliferation, invasion, and metastasis in Table 1

cervical cancer through SPARC [11]. However, the upstream regulatory The clinical characteristics of 44 patients diagnosed with
mechanisms of miR-29c-3p have not been reported. Long non-coding cervical cancer.
RNAs (lncRNAs), typical non-coding RNAs with a length of more than Total number N = 44
200 nucleotides, control gene expression via functioning as competing Age (Years)
endogenous RNAs (ceRNAs) for miRNAs, thus regulating cervical cancer >60 22
progression [12,13]. We hypothesized that lncRNA might be involved in ≤60 22
the regulation of miR-29c-3p-mediated anti-tumor activity in cervical HPV status
Positive 28
cancer.
Negative 16
LncRNA Endogenous Bornavirus Like Nucleoprotein 3, Pseudogene Histology
(EBLN3P) is a novel lncRNA that is highly expressed in cancers, and Squamous cell cancer 30
emerging evidence has suggested EBLN3P as an oncogene. Xu and col­ Adenocarcinoma 14
leagues found that EBLN3P promoted colorectal cancer progression via Stage
I-II 18
sponging miR-323a-3p and regulating UHMK1 expression [14]. More­ III-IV 26
over, EBLN3P facilitated cell migration and invasion in liver cancer Tumor size (mm3)
through the miR-144-3p-DOCK4 axis [15]. However, whether it could ≤4.0 24
take actions on the development of cervical cancer remains largely un­ >4.0 20
known. We observed increased EBLN3P expression in cervical cancer
patients in preliminary assays and identified a potential interaction
2.2. Cell culture
between EBLN3P and miR-29c-3p through bioinformatics analysis.
Therefore, we hypothesized that EBLN3P might be implicated in the
The ectocervical cell line Ect1/E6E7 and cervical cancer cell lines C-
regulation of cervical cancer progression via interacting with
33A, HeLa, CaSki and SiHa were purchased from the American Type
miR-29c-3p. Besides sponging miRNAs, lncRNAs regulate gene expres­
Culture Collection (Manassas, VA, USA) and maintained in HyClone
sion through various mechanisms, such as interacting with RNA-binding
DMEM (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal
proteins to modulate RNA splicing, stability and translation, which is
bovine serum (ScienCell, Carlsbad, CA, USA). Cells at Passage 4–10 were
linked to the subcellular localization of lncRNAs [16]. EBLN3P might
used for cell assays. For actinomycin D (Act D) treatment, C-33A and
regulate the expression of a specific downstream gene via various
SiHa cells (1 × 106 cells) were seeded in 6-well plates and treated with
mechanisms, but it needs further investigation.
Act D (Sigma, St. Louis, MO, USA) at 5 μg/mL for 0, 2, 4, 6 or 8 h and
The regulator of chromosome condensation 2 (RCC2) is a highly
collected for examining RCC2 mRNA stability through quantitative PCR.
conserved nuclear protein that functions as a key regulator of mitosis
and cytoplasmic division in cell cycle [17]. Importantly, growing evi­
2.3. Cell transfection
dence has shown that RCC2 plays an oncogenic role in many cancers,
such as breast cancer, lung adenocarcinoma, colorectal cancer, and
The coding region for RCC2 and ALKBH5 were amplified through
ovarian cancer, by inducing drug resistance in tumor cells, facilitating
PCR and inserted into the pcDNA3.1 vector (Thermo Fisher Scientific,
metastasis and promoting cancer progression [17]. Intriguingly, a recent
Waltham, MA, USA) for RCC2 and ALKBH5 overexpression. MiR-29c-3p
study has reported that lncRNA HOTAIR promotes RCC2 expression to
mimics, miR-29c-3p inhibitor and negative controls (NC mimics and
accelerate the progression of cervical cancer via acting as a miR-331-3p
inhibitor) were provided by RiboBio (Guangzhou, Guangdong, China).
sponge [18], demonstrating the oncogenic activity of RCC2 in cervical
The TAF15 shRNAs (sh-TAF15#1 and #2) and scramble control (sh-NC)
cancer. In the preliminary experiment, we identified a potential inter­
were provided by Genepharma (Shanghai, China) and inserted into the
action between miR-29c-3p and RCC2, raising a possibility that RCC2
pLKO.1 puro vector (Addgene, Watertown, MA, USA). Cells (1 × 106
might be targeted by miR-29c-3p to regulate the progression of cervical
cells) were seeded in 6-well plates and transfected with these mimics,
cancer.
inhibitor and constructs using Lipofectamine 3000 (Thermo Fisher Sci­
In this study, we firstly identified a reciprocal negative regulation
entific) following the manual. The EBLN3P shRNA (sh-EBLN3P) and
between EBLN3P and miR-29c-3p in cervical cancer cells. EBLN3P was
scramble control (sh-NC) lentiviral particles were designed and pack­
highly expressed in cervical cancer, and knockdown of EBLN3P
aged by Genepharma. Cells (1 × 106 cells) were seeded in 6-well plates,
repressed proliferation, migration, and invasion by upregulating miR-
and lentiviral particles were added into cells at the multiplicity of
29c-3p. EBLN3P promoted cervical cancer progression by upregulating
infection of 50. Cells were collected after 72 h for subsequent assays.
RCC2 expression via sponging miR-29c-3p and interacting with TAF15.
Our findings provide a novel mechanistic insight to the pathogenesis of
2.4. Reverse transcription-quantitative PCR (RT-qPCR)
cervical cancer and potential therapeutic targets for cervical cancer.
Tumor and adjacent normal tissues were homogenized in Trizol re­
2. Methods
agent (Thermo Fisher Scientific) and centrifugated for removing insol­
uble debris. Ect1/E6E7, C-33A, HeLa, CaSki and SiHa cells (1 × 106
2.1. Clinical specimens
cells) with indicated treatment were washed and resuspended in Trizol
reagent. Total RNA was isolated from tissues and cells using PureLink
Patients were firstly diagnosed with cervical cancer at Guizhou
RNA Mini Kit (Thermo Fisher Scientific) and processed to reverse tran­
Provincial People’s Hospital from June 2019 to June 2021, and tumor
scription for cDNA synthesis with QuantiTect Reverse Transcription Kit
and adjacent normal tissues were collected from these patients (N = 44)
(Qiagen, Venlo, Netherlands). MiR-29c-3p expression was analyzed with
for RNA extraction and quantitative PCR analysis. Patients diagnosed
miRCURY LNA miRNA PCR Starter Kit (Qiagen) and normalized to U6.
with cervical cancer that received treatment before surgery were
The expression of EBLN3P, RCC2 and TAF15 was determined with
excluded in this study. All patients provided written informed consent,
quantitative PCR and normalized to GAPDH. The 2− ΔΔCt method was
and our study was approved by the Ethics Committee of Guizhou Pro­
used for calculation. Primers were showed in Table 2.
vincial People’s Hospital. The clinical characteristics of patients were
summarized in Table 1.

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T. Zou et al. Archives of Biochemistry and Biophysics 755 (2024) 109980

Table 2 imaged with Olympus BX51 microscope and counted.

The primers utilized for the detection of qRT-PCR assay were listed.
EBLN3P Forward: 5′-CAGACTAAAGGATCAAGCGAGA-3′ 2.9. RNA immunoprecipitation (RIP) assay
Reverse: 5′-ATCAATTGCCACAGGTTGAAGA-3′
miR-29c-3p Forward: 5′-ACACTCCAGCTGGGTAGCACCATTTGAAAT-3′ Magna RIP kit was obtained from Merck (Darmstadt, Germany), and
Reverse: 5′-TGGTGTCGTGGAGTCG-3′
RIP assays were conducted following the manual. Briefly, C-33A and
RCC2 Forward: 5′-TTTTCTCAGAGCAGGTCGCC-3′
Reverse: 5′-TTCGGGGGTTGTATTCTGGC-3′ SiHa cells (5 × 106 cells) were lysed in 1 mL RIP lysis buffer and cen­
TAF15 Forward: 5′-GGGAGCACAGTGTCATTT-3′ trifugated for collecting cell lysates. Cell lysates were aliquoted into two
Reverse: 5′-ATTTCCGCATGACGGATTAG-3′ fractions of 500 μL each (for RIP and normal IgG). The rabbit anti-Ago2
U6 Forward: 5′-CTCGCTTCGGCAGCACA-3′ (5 μg, ab186733, Abcam), anti-TAF15 (5 μg, PA5-27845, Thermo Fisher
Reverse: 5′-AACGCTTCACGAATTTGCGT-3′
GAPDH Forward: 5′-ATGGGGAAGGTGAAGGTCGGAG-3′
Scientific), anti-ALKBH5 (5 μg, MA5-51412, Thermo Fisher Scientific),
Reverse: 5′-GATGACAAGCTTCCCGTTCTCAGC-3′ anti-YTHDF2 (5 μg, ab220163, Abcam) and normal rabbit IgG (5 μg,
ab37415, Abcam) were mixed with 50 μL protein A/G magnetic beads
for preparing the magnetic bead-antibody complexes. The RNA-protein
2.5. Western blot complexes were incubated with the magnetic bead-antibody complexes
overnight at 4 ◦ C and immunopreciated by magnetic bead-antibody
Protein was extracted from C-33A and SiHa cells (5 × 106 cells) with complexes. The beads were washed with RIP Wash Buffer, and RNA
Protein Extraction Kit (Abcam, Cambridge, UK) and quantified using was extracted by adding 400 μL of phenol:chloroform:isoamyl alcohol
BCA Protein Quantification Kit (Vazyme, Nanjing, Jiangsu, China). solution. The enrichment of EBLN3P, miR-29c-3p and RCC2 was eval­
Then, 30 μg of protein was electrophoresed in 12% Bis-Tris gel and uated by RT-qPCR.
transferred to PVDF membranes. Subsequently, membranes were
blocked in 10% non-fat milk solution, washed, and incubated with 2.10. RNA pull-down assay
rabbit anti-RCC2 (ab70787, 1:2000, Abcam), anti-phosphorylated JNK
(p-JNK, ab4821, 1:1000, Abcam), anti-JNK (ab112501, 1:1000, Abcam), Biotinylated EBLN3P (Bio-EBLN3P), miR-29c-3p (Bio-miR-29c-3p)
or anti-GAPDH (ab9485, 1:2500, Abcam) overnight. Membranes were and negative control (Bio-NC) probes were synthesized by Sangon
washed and incubated with a goat anti-rabbit HRP secondary antibody (Shanghai, China). C-33A and SiHa cells (5 × 106 cells) were lysed in 1
(ab6721, 1:20000, Abcam) for 1 h. Bands were visualized by adding ECL mL lysis buffer and centrifugated for collecting cell lysates. Cell lysates
substrate (Beyotime, Shanghai, China) and analyzed with ImageJ were aliquoted into two fractions of 500 μL each (for pull-down and
software. control probes). Biotinylated probes were mixed with the lysates and
incubated for 6 h, and streptavidin magnetic beads (50 μL) were added
2.6. Cell proliferation and incubated for additional 2 h at 4 ◦ C. Beads were washed with wash
buffer (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl) and RNA-protein
The proliferative capacity of C-33A and SiHa cells was evaluated complexes pulled down by the beads were subjected for RT-qPCR
with CCK-8 and colony formation assays. Briefly, cells (5 × 104 cells) analysis of miR-29c-3p and EBLN3P and Western blot analysis of
were treated as indicated in 96-well plates, and medium was removed. A TAF15 (1:5000, ab134916, Abcam), ALKBH5 (1:2000, MA5-51412,
pre-prepared mixture of fresh culture medium (100 μL each well) and Thermo Fisher Scientific) and YTHDF2 (1:1000, ab220163, Abcam).
CCK-8 (10 μL each well, Vazyme) was added into each well, and cells
were further incubated for 2 h. The absorbance at 450 nm was 2.11. Dual-luciferase reporter assay
measured. For the colony formation assay, after indicated treatment,
cells were detached, and single cell suspension was prepared. Subse­ The wildtype and mutant binding sites for miR-29c-3p in EBLN3P
quently, cells (1 × 103 cells) were plated into 6-well plates and incu­ (EBLN3P-WT) and RCC2 (RCC2-WT) and mutant sites (EBLN3P-MUT
bated for 7 days for colony formation. Colonies were washed, stained in and RCC2-MUT) were inserted into the pmirGLO vector (Promega,
a mixture of glutaraldehyde (6%, Sigma) and crystal violet (0.5%, Madison, WI, USA). Cells (1 × 106 cells) were seeded in 6-well plates and
Sigma) for 20 min and counted with the eCount Colony Counter (VWR, co-transfected with miR-29c-3p mimics and the EBLN3P or RCC2 re­
Radnor, PA, USA) under a Leica stereomicroscope (Leica, Wetzlar, porter and harvested after 48 h for analyzing the luciferase activity with
Germany). Dual-Glo Luciferase Assay System (Promega). NC mimics was used as the
negative control.
2.7. Wound healing assay
2.12. Combined fluorescent in situ hybridization (FISH) and
For determining migrative capacity of C-33A and SiHa cells, cells (1 immunofluorescence (IF) staining
× 105 cells) were seeded in 6-well plates and cultured to form a
monolayer. Subsequently, medium was removed, and cells were incu­ SiHa cells (1 × 104 cells) were seeded on coverslips, fixed in 4%
bated in serum-free medium for 12 h for starvation. A scratch of 1 mm formaldehyde and permeabilized in 0.5% Triton X-100. Then, cells were
was made on the monolayer, and cells were cultured for additional 24 h. rinsed and incubated in proteinase K (15 μg/μL, Thermo Fisher Scien­
Wound healing was imaged with Olympus BX51 microscope (Olympus, tific) for 30 min. After prehybridization, cells were hybridized with the
Tokyo, Japan) and analyzed with Image J software. Alexa Fluor 555-conjugated EBLN3P probe at 50 nM overnight. After
wash, cells were incubated with a rabbit anti-TAF15 antibody (1:250,
2.8. Transwell assay ab134916, Abcam) for 1 h, and an Alexa Fluor 488-conjugated goat anti-
rabbit secondary antibody (Thermo Fisher Scientific) was added to
The matrigel (Corning, Corning, NY) was thawed and mixed with visualize TAF15. The nuclei were stained with DAPI (Beyotime), and
serum-free culture medium. The mixture was added to 24-mm Transwell cells were mounted for imaging with Leica TCS SP5 confocal system.
inserts (Corning), and the inserts were placed into 6-well plates. Com­
plete culture medium was added into wells, and cells (1 × 105 cells) 2.13. A subcutaneous tumor xenograft mouse model
were suspended in serum-free medium and seeded onto the upper part of
the inserts. After 24 h, cells that invaded into the lower part were fixed BALB/c nude mice (male, 4-week-old) were obtained from SJA
and stained with crystal violet (0.5%) for 20 min, and colonies were Laboratory Animal (Changsha, Hunan, China) and blindly divided into

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T. Zou et al. Archives of Biochemistry and Biophysics 755 (2024) 109980

sh-NC and sh-EBLN3P groups (N = 6 per group). SiHa cells were 2.15. Statistical analysis
transfected with sh-NC or sh-EBLN3P through lentivirus particles and
collected for subcutaneous injection. Cells were washed, resuspended in SPSS version 22.0 (SPSS, IL, USA) was used for statistical analysis.
PBS, and subcutaneously injected under the left armpit (1 × 107 cells in Data from at least three independent experiments was expressed as
100 μL each mouse). The length and width of subcutaneous tumors were mean ± standard deviation (SD). There were at least three biological
measured using a vernier caliper, and the volume was calculated with replicates in each independent experiment. Spearman correlation anal­
the formula length × width2/2. After 6 weeks, mice were sacrificed, and ysis was applied to analyze the correlation among EBLN3P, miR-29c-3p
tumors were excised for imaging and IHC staining. Animal procedures and RCC2. The determination of statistical differences between two
were conducted in accordance with the Institutional Animal Care groups or among multiple groups was analyzed using the Student’s t-test
guidelines of Guizhou Provincial People’s Hospital and approved by the and one-way analysis of variance (ANOVA) with GraphPad Prism 8.0
Animal Care and Use Committee of Guizhou Provincial People’s (GraphPad Software, San Diego, CA, USA). The normality of distribution
Hospital. was checked using the SPSS Software, and Tukey’s Honest Significant
Difference (HSD) post hoc test was used for one-way ANOVA. P < 0.05
2.14. Immunohistochemistry (IHC) staining suggested statistically significance at a statistical level.

Mice were sacrificed, and tumors were gently excised and fixed in 4% 3. Results
formaldehyde overnight. Tissues were washed and dehydrated in
increased concentrations of ethanol solution (70%, 80%, 95% and 3.1. Reciprocal negative regulation between miR-29c-3p and EBLN3P in
100%). After clearing, tissues were embedded in paraffin and sectioned cervical cancer cells
at 5 μm. Sections were deparaffinized and rehydrated prior to antigen
retrieval in a microwave. After inactivation of endogenous peroxidase in To explore the implication of miR-29c-3p, EBLN3P and RCC2 in
H2O2 solution, sections were blocked and incubated with a rabbit anti- cervical cancer, we analyzed the expression of hsa-miR-29c-3p, EBLN3P,
RCC2 antibody (1:100, ab154705, Abcam) overnight. Then, sections and RCC2 in cervical squamous cell carcinoma (CESC) from TCGA
were washed and incubated with an HRP-conjugated secondary anti­ database and observed decreased hsa-miR-29c-3p expression and
body for 1 h. DAB peroxidase substrate (Beyotime) was added for signal increased expression of EBLN3P and RCC2 in CESC (Supplementary
visualization. Sections were counterstained with hematoxylin (Sigma) Figs. 1A–C). Moreover, we found that RCC2 was highly expressed in
and mounted for imaging with Olympus BX51 microscope. various human cancers (Supplementary Fig. 1D). As demonstrated in
Starbase (https://starbase.sysu.edu.cn/) database, a potential binding
site for miR-29c-3p in EBLN3P was observed (Fig. 1A). Overexpression

Fig. 1. Reciprocal negative regulation between miR-29c-3p and EBLN3P in cervical cancer cells. (A) A potential binding site for miR-29c-3p in EBLN3P. (B)
The luciferase activity of the wildtype and mutant EBLN3P reporters in NC mimics or miR-29c-3p mimics-transfected SiHa cell. (C) The enrichment of miR-29c-3p by
biotinylated NC or EBLN3P probe. (D) The enrichment of EBLN3P by biotinylated NC, wildtype or mutant miR-29c-3p prob. (E–G) The expression of EBLN3P and
miR-29c-3p in C-33A and SiHa cells transfected with NC mimics, miR-29c-3p mimics, sh-NC or sh-EBLN3P was analyzed via RT-qPCR. Results were represented as
mean ± SD from three independent experiments. ***P < 0.001.

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of miR-29c-3p largely reduced the luciferase activity of the wildtype highly enriched by the EBLN3P probe. Moreover, the EBLN3P was also
EBLN3P reporter but did not affect the activity of the mutant EBLN3P significantly enriched by the ALKBH5 and YTHDF2 antibodies (Sup­
reporter (Fig. 1B). Moreover, miR-29c-3p was significantly enriched by plementary Fig. 2D). Furthermore, we found decreased ALKBH5
biotinylated-EBLN3P (Fig. 1C). EBLN3P was efficiently pulled down by expression in cervical cancer tissues, and the expression of ALKBH5 and
biotinylated-miR-29c-3p, but biotinylated-mutant miR-29c-3p failed to EBLN3P was negatively correlated (Supplementary Figs. 2E and F).
enrich EBLN3P (Fig. 1D). miR-29c-3p was overexpressed and EBLN3P EBLN3P was downregulated in ALKBH5-overexpressing C-33A and SiHa
was knocked down in C-33A and SiHa cells through transfection of miR- cells (Supplementary Figs. 2G and H). Overexpression of ALKBH5
29c-3p mimics and sh-EBLN3P. We found that overexpression of miR- accelerated EBLN3P decay in C-33A and SiHa cells (Supplementary
29c-3p repressed EBLN3P expression, and knockdown of EBLN3P Fig. 2I). These data suggested that ALKBH5 might impair EBLN3P sta­
enhanced miR-29c-3p expression in C-33A and SiHa cells (Fig. 1E–G). bility to reduce its expression in cervical cancer.
These observations suggested a reciprocal negative regulation between
miR-29c-3p and EBLN3P in cervical cancer cells. 3.3. Depletion of miR-29c-3p abrogated EBLN3P silencing-mediated
suppression of proliferation, migration, and invasion of C-33A and SiHa
cells
3.2. EBLN3P was highly expressed and miR-29c-3p was downregulated in
cervical cancer
EBLN3P and miR-29c-3p were knocked down in C-33A and SiHa cells
to further explore their involvement in the regulation of cervical cancer
To explore the roles of EBLN3P and miR-29c-5p in cervical cancer,
cell malignancy. EBLN3P was significantly knocked down through sh-
we collected tumor tissues from cervical cancer patients and examined
EBLN3P transfection, but its expression was restored by depletion of
their expression. We found that EBLN3P was highly expressed in cervical
miR-29c-3p (Fig. 3A). miR-29c-3p was upregulated in EBLN3P-silencing
cancer tissues (Fig. 2A and B), and cervical cancer tissues showed
cells, and its expression was reduced by miR-29c-3p knockdown
decreased miR-29c-3p expression compared to adjacent normal tissues
(Fig. 3B). We analyzed cell proliferation through CCK-8 and colony
(Fig. 2C). Furthermore, miR-29c-3p expression was negatively corre­
formation assays. CCK-8 assays showed that silencing of EBLN3P
lated with EBLN3P expression in cancer tissues (Fig. 2D). In addition,
inhibited C-33A and SiHa cell proliferation, but the suppressive effect
increased EBLN3P expression was observed in cervical cancer cell lines
was abolished by miR-29c-3p knockdown (Fig. 3C). Moreover, the
including C-33A, HeLa, CaSki and SiHa cells (Fig. 2E). These results
colony-forming capacity of EBLN3P-silencing C-33A and SiHa cells was
showed increased EBLN3P expression and decreased miR-29c-3p
largely impaired, which was reversed by simultaneous depletion of miR-
expression in cervical cancer.
29c-3p (Fig. 3D).
N6-methyladenosine (m6A) modification is the most widespread
Furthermore, silencing of EBLN3P significantly inhibited migration
RNA modification that affects RNA stability, translation, slicing and
and invasion, whereas simultaneous depletion of miR-29c-3p abrogated
translocation [19]. The m6A modification of EBLN3P was predicted
these EBLN3P silencing-mediated suppressive effects (Fig. 4A and B).
using the SRAMP prediction server (http://www.cuilab.cn/sramp/,
Hence, our findings suggested that the tumor suppressive functions
Supplementary Fig. 2A). M6A is modified by the m6A writers, recog­
induced by EBLN3P knockdown might partially dependent on miR-29c-
nized by the m6A readers, and removed by the m6A erasers [19]. The
3p in cervical cancer.
m6A reader YTHDF2 and eraser ALKBH5 were predicted to be involved
in the m6A modification of EBLN3P (Supplementary Fig. 2B). Then, we
performed RNA pull-down and RIP assays to investigate their interac­
tion. As shown in Supplementary Fig. 2C, ALKBH5 and YTHDF2 could be

Fig. 2. EBLN3P was highly expressed and miR-29c-3p was downregulated in cervical cancer. (A–C) RT-qPCR analysis of EBLN3P and miR-29c-3p in tumor and
adjacent normal tissues from 44 patients with cervical cancer. The log2 of fold change was shown in B. (D) The expression of EBLN3P and miR-29c-3p was negatively
correlated in cervical cancer. (E) RT-qPCR analysis of EBLN3P in Ect1/E6E7, C-33A, HeLa, CaSki and SiHa cells. Results were represented as mean ± SD from three
independent experiments. **P < 0.01, ***P < 0.001.

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Fig. 3. Depletion of miR-29c-3p abrogated EBLN3P silencing-mediated anti-proliferative effects. C-33A and SiHa cells were divided into sh-NC + inhibitor NC,
sh-EBLN3P + inhibitor NC and sh-EBLN3P + miR-29c-3p inhibitor groups. (A and B) RT-qPCR analysis of EBLN3P and miR-29c-3p. (C and D) Cell proliferation was
examined with CCK-8 and colony formaation assays. Results were represented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P
< 0.001.

3.4. EBLN3P functioned as a ceRNA to promote RCC2 expression as a ceRNA for miR-29c-3p and relieved miR-29c-3p-mediated sup­
pression of RCC2 expression in cervical cancer.
We identified a potential binding site for miR-29c-3p in the 3′-un­
translated region of RCC2 mRNA (Fig. 5A), and overexpression of miR- 3.5. EBLN3P interacted with TAF15 to maintain RCC2 expression
29c-3p greatly reduced the luciferase activity of the wildtype RCC2 re­
porter but did not affect the activity of the mutant RCC2 reporter Besides acting as a ceRNA, lncRNAs can regulate gene expression via
(Fig. 5B). Moreover, RCC2 expression was suppressed by overexpression interacting with RNA binding proteins [20]. Although both cytoplasmic
of miR-29c-3p but promoted by knockdown of miR-29c-3p (Fig. 5C). RIP and nuclear localization of EBLN3P were observed, EBLN3P showed
assays showed that EBLN3P, miR-29c-3p and RCC2 mRNA were simul­ predominant cytoplasmic localization in C-33A and SiHa cells (Fig. 6A).
taneously enriched by the anti-Ago2 antibody (Fig. 5D). Furthermore, RNA pull-down assays showed that TAF15 could be efficiently pulled
knockdown of EBLN3P reduced RCC2 expression, but it was reversed by down by the sense EBLN3P probe, but the antisense EBLN3P probe failed
miR-29c-5p knockdown (Fig. 5E and F). In addition, RCC2 expression to enrich TAF15 (Fig. 6B). Moreover, both EBLN3P and RCC2 mRNA
was increased in tumor tissues (Fig. 5G). RCC2 expression was nega­ could be enriched by the anti-TAF15 antibody in C-33A and SiHa cells
tively correlated with miR-29c-3p expression but positively correlated (Fig. 6C), suggesting that the interaction of EBLN3P and TAF15 might
with EBLN3P expression in tumor tissues (Fig. 5H and I). Compared to regulate RCC2 expression. Furthermore, we confirmed that EBLN3P and
Ect1/E6E7 cells, C-33A, HeLa, CaSki and SiHa cells showed elevated TAF15 were colocalized in the cytoplasm of SiHa cells (Fig. 6D). C-33A
RCC2 expression. Collectively, these data suggested that EBLN3P acted and SiHa cells transfected with sh-TAF15 showed decreased expression

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Fig. 4. Depletion of miR-29c-3p abrogated EBLN3P silencing-mediated suppression of migration and invasion. C-33A and SiHa cells were divided into sh-NC
+ inhibitor NC, sh-EBLN3P + inhibitor NC and sh-EBLN3P + miR-29c-3p inhibitor groups. (A) The migration of C-33A and SiHa cells was determined via wound
healing assays. (B) Transwell assays were applied to analyze cell invasion. Results were represented as mean ± SD from three independent experiments. *P < 0.05,
**P < 0.01, ***P < 0.001.

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Fig. 5. EBLN3P functioned as a ceRNA to promote RCC2 expression. (A) A potential binding site for miR-29c-3p in the 3′-untranslated region of RCC2 mRNA. (B)
The luciferase activity of the wildtype and mutant RCC2 reporters in NC mimics or miR-29c-3p mimics-transfected SiHa cells. (C) RT-qPCR analysis of RCC2 in C-33A
and SiHa cells transfected with NC mimics, miR-29c-3p mimics, inhibitor NC or miR-29c-3p inhibitor. (D) RIP assays for analyzing the enrichment of EBLN3P, miR-
29c-3p and RCC2 by the anti-Ago2 antibody. (E and F) C-33A and SiHa cells were divided into sh-NC + inhibitor NC, sh-EBLN3P + inhibitor NC and sh-EBLN3P +
miR-29c-3p inhibitor groups. The expression of RCC2 was examined through RT-qPCR and Western blot. (G) RCC2 in tumor and adjacent normal tissues from
patients with cervical cancer was examined by RT-qPCR. (H and I) RCC2 expression was negatively correlated with miR-29c-3p expression but positively correlated
with EBLN3P expression in cervical cancer. (J) RT-qPCR analysis of RCC2 in Ect1/E6E7, C-33A, HeLa, CaSki and SiHa cells. Results were represented as mean ± SD
from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig. 6. EBLN3P interacted with TAF15 to maintain RCC2 expression. (A) The abundance of EBLN3P, U6 snRNA and GAPDH in the nucleus and cytoplasm from
C-33A and SiHa cells was determined with RT-qPCR. (B) TAF15 was pulled down by the sense or antisense EBLN3P probe and detected through Western blot. (C) RIP
assays for analyzing the enrichment of EBLN3P and RCC2 by the anti-TAF15 antibody. (D) Combined FISH and IF staining for analyzing the colocalization of EBLN3P
(red) and TAF15 (green) in SiHa cells. The nuclei were stained with DAPI (blue). Scale bar, 10 μm. (E–G) The expression of TAF15 and RCC2 in C-33A and SiHa cells
transfected with sh-NC, sh-TAF15#1 or sh-TAF15#2 was analyzed through RT-qPCR and Western blot. (H) RCC2 mRNA stability was detected by RT-qPCR in C-33A
and SiHa cells after actinomycin D treatment at 0, 2, 4, 6 and 8 h. Results were represented as mean ± SD from three independent experiments. *P < 0.05, **P <
0.01, ***P < 0.001.

of TAF15 and RCC2 (Fig. 6E–G). Knockdown of TAF15 and EBLN3P 3.6. MiR-29c-3p repressed proliferation, migration, and invasion through
accelerated the decay of RCC2 mRNA in C-33A and SiHa cells in downregulation of RCC2
response to actinomycin D (Fig. 6H). These observations implied that
EBLN3P promoted RCC2 expression via maintaining the stability of To further investigate whether miR-29c-3p regulates the malignancy
RCC2 mRNA by interacting with TAF15 in cervical cancer. of cervical cancer cells, miR-29c-3p and RCC2 were overexpressed in

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T. Zou et al. Archives of Biochemistry and Biophysics 755 (2024) 109980

SiHa cells through transfection. RCC2 expression was significantly inhibition of migration and invasion in SiHa cells (Fig. 7C and D). Be­
suppressed by overexpression of miR-29c-3p, but it was restored by sides, overexpression of miR-29c-3p reduced the abundance of RCC2
RCC2 overexpression (Fig. 7A). Overexpression of RCC2 promoted SiHa and phosphorylated JNK in SiHa cells, which was reversed by over­
cell proliferation, and miR-29c-3p overexpression-mediated inhibition expression of RCC2 (Fig. 7E). In conclusion, miR-29c-3p inhibited ma­
of cell proliferation was abrogated by overexpression of RCC2 (Fig. 7B). lignancy in cervical cancer by downregulating RCC2.
Furthermore, cell migration and invasion were reduced by over­
expression of miR-29c-3p but enhanced by overexpression of RCC2, and
RCC2 overexpression reversed miR-29c-3p overexpression-mediated

Fig. 7. MiR-29c-3p repressed proliferation, migration, and invasion through downregulation of RCC2 in cervical cancer cells. SiHa cells were divided into
NC mimics, miR-29c-3p mimics, NC mimics + RCC2 and miR-29c-3p mimics + RCC2 groups. (A) RT-qPCR analysis of RCC2. (B) Cell proliferation was examined with
CCK-8 assay. (C) Cell migration was determined via wound healing assays. (D) Transwell assays were applied to analyze cell invasion (n = 3). (E) RCC2, p-JNK and
JNK were detected through Western blot. Results were represented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

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3.7. Silencing of EBLN3P repressed tumor growth in mice largely depends on the stage of cancer and significantly drops for pa­
tients with distant metastasis cancer [24]. Thus, exploring the molecular
In order to further testify the modulatory role of EBLN3P in cervical mechanisms underlying the regulation of cervical cancer progression
cancer tumorigenesis, EBLN3P-silencing SiHa cells were subcutaneously contributes to understanding its pathogenesis and seeking potential
injected into nude mice for in vivo confirmation. The results indicated therapeutic targets. We previously reported that miR-29c-3p was
that EBLN3P depletion resulted in a remarkable reduction in the volume downregulated in cervical cancer, and miR-29c-3p suppressed prolifer­
and size of tumors, as compared with the control group (Fig. 8A and B). ation, migration, invasion, and metastasis but enhanced apoptosis in
As expected, silencing of EBLN3P inhibited the expression of EBLN3P cervical cancer via repressing SPARC expression [11]. However, the
and RCC2 but enhanced miR-29c-3p expression (Fig. 8C–E). As illus­ mechanism by which miR-29c-3p is regulated in cervical cancer is un­
trated in Fig. 8F, the levels of RCC2 examined by IHC staining in EBLN3P known. Here, we reported increased EBLN3P expression and a reciprocal
silencing group were lower than control group, which was consistent negative regulation between EBLN3P and miR-29c-3p in cervical cancer.
with the results from Western blot analysis (Fig. 8G). Briefly, these Silencing of EBLN3P inhibited cervical cancer cell proliferation,
findings demonstrated a promotive regulator of EBLN3P in the tumori­ migration, and invasion and reduced tumor growth by upregulating
genesis of cervical cancer in vivo. miR-29c-3p and downregulating RCC2. Mechanically, EBLN3P pro­
moted RCC2 expression through various mechanisms including acting as
4. Discussion a ceRNA for miR-29c-3p to relieve its suppression of RCC2 and inter­
acting with TAF15 to stabilize RCC2 mRNA stability.
Cervical cancer is a major type of gynecologic cancer that poses a LncRNA EBLN3P is a pseudogene of EBLN3 that is located on chro­
serious threat to women’s health worldwide. Thanks to the development mosome 9 and plays vital roles in regulating the progression of various
of HPV vaccines, early diagnosis and therapeutics, the incidence of cancers, but its mechanisms have not been well documented due to
cervical cancer constantly decreases, and the survival rate is increasing, limited studies. Yang et al. found that EBLN3P was overexpressed in T-
but the incidence and mortality still remain high in developing countries cell acute lymphoblastic leukemia, and its silencing repressed prolifer­
[21,22]. Patients in low-income countries often are diagnosed with ation, migration, and invasion of Jurcat cells but enhanced cell apoptosis
cervical cancer at advanced stages that greatly raises the difficulty for by sponging miR-655-3p [25]. EBLN3P facilitated osteosarcoma pro­
treatment and leads to poor prognosis [23]. The outcome of treatment gression by promoting proliferation, migration, and invasion via

Fig. 8. Silencing of EBLN3P repressed tumor growth in mice. SiHa cells were transfected with sh-NC or sh-EBLN3P and injected into nude mice, (N = 6 per
group). (A) Tumor volume. (B) The record of tumor weight, and tumors were excised and imaged. (C–E) RT-qPCR analysis of EBLN3P, miR-29c-3p and RCC2 in
tumor. (F) IHC staining of RCC2. (G) Detection of RCC2 protein levels by Western blot assay. Values were represented as mean ± SD. *P < 0.05, ***P < 0.001.

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sponging miR-224-5p and upregulating Rab10 [26]. Other studies have Institutional Animal Care guidelines of Guizhou Provincial People’s
suggested EBLN3P as an oncogene in colorectal cancer and hepatocel­ Hospital and approved by the Animal Care and Use Committee of
lular carcinoma via targeting miRNAs [14,15]. In consistence, we firstly Guizhou Provincial People’s Hospital.
reported increased EBLN3P expression in cervical cancer, and EBLN3P
promoted the progression of cervical cancer dependent on sponging 6. Consent for publication
miR-29-3p and subsequent RCC2 upregulation, supporting the notion
that EBLN3P was an oncogene in cervical cancer. According to current All participants were informed and gave written consent.
studies, functioning as a ceRNA to sponge miRNAs is the primary
mechanism by which EBLN3P exerts its effects [27,28]. Besides CRediT authorship contribution statement
sponging miRNAs, interacting with RNA-binding proteins to regulate
mRNA stability is also a major mechanism by which lncRNAs control the Ting Zou: Writing – original draft, Methodology, Data curation. Yan
expression of target genes [29,30]. However, whether EBLN3P regulates Gao: Formal analysis. Mingrong Qie: Conceptualization, Supervision,
gene expression through this mechanism has not been characterized. Writing – review & editing.
Intriguingly, we found colocalization of EBLN3P and a RNA binding
protein TAF15 in the cytoplasm, indicating a potential interaction.
Further analysis showed the interaction of EBLN3P and TAF15 in cer­ Declaration of competing interest
vical cancer cells, and EBLN3P stabilized RCC2 mRNA stability via
interacting with TAF15. To the best of our knowledge, it is the first time The authors declare that they have no conflict of interest.
to prove that EBLN3P simultaneously functions as a ceRNA and interacts
with TAF15 to affect gene expression. Acknowledgement
RCC2 is an important regulator of cell cycle and mitosis that belongs
to the regulator of chromatin condensation 1 family [31]. RCC2 has been This study is supported by Youth Foundation of Guizhou Provincial
reported to be overexpressed in cancers and accelerate cancer progres­ People’s Hospital (GZSYQN[2021]06), Foundation of Guizhou Provin­
sion [17]. RCC2 was upregulated in esophageal cancer and promoted its cial Health Commission (GZWKJ2021-317) and Guizhou Provincial
proliferation, migration, and growth through Sox2 [32]. Several reports Science and Technology Projects ([2021]450).
have suggested that abnormal RCC2 expression is essential to the pro­
gression of breast cancer [33], colon cancer [34] and lung cancer [35]. Appendix A. Supplementary data
Importantly, a recent study reported that RCC2 expression was pro­
moted by lncRNA HOTAIR, and RCC2 significantly enhanced the Supplementary data to this article can be found online at https://doi.
growth, migration, and invasion of cervical cancer cells [36]. In this org/10.1016/j.abb.2024.109980.
study, we found that EBLN3P upregulated RCC2 by sponging
miR-29c-3p, and overexpression of RCC2 abrogated miR-29c-3p over­ References
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