- Email: [email protected]
EBF1 feedback loop contributes to the progression of bladder cancer
Journal Pre-proof TMPO-AS1/miR-98-5p/EBF1 feedback loop contributes to the progression of bladder cancer Hua Luo (Conceptualization) (Methodology) (Software), Lu Yang (Investigation) (Resources), Chen Liu (Visualization) (Supervision) (Data curation), Xiaobo Wang (Formal analysis) (Writing - original draft), Qiang Dong (Writing - review and editing), Liangren Liu (Project administration) (Validation), Qiang Wei (Funding acquisition)
PII:
S1357-2725(20)30019-4
DOI:
https://doi.org/10.1016/j.biocel.2020.105702
Reference:
BC 105702
To appear in:
International Journal of Biochemistry and Cell Biology
Received Date:
23 August 2019
Revised Date:
20 January 2020
Accepted Date:
28 January 2020
Please cite this article as: Luo H, Yang L, Liu C, Wang X, Dong Q, Liu L, Wei Q, TMPO-AS1/miR-98-5p/EBF1 feedback loop contributes to the progression of bladder cancer, International Journal of Biochemistry and Cell Biology (2020), doi: https://doi.org/10.1016/j.biocel.2020.105702
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
TMPO-AS1/miR-98-5p/EBF1 feedback loop contributes to the progression of bladder cancer
Hua Luo1, 2, Lu Yang1, Chen Liu2, Xiaobo Wang2, Qiang Dong1, Liangren Liu1, Qiang Wei1, *
1
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Department of Urology, Institute of Urology, West China Hospital of
Sichuan University, No.37 Guoxue Alley, Chengdu, 610000, Sichuan, P.R. China. 2
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Deyang, 618000, Sichuan, P.R. China.
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Department of Urology, the Second Peoples Hospital of Deyang City,
*
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Corresponding to: Qiang Wei, Department of Urology, Institute of
Urology, West China Hospital of Sichuan University, No.37 Guoxue
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Alley, Chengdu, 610000, Sichuan, P.R. China.
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Email: [email protected].
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Highlights
Overexpressed TMPO-AS1 in BCa predicts a poor prognosis TMPO-AS1 promotes cell proliferation, migration, invasion and inhibits cell apoptosis in BCa The promoter of TMPO-AS1 binds to EBF1 TMPO-AS1 sponged miR-98-5p in BCa
MiR-98-5p targets to EBF1 TMPO-AS1/miR-98-5p/EBF1 axis promotes BCa progression
Abstract As reported in numerous studies, long non-coding RNAs (lncRNAs) exert significant effect on the regulation of tumor development. LncRNA
ro of
TMPO antisense RNA 1 (TMPO-AS1) has been confirmed to be
implicated in the development of several cancers. However, its clinical
significance is still largely unknown in bladder cancer (BCa). In this
-p
study, high expression of TMPO-AS1 was revealed in BCa tissues and
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cell lines, and TMPO-AS1 predicted poor prognosis. Moreover, TMPO-AS1 facilitated cell growth. Additionally, TMPO-AS1 also
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boosted the migration and invasion of BCa cells. Mechanistically,
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overexpressed EBF transcription factor 1 (EBF1) in BCa cell was verified to promote the transcription of TMPO-AS1. Later, we found that could sponge miR-98-5p.
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TMPO-AS1 was a cytoplasmic RNA and
Besides, it was validated that EBF1 is a target gene of miR-98-5p and
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negatively correlated with miR-98-5p in terms of expression level. According to the results of rescue experiments, we observed that EBF1 overexpression restored the repressive effect of TMPO-AS1 silencing on BCa development. Our research is the first to disclose the biological role and molecular mechanism of TMPO-AS1 in BCa, and TMPO-AS1 might
be identified as a new therapeutic target for BCa patients.
Keywords: TMPO-AS1; miR-98-5p; EBF1; bladder cancer
1. Introduction As a most commonly occurring cancer, bladder cancer (BCa) ranks the
ro of
tenth among the malignancies worldwide, the sixth in men and the 17th in
women in terms of incidence rate (Bray et al., 2018). Importantly, there is a notable increase in its incidence and mortality every year (Berdik, 2017;
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Dy et al., 2017). In despite of the improved medical level over the past
re
decades, the patients who are diagnosed with BCa still have an unimproved prognosis (Babjuk, 2017; Humphrey et al., 2016). The stage
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of disease is mainly responsible for the bad prognosis, but specific
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symptoms for BCa will not emerged in patients with BCa at early stage (Abufaraj et al., 2018; Babjuk, 2018). Therefore, finding the promising
BCa.
ur
therapeutic strategies is crucial for improving the clinical outcomes of
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Long non-coding RNAs (lncRNAs), longer than 200 nucleotides, are of importance in stabilizing their downstream mRNAs (Pefanis et al., 2015; Shi et al., 2013). The significance of lncRNAs has been highlighted with the booming growth of RNA genomics that have been understood in multiple human diseases, particularly in cancers (Cheng et al., 2013; Ma
et al., 2018). An increasing number of studies have indicated the regulatory role of lncRNAs in cancers (Andrew et al., 2015; Liu et al., 2017). For example, TUG1 is identified as a therapeutic biomarker in osteosarcoma and participates in the progression of osteosarcoma (Xie et al., 2018). HOXD-AS1 suppresses RUNX3 expression and thereby facilitates the proliferation and invasion of melanoma cells (Zhang, H. et
ro of
al., 2017). SUMO1P3 is an oncogene in colon cancer and drives its tumor
growth (Zhang, L.M. et al., 2017). Numerous studies have reported that lncRNAs, like ZEB2-AS1, MALAT1, DUXAP10, are closely involved in
-p
BCa development (Lv et al., 2018; Wu et al., 2017; Xie et al., 2017).
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Although TMPO-AS1 has been confirmed to exert oncogenic property in prostate cancer (Huang et al., 2018), the potential mechanismof
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TMPO-AS1 in BCa is still unclear and thus worth exploring.
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In this study, it was discovered that the transcription of TMPO-AS1 was activated by EBF1 protein. Besides, TMPO-AS1 sponged miR-98-5p to
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liberate EBF1 mRNA, thereby increasing EBF1 protein and promoting TMPO-AS1
transcription.
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TMPO-AS1/miR-98-5p/EBF1 progression of BCa.
2. Materials and methods 2.1 Tissue collection
This
discovery
feedback
loop
uncovered contributes
to
that the
40 fresh BCa tissues and paired adjacent normal (AN) tissues were obtained from patients hospitalized at West China Hospital of Sichuan University from 2013 to 2015. In a previous research, Aran et al strongly suggested that normal adjacent tumor tissue samples must be collected >2 cm from the tumor margin and/or must not contain tumor (Aran et al., 2017). Herein, the samples at 5cm from BCa tumor margin
ro of
were obtained as AN tissues which were then confirmed by three
histopathologists. All patients did not receive any preoperative treatment.
Tumor and AN specimen from resection surgery were instantly frozen at
-p
liquid nitrogen and then preserved at -80oC freezer until use. Written
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informed consents were obtained from all participants. This research was
of Sichuan University.
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2.2 Cell lines
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approved and supervised by the ethics committee of West China Hospital
Human normal bladder cell SV‑ HUC‑ 1 and human BCa cell lines (T24,
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UMUC3, 5637, J82) were acquired commercially from Shanghai Institute of Cell Biology (Shanghai, China) and kept under 5% CO2 at 37oC. All
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cells were incubated routinely in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco), 100 IU/mL penicillin and 100 IU/mL streptomycin. 2.3 Quantitative real-time PCR (qRT-PCR) TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was
employed to extract total RNA and whole procedure was conducted following the user guide. RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA) was applied for reverse-transcription from total RNA to cDNA. SYBR Green PCR Master Mix (Takara) was employed for quantitative PCR on Step-One Plus System (Applied Biosystems, Foster City, CA, USA). GAPDH was an internal control for lncRNA and
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mRNA, while U6 was an endogenous reference for miRNA. The relative quantification method (2-ΔΔCT) was applied for determination of quantitative variation.
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2.4 Transfection
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TMPO-AS1 or EBF1-specific short hairpin RNAs (shRNAs) and negative control (sh-NC) were constructed by RiboBio (Guangzhou,
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China). The whole length of EBF1 was sub-cloned into pcDNA3.1 vector
na
so that EBF1 was overexpressed. Empty vector acted as the negative control for pcDNA3.1/EBF1. To overexpress miR-98-5p, miR-98-5p
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construct were synthesized by GeneCopoecia (Guangzhou, China) with NC construct as negative control. Transfection was performed in UMUC3
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and J82 cells using Lipofectamine2000 kit (Invitrogen, Carlsbad, CA, USA) for 48 h. 2.5 CCK-8 assay Transfected UMUC3 and J82 cells were reaped and put into 96-well plates (2 × 103 cells/well), followed by cultivation for 0, 24, 48, 72 or 96
h. Later, 10 μL of Cell Counting Kit 8 reagent (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added and then cells were cultivated at 37oC. After 4 h of incubation, cell viability was estimated via examining the absorbance at 450 nm with a microplate reader. And then cell viability was estimated. 2.6 Colony formation assay
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Plate clone formation assay was applied to analyze the proliferative ability of indicated cells. UMUC3 and J82 cells (500 cells per well) treated with indicated transfection plasmids were harvested and planted
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into 6-well plates. The plates were cultured at 37oC for 14 days. Then,
2.7 TUNEL staining
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counting and recording manually.
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colonies were subjected to 0.1% crystal violet in methanol, followed by
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Transfected cells were fixed with 1% formaldehyde and permeabilized with Triton X-100. Then, cells were treated with dUTP-end labeling from
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Clontech (Mountain View, CA, USA). After nucleus staining with DAPI, cells were observed by fluorescence microscope (NIKON, Tokyo, Japan).
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Red represented apoptotic cells, blue represented the nuclei of total cells. The rate of apoptosis was calculated as TUNEL positive cells/total cells by Image pro-plus. 2.8 Wound-healing assay UMUC3 and J82 cells were first seeded in 96-well plates all night at 37oC.
The wound was scratched by sterile pipette tip. Subsequently, cells were washed in phosphate-buffered saline (PBS) and detected after 36 h. The change of scratch and the percentage of wound healing were calculated by Image J. 2.9 Transwell invasion assay 48 h post-transfection, cells were put into the top chamber of inserts with
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24 well plates and 8-μm pore polycarbonate filters (Corning Incorporated, Corning, NY, USA) coated with Matrigel membrane (BD Biosciences,
Franklin Lakes, NJ, USA). The invaded cells were then dyed in crystal
-p
violet and visualized through microscope (magnification × 200). Finally,
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stained cells were manually counted in five random fields and an average number was calculated using Image J software.
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2.10 Bioinformatics analysis
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To predict the upstream transcription factor of TMPO-AS1, we used UCSC (http://genome.ucsc.edu/) for searching the potential transcription
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factor that could bind to TMPO-AS1 promoter and JASPAR (http://jaspar.genereg.net/) for predicting binding sites. To predict the
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TMPO-AS1-binding
miRNAs,
we
applied
starBase
(http://starbase.sysu.edu.cn/index.php), resulting in a series of miRNAs that could interact with TMPO-AS1. 2.11 Luciferase reporter assay The wild-type or mutation of EBF1 binding sites to TMPO-AS1 promoter
was separately cloned to pGL3 luciferase reporter vector (Promega Corporation, Madison, WI, USA). J82 and UMUC3 cells in 96-well plates were co-transfected with above vectors and sh-EBF1#1/2 or sh-NC for 48 h. The reporter vectors pmirGLO-TMPO-AS1-WT and pmirGLO-EBF1-WT were generated via sub-cloning the predictive interacting sequences of miR-98-5p in the sequence of TMPO-AS1 or
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EBF1 into pmirGLO Luciferase miRNA Target Expression Vector
(Promega). Their mutant vectors were formed using the point mutations
of miR-98-5p seed region binding sites. These reporter vectors were
-p
transfected into cells with indicated transfection plasmids for 48 h. After
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transfection, the Luciferase Reporter Assay System (Promega) was applied. Relative luciferase activity was normalized to the Renilla
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2.12 ChIP assay
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luciferase internal control.
ChIP assay was conducted utilizing Magna ChIP and EZ-Magna ChIP Kit
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(Millipore) following the manufacturer’s instruction. Briefly, UMUC3 and J82 cells were fixed by formaldehyde and then treated with glycine to
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form DNA-protein cross-links. Afterwards, cells were lysed in lysis Buffer and then sonicated to break the DNA chains into small fragments and then antibodies against EBF1 and control normal immunoglobulin G (IgG)
(Millipore,
Bedford,
MA,
USA)
were
utilized
for
immunoprecipitation with the sonicated DNA. At length, precipitated
chromatin DNA was analyzed with qRT-PCR. 2.13 Nucleus-cytoplasm separation A PARIS™ kit (Thermo Fisher Scientific) was used for total RNA isolation. To separate nuclear and cytoplasmic fraction, RNAs from J82 and UMUC3 cells were isolated with Cytoplasmic and Nuclear RNA Purification Kit (Norgenbiotek, ON, Canada). RNA extracted from
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nuclear or cytoplasmic fractions was reversely transcribed into cDNA and then analyzed through qRT-PCR as above described. GAPDH and U6 RNA were tested as the control for cytoplasmic RNA and nuclear RNA.
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2.14 RIP assay
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Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit from Millipore was applied for this experiment based on the guidebook. Briefly,
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cells were lysed in a complete RIP buffer, and then cell extraction was
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cultured with magnetic beads bounded with anti-Ago2 (required for RISC) or anti-IgG antibody which was taken as control (Millipore). The
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retrieved RNA was assayed via qRT-PCR. 2.15 RNA pull-down
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Pierce™ RNA 3' End Desthiobiotinylation Kit (Thermo 20163) was used to label the biotinylation for RNAs. To test the interaction between miR-98-5p and TMPO-AS1 or EBF1, cells were transfected with 50 nM biotinylated miR-98-5p-WT or miR-98-5p-Mut (Bio-miR-98-5p-WT or Bio-miR-98-5p-Mut). 48 hours later, cells were harvested and subjected
to PBS washing. Afterwards, cells were lysed using a specific lysis buffer (Ambion, Austin, Texas, USA) for about 10 minutes. Then, M-280 streptavidin magnetic beads (S3762, Sigma, USA) were applied to incubate the lysate. Next, lysate was subjected to pre-coating of RNase-free BSA and yeast tRNA (TRNABAK-RO, Sigma, USA) to prevent non-specific binding of RNA and protein complexes, and
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followed by cultivation for 3 h at 4°C. Afterwards, these beads were washed by lysis buffer and low salt buffer. After purification of bound RNAs with Trizol, RT-qPCR was applied to determine RNA expression.
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2.16 Statistical analysis
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The data were obtained and then processed by SPSS 19.0 statistical software. Later, the processed data was imported to GraphPad Prism 6.0
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to generate graph. Overall survival of BCa patients was plotted by
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Kaplan-Meier method. Pearson’s correlation method was used for gene expression correlation. Continuous variables from three replications were
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exhibited as mean ± SD. Significance of differences between groups was evaluated by Student’s t test or one-way ANOVA, with the threshold of
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p-value less than 0.05.
3. Results 3.1 TMPO-AS1 predicts poor prognosis in BCa In order to determine the biological role of TMPO-AS1 in BCa, qRT-PCR
was utilized to examine TMPO-AS1 expression in two types of tissues. The results displayed that TMPO-AS1 expression was remarkably higher in tumor tissues (Fig. 1A). Then, TMPO-AS1 expression in BCa cell lines (T24, UMUC3, 5637, J82) was also examined, and SV-HUC-1 cell was treated as control. Data from qRT-PCR uncovered high expression of TMPO-AS1 was in BCa cells (Fig. 1B). According to clinical analysis
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shown in Table 1, there was a close association between TMPO-AS1 and
tumor size, tumor stage and tumor grade. Further, the analysis of survival
rate unveiled that BCa patients with high level of TMPO-AS1 presented a
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worse prognosis than those with low level of TMPO-AS1 (Fig. 1C).
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Besides, proportional hazards method analysis indicated an independent prognostic significance of TMPO-AS1 expression (Table 2). In a word,
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TMPO-AS1 predicts poor prognosis in BCa.
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3.2 TMPO-AS1 drives cell growth, migration and invasion in BCa In order to understand the function of TMPO-AS1 in BCa cell growth,
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loss-of-function assays were applied. Firstly, shRNAs targeting TMPO-AS1 (sh-TMPO-AS1#1/2) and negative control (sh-NC) were
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transfected into the UMUC3 and J82 cells. The decreased expression of TMPO-AS1 in the above cells was examined by qRT-PCR (Fig. 2A). The effect of TMPO-AS1 on the proliferation of UMUC3 and J82 cells was then analyzed by CCK-8 and colony formation assays. Results showed that TMPO-AS1 silencing notably suppressed BCa cell proliferative
ability (Fig. 2B-C). Next, data from TUNEL assay indicated that TMPO-AS1 depletion induced an increase in apoptosis rate (Fig. 2D). Later, wound healing assay demonstrated a reduced migration rate of BCa cells after knockdown of TMPO-AS1 (Fig. 2E). Subsequently, transwell assay was conducted to further evaluate the influence of TMPO-AS1 on the invasion. As expected, the number of invaded cells was notably
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reduced after the transfection of TMPO-AS1-specific shRNAs (Fig. 2F). Conclusively, TMPO-AS1 functions in BCa by promoting cell proliferation, migration and invasion.
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3.3 EBF1 protein binds to TMPO-AS1 promoter
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Based on bioinformatics analysis, we found that EBF1 might be an upstream gene of TMPO-AS1, and EBF1 has been reported as a
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transcription factor (Belarbi et al., 2018). Hence, we hypothesized that
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EBF1 might promote the transcription of TMPO-AS1 in BCa. To verify our hypothesis, EBF1 expression in BCa was firstly detected. As expected,
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EBF1 expression was quite high in BCa tissues and cells (Fig. 3A-B). Besides, we found that there was a positive relationship between
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TMPO-AS1 and EBF1 through Pearson’s correlation analysis (Fig. 3C). Further, we investigated the effect of TMPO-AS1 on EBF1 expression, the results depicted that EBF1 expression was significantly diminished in sh-TMPO-AS1-transfected BCa cells (Fig. 3D). Later, we knocked down EBF1 expression for the follow-up experiments. The results displayed
that EBF1 expression was markedly reduced with the transfection of sh-EBF1#1/2 (Fig. 3E). Subsequently, data from luciferase reporter assay revealed the repressive impact of EBF1 knockdown on the transcriptional activity of TMPO-AS1 promoter (Fig. 3F). According to ChIP assay, only the -1500~-2000 bp (upstream of the initiation start site) of TMPO-AS1 promoter region was revealed to be significantly enriched in EBF1
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antibody group (Fig. 3G). Then, the binding sequences between
TMPO-AS1 promoter and EBF1 protein were predicted by JASPAR and UCSC database. As shown in Fig. 3H, the predicted binding sites were at
-p
-1636~1646 bp upstream transcription start site (TSS). Besides, data from
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luciferase reporter assay suggested that the luciferase activity of wide type TMPO-AS1 promoter was evidently decreased upon EBF1
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knockdown, while no significant change was observed in the group of
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mutant TMPO-AS1 promoter (Fig. 3I). All the data confirmed that EBF1 protein binds to TMPO-AS1 promoter and acts as a transcription
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activator.
3.4 TMPO-AS1 sponges miR-98-5p in BCa
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To examine the potential mechanism by which TMPO-AS1 functioned in BCa, we firstly performed nucleus-cytoplasm separation assay, and observed that TMPO-AS1 was mainly distributed in cytoplasm, suggesting the regulation of TMPO-AS1 at post-transcriptional level (Fig. 4A). Increasing studies have manifested that lncRNA regulates cancer
progression through serving as a competitive endogenous RNA (ceRNA) (Cheng et al., 2017). Thus, starBase was used to search miRNAs that potentially bind with TMPO-AS1. Ten candidates (let-7i-5p, miR-370-5p, miR-4500,
let-7g-5p,
miR-670-3p,
miR-873-3p,
miR-98-5p,
miR-3150b-3p, miR-4784 and miR-383-5p) were screened out and subjected to RNA pull down assay. It showed that miR-98-5p was
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markedly enriched in the TMPO-AS1 probe compared to other miRNAs
(Fig. 4B). Therefore, miR-98-5p was used to conduct the following
experiments. As illustrated in Fig. 4C, a considerably decreased
-p
expression of miR-98-5p was found in BCa tissues and miR-98-5p
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expression was negatively associated with TMPO-AS1 expression. Moreover, miR-98-5p was down-regulated in BCa cells (Fig. 4D). In
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addition, miR-98-5p expression was signally elevated by silenced
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TMPO-AS1 (Fig. 4E). Furthermore, starBase predicted the binding sites of miR-98-5p on TMPO-AS1 (Fig. 4F). After miR-98-5p expression was
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increased by miR-98-5p construct in BCa cells (Fig. 4G), the luciferase activity of pmirGLO-TMPO-AS1-WT was evidently restrained, but the
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luciferase activity of pmirGLO-TMPO-AS1-Mut was not affected (Fig. 4H). Lastly, data from RNA pull down assay displayed that TMPO-AS1 exceedingly enriched by bio-miR-98-5p-WT compared with bio-NC and bio-miR-98-5p-Mut, further verifying the direct combination between TMPO-AS1 and miR-98-5p (Fig. 4I). In brief, TMPO-AS1 sponges
miR-98-5p in BCa. 3.5 MiR-98-5p targets EBF1 mRNA To further support the ceRNA mechanism of TMPO-AS1, we applied RIP assay and observed that TMPO-AS1, miR-98-5p and EBF1 mRNA bound to RISC complex in BCa cells (Fig. 5A). The binding site of miR-98-5p on EBF1 mRNA was presented in Fig. 5B. Additionally, TMPO-AS1
shown
in
Fig.
5D,
the
decreased
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expression was up-regulated for the subsequent assays (Fig. 5C). As luciferase
activity
of
pmirGLO-EBF1-WT caused by miR-98-5p upregulation was restored by
-p
the transfection of pcDNA3.1/TMPO-AS1, while no notable changes
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were found in that of pmirGLO-EBF1-Mut. Results of RNA pull down assay further unveiled the combination between miR-98-5p and EBF1
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mRNA (Fig. 5E). Later, the negative expression association between
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miR-98-5p and EBF1 was unveiled by Pearson’s correlation analysis (Fig. 5F). To further confirm this, qRT-PCR was utilized and data revealed that
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overexpressing miR-98-5p significantly down-regulated the expression of EBF1 (Fig. S1A). Then, EBF1 expression in UMUC3 and J82 cells was
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up-regulated for further study by transfecting pcDNA3.1/EBF1 (Fig. S1B). Results of colony formation presented that EBF1 overexpression rescued the inhibitive effect of up-regulated miR-98-5p on the proliferative capacity of BCa cells (Fig. S1C). Through TUNEL assay, we observed that the induced apoptosis caused by miR-98-5p overexpression
was reserved by transfecting pcDNA3.1/EBF1 (Fig. S1D). Furthermore, transwell assay indicated that overexpressed EBF1 recovered miR-98-5p overexpression-mediated repressive function on BCa cell invasion (Fig. S1E). All these findings indicated that miR-98-5p targets EBF1 mRNA. 3.6
TMPO-AS1/miR-98-5p/EBF1
axis
promotes
BCa
cell
role
of
proliferation, migration and invasion last,
we
planned
to
investigate
the
ro of
At
TMPO-AS1/miR-98-5p/EBF1 axis in the progression of BCa. And rescue
experiments were designed to reveal the effect of the above axis. Data
-p
from CCK-8 and colony formation manifested that EBF1 overexpression
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counteracted the repressed proliferation in TMPO-AS1-silenced cells (Fig. 6A-B). According to TUNEL assay, it was observed that TMPO-AS1
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silencing-mediated promotion on cell apoptosis was recovered by
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overexpression of EBF1 (Fig. 6C). Additionally, wound healing assay suggested that the suppressed migration caused by knockdown of
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TMPO-AS1 was rescued by the transfection of pcDNA3.1/EBF1 (Fig. 6D). According to transwell assay, it was discovered that up-regulated
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EBF1 reversed the restraining effect of silenced TMPO-AS1 on cell migration (Fig. 6E). Taken together, lncRNA TMPO-AS1 sponges miR-98-5p to liberate EBF1 mRNA, and thus increases EBF1 protein and enhances TMPO-AS1 transcription. This TMPO-AS1/miR-98-5p/EBF1 feedback loop promotes BCa cell progression (Fig. 7).
4. Discussion In recent years, accumulating evidence has implied that lncRNAs modulate gene expression in multiple biological processes, including chromatin modification, transcriptional and posttranscriptional regulation (Hon et al., 2017; Meng et al., 2015). For instance, HOTAIR and Xist
ro of
restrain gene regulation via interaction with PRC2 (Fu et al., 2017;
Yildirim et al., 2013). Additionally, increasing studies have indicated that lncRNA regulates cancer progression by acting as a ceRNA (Cheng et al., For
example,
LINC00460
functions
as
-p
2017).
a
ceRNA
in
re
nasopharyngeal carcinoma by sponging miR-149-5p and up-regulating IL6 to facilitate the tumorigenesis of nasopharyngeal carcinoma (Kong et
lP
al., 2018). LncRNA MYOSLID functions as a ceRNA in gastric cancer
na
and boosts cancer progression by targeting miR-29c-3p/MCL-1 axis (Han et al., 2019). Existing reports have verified the oncogenic role that
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TMPO-AS1 paly in prostate cancer (Huang et al., 2018), whereas its role in BCa has not been identified. Our research displayed that TMPO-AS1
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expression was up-regulated in BCa tissues and cells, and it predicted poor prognosis. Furthermore, TMPO-AS1 was validated to boost cell proliferation, migration, invasion and restrained cell apoptosis in BCa cells. All the above results confirmed that BCa progression was promoted by TMPO-AS1.
EBF transcription factor 1 (EBF1) has been reported as a transcription factor in a variety of diseases. For example, EBF1 has been certific ated to enhance the proliferation and hamper the apoptosis of bone marrow CD34+ cells in myelodysplastic syndrome via negatively regulating mitogen-activated protein kinase (Hou et al., 2018). Further, as a transcription factor, EBF1 protein has been revealed to transcriptionally
ro of
activate the expression of Aryl hydrocarbon receptor (AHR) to impair
human B lymphopoiesis (Li et al., 2017). This research revealed that
EBF1 was overexpressed in BCa tissues and cells. In addition, the
-p
expression of EBF1 was positively correlated with that of TMPO-AS1.
re
Importantly, EBF1 combined with TMPO-AS1 promoter to promote TMPO-AS1 transcription. To sum up, EBF1 functioned as a transcription
lP
factor in BCa and transcriptionally activated the transcription of
na
TMPO-AS1.
MicroRNAs (miRNAs) are a groups of small RNAs that cannot code
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proteins whereas bind to 3′ UTR of target genes to regulate target gene expression in human cancers (Stark et al., 2005; Tian et al., 2012). For
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instance, miR-675 binds to RB1 and negatively modifies its expression to promote glioma cell proliferation (Zheng et al., 2017). MiR-381 down-regulates the expression of SOX4 to suppress cell migration and invasion in gastric cancer (Zhang, M. et al., 2017). Interestingly, miRNAs are also sponged by lncRNAs and targets mRNAs. As reported, CRNDE
sponges miR-384 and up-regulates IRS1 to enhance cell proliferation and metastasis in pancreatic cancer (Wang et al., 2017). PCAT-1 sponges miR-129-5p to upregulate HMGB1 to facilitate the invasion and metastasis of hepatocellular carcinoma cells (Zhang, D. et al., 2017). MiR-98-5p has been validated as an anti-tumor gene in many cancers, such as pancreatic ductal adenocarcinoma (Fu et al., 2018), ovarian
ro of
cancer (Yang et al., 2018), hepatocellular carcinoma (Jiang et al., 2017) and so on. However, the role of miR-98-5p has not been studied in BCa.
In present study, miR-98-5p was sponged by TMPO-AS1, and showed a
-p
negative expression association with TMPO-AS1. Furthermore, EBF1
re
mRNA was uncovered as a target gene of miR-98-5p and present a reverse expression relationship with miR-98-5p. In line with rescue
lP
assays, overexpressed EBF1 could recover the promotion caused by
na
silenced TMPO-AS1 on BCa cell growth. In conclusion, this study unveiled that TMPO-AS1 is a tumor facilitator
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in the development of BCa and transcriptionally activated by EBF1 protein. Meanwhile, TMPO-AS1 contributes to BCa cell growth by
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sponging miR-98-5p and upregulating EBF1. This discovery indicated that TMPO-AS1/miR-98-5p/EBF1 positive feedback loop might be helpful for the treatment of patients suffered from BCa.
Availability of data
Not applicable.
Consent for publication The publication of this worked has received the permission of all authors.
Author’s contribution
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Hua Luo: Conceptualization, Methodology, Software; Lu Yang: Investigation, Resources;
Chen Liu: Visualization, Supervision, Data curation;
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Qiang Dong: Writing - review & editing;
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Xiaobo Wang: Formal analysis, Writing - original draft;
Liangren Liu: Project administration, Validation;
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Qiang Wei: Funding acquisition.
Conflicts of interests
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None exist. Funding
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The project of Sichuan Deyang science and technology bureau (2017SZ097-1)
Acknowledgement
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We appreciate all participants involved in this experiment.
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Figure legends Figure 1 TMPO-AS1 predicts poor prognosis in BCa. (A) The expression of TMPO-AS1 in BCa tissues and paired non-tumor tissues. (B) TMPO-AS1 expression in BCa cell lines (T24, UMUC3, 5637, J82) and normal bladder cell line (SV-HUC-1). (C) The correlation between
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TMPO-AS1 expression and overall survival rate was assessed by
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Kaplan-Meier survival analysis (P = 0.013). **P < 0.01.
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Figure 2 TMPO-AS1 drives cell proliferation, migration and invasion, and inhibits cell apoptosis in BCa.
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(A) The transfection efficiency of sh-TMPO-AS1#1/#2 relative to sh-NC was showed. (B-C) The effect of TMPO-AS1 silencing on
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cell proliferation was unveiled by CCK-8 and colony formation. (D) TUNEL assay indicated the role of TMPO-AS1 knockdown in cell apoptosis. Green, apoptotic cells; blue, DAPI stained nuclei. Scale bar = 100μm. (E) Wound healing assay demonstrated the migration caused by knockdown of TMPO-AS1. Scale bar =
100μm. (F) The effect of TMPO-AS1 depletion on cell invasion was displayed through transwell assay. Scale bar = 50μm. **P <
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0.01.
Figure 3 EBF1 protein binds to TMPO-AS1 promoter. (A) EBF1 mRNA expression in BCa tissues and matched normal tissues. (B) EBF1 mRNA expression in BCa cell lines (T24, UMUC3, 5637, J82) and
normal cell line (SV-HUC-1). (C) Expression relation between TMPO-AS1 and EBF1 mRNA in BCa samples was showed by Pearson’s correlation analysis. (D) EBF1 mRNA expression in sh-TMPO-AS1#1/2 transfected cells was determined. (E) EBF1 mRNA expression was examined after transfecting with sh-EBF1#1/#2. sh-NC was used as negative control. (F) The luciferase reporter assay was conducted to
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determine the interaction between EBF1 mRNA and five section of
TMPO-AS1 promoter from -2000bp of TSS. (G) ChIP assay confirmed
the combination between EBF1 protein and TMPO-AS1 promoter. (H)
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The DNA motif of EBF1 and binding site between EBF1 and TMPO-AS1
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promoter was showed. (I) Luciferase reporter assay further verified that
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EBF1 mRNA interacted with TMPO-AS1 promoter. **P < 0.01.
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Figure 4 TMPO-AS1 sponges miR-98-5p in BCa. (A) The location of
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TMPO-AS1 was certified via nucleus-cytoplasm separation assay. (B) RNA pull down assay was employed to screen out the potential miRNAs interacted with TMPO-AS1 by using control probe or TMPO-AS1 probe to assess the relative enrichment of miRNAs. (C) The expression of miR-98-5p in BCa tissues and the association with TMPO-AS1 was
respectively determined by qRT-PCR and Pearson’s correlation analysis. (D) MiR-98-5p expression in BCa cell lines was evaluated by qRT-PCR. (E) MiR-98-5p expression in cells transfected with sh-TMPO-AS1 was displayed. (F) The binding sites between TMPO-AS1 and miR-98-5p were predicted by starBase. (G) The transfection efficiency of miR-98-5p construct was assessed. (H) Luciferase reporter assay confirmed the
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combination between TMPO-AS1 and miR-98-5p. (I) Interaction between TMPO-AS1 and miR-98-5p was confirmed by RNA pull down
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assay. *P < 0.05, **P < 0.01.
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Figure 5 MiR-98-5p targets EBF1 mRNA. (A) Ago2-RIP assay validated that TMPO-AS1, miR-98-5p and EBF1 mRNA were enriched in RISC. (B) The binding site between miR-98-5p and EBF1 mRNA was predicted. (C) TMPO-AS1 expression was tested in cells transfected with pcDNA3.1/TMPO-AS1. (D) The interaction between miR-98-5p and TMPO-AS1 (or EBF1) was implied with luciferase reporter assay. (E)
RNA pull down assay verified the interaction between miR-98-5p and EBF1 mRNA. (F) Negative expression association between miR-98-5p and EBF1 mRNA in BCa samples was showed by Pearson’s correlation
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analysis. **P < 0.01.
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Figure 6 TMPO-AS1/miR-98-5p/EBF1 axis promotes BCa cell proliferation, migration and invasion. (A-B) Cell proliferation was estimated via CCK-8 and colony formation. (C) TUNEL assay was conducted to estimate cell apoptosis. Green, apoptotic cells; blue, DAPI stained nuclei. Scale bar = 100μm. (D) Wound healing assay was employed to evaluate cell migration. Scale bar = 100μm. (E) Transwell
was utilized to analyze cell invasion. Scale bar = 50μm. *P < 0.05, **P <
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0.01.
Figure 7 Role of TMPO-AS1/miR-98-5p/EBF1 feedback loop in BCa was illustrated.
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Table1. Correlation between lncRNA TMPO-AS1 Expression and Clinical Features. (n=40) lncRNA TMPO-AS1
Variable
low
high
<60
12
15
≥60
8
5
Male
15
13
Female
5
7
Tumor size (cm) <4
13
3
≥4
7
17
15 5
5 15
Low
10
2
High
10
18
P-value
Age 0.501
Sex
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0.731
0.003**
Tumor stage
Tumor grade
0.004**
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<=T1 T2-T4
0.014*
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Low/high by the sample median. Pearson χ2 test. *P<0.05, **P<0.01 was considered to be statistically significant.
Table 2. Multivariate analysis of prognostic parameters in patients with BCa by Cox regression analysis Category
HR
Age
<60
0.796
CI(95%)
P-value
0.427-1.48
0.473
0.879
0.451-1.71
0.704
0.878
0.401-1.92
2.686
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Variable
≥60 Male
Sex
Tumor Size
<4
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Female
≥4 Tumor Stage
≤T1
Low
0.495
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Tumor Grade
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T2-T4
1.192-6.05
0.745
0.017*
0.232-1.05
0.069
1.27-6.121
0.011*
High Low
2.788
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TMPO-AS1
High
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Proportional hazards method analysis showed a positive, independent prognostic
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importance of TMPO-AS1 expression (P = 0.011*), in addition to the independent prognostic impact of Tumor Stage (P =0.017*). *P < 0.05 was considered statistically significant.
PII:
S1357-2725(20)30019-4
DOI:
https://doi.org/10.1016/j.biocel.2020.105702
Reference:
BC 105702
To appear in:
International Journal of Biochemistry and Cell Biology
Received Date:
23 August 2019
Revised Date:
20 January 2020
Accepted Date:
28 January 2020
Please cite this article as: Luo H, Yang L, Liu C, Wang X, Dong Q, Liu L, Wei Q, TMPO-AS1/miR-98-5p/EBF1 feedback loop contributes to the progression of bladder cancer, International Journal of Biochemistry and Cell Biology (2020), doi: https://doi.org/10.1016/j.biocel.2020.105702
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
TMPO-AS1/miR-98-5p/EBF1 feedback loop contributes to the progression of bladder cancer
Hua Luo1, 2, Lu Yang1, Chen Liu2, Xiaobo Wang2, Qiang Dong1, Liangren Liu1, Qiang Wei1, *
1
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Department of Urology, Institute of Urology, West China Hospital of
Sichuan University, No.37 Guoxue Alley, Chengdu, 610000, Sichuan, P.R. China. 2
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Deyang, 618000, Sichuan, P.R. China.
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Department of Urology, the Second Peoples Hospital of Deyang City,
*
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Corresponding to: Qiang Wei, Department of Urology, Institute of
Urology, West China Hospital of Sichuan University, No.37 Guoxue
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Alley, Chengdu, 610000, Sichuan, P.R. China.
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Email: [email protected].
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Highlights
Overexpressed TMPO-AS1 in BCa predicts a poor prognosis TMPO-AS1 promotes cell proliferation, migration, invasion and inhibits cell apoptosis in BCa The promoter of TMPO-AS1 binds to EBF1 TMPO-AS1 sponged miR-98-5p in BCa
MiR-98-5p targets to EBF1 TMPO-AS1/miR-98-5p/EBF1 axis promotes BCa progression
Abstract As reported in numerous studies, long non-coding RNAs (lncRNAs) exert significant effect on the regulation of tumor development. LncRNA
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TMPO antisense RNA 1 (TMPO-AS1) has been confirmed to be
implicated in the development of several cancers. However, its clinical
significance is still largely unknown in bladder cancer (BCa). In this
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study, high expression of TMPO-AS1 was revealed in BCa tissues and
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cell lines, and TMPO-AS1 predicted poor prognosis. Moreover, TMPO-AS1 facilitated cell growth. Additionally, TMPO-AS1 also
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boosted the migration and invasion of BCa cells. Mechanistically,
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overexpressed EBF transcription factor 1 (EBF1) in BCa cell was verified to promote the transcription of TMPO-AS1. Later, we found that could sponge miR-98-5p.
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TMPO-AS1 was a cytoplasmic RNA and
Besides, it was validated that EBF1 is a target gene of miR-98-5p and
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negatively correlated with miR-98-5p in terms of expression level. According to the results of rescue experiments, we observed that EBF1 overexpression restored the repressive effect of TMPO-AS1 silencing on BCa development. Our research is the first to disclose the biological role and molecular mechanism of TMPO-AS1 in BCa, and TMPO-AS1 might
be identified as a new therapeutic target for BCa patients.
Keywords: TMPO-AS1; miR-98-5p; EBF1; bladder cancer
1. Introduction As a most commonly occurring cancer, bladder cancer (BCa) ranks the
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tenth among the malignancies worldwide, the sixth in men and the 17th in
women in terms of incidence rate (Bray et al., 2018). Importantly, there is a notable increase in its incidence and mortality every year (Berdik, 2017;
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Dy et al., 2017). In despite of the improved medical level over the past
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decades, the patients who are diagnosed with BCa still have an unimproved prognosis (Babjuk, 2017; Humphrey et al., 2016). The stage
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of disease is mainly responsible for the bad prognosis, but specific
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symptoms for BCa will not emerged in patients with BCa at early stage (Abufaraj et al., 2018; Babjuk, 2018). Therefore, finding the promising
BCa.
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therapeutic strategies is crucial for improving the clinical outcomes of
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Long non-coding RNAs (lncRNAs), longer than 200 nucleotides, are of importance in stabilizing their downstream mRNAs (Pefanis et al., 2015; Shi et al., 2013). The significance of lncRNAs has been highlighted with the booming growth of RNA genomics that have been understood in multiple human diseases, particularly in cancers (Cheng et al., 2013; Ma
et al., 2018). An increasing number of studies have indicated the regulatory role of lncRNAs in cancers (Andrew et al., 2015; Liu et al., 2017). For example, TUG1 is identified as a therapeutic biomarker in osteosarcoma and participates in the progression of osteosarcoma (Xie et al., 2018). HOXD-AS1 suppresses RUNX3 expression and thereby facilitates the proliferation and invasion of melanoma cells (Zhang, H. et
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al., 2017). SUMO1P3 is an oncogene in colon cancer and drives its tumor
growth (Zhang, L.M. et al., 2017). Numerous studies have reported that lncRNAs, like ZEB2-AS1, MALAT1, DUXAP10, are closely involved in
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BCa development (Lv et al., 2018; Wu et al., 2017; Xie et al., 2017).
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Although TMPO-AS1 has been confirmed to exert oncogenic property in prostate cancer (Huang et al., 2018), the potential mechanismof
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TMPO-AS1 in BCa is still unclear and thus worth exploring.
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In this study, it was discovered that the transcription of TMPO-AS1 was activated by EBF1 protein. Besides, TMPO-AS1 sponged miR-98-5p to
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liberate EBF1 mRNA, thereby increasing EBF1 protein and promoting TMPO-AS1
transcription.
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TMPO-AS1/miR-98-5p/EBF1 progression of BCa.
2. Materials and methods 2.1 Tissue collection
This
discovery
feedback
loop
uncovered contributes
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that the
40 fresh BCa tissues and paired adjacent normal (AN) tissues were obtained from patients hospitalized at West China Hospital of Sichuan University from 2013 to 2015. In a previous research, Aran et al strongly suggested that normal adjacent tumor tissue samples must be collected >2 cm from the tumor margin and/or must not contain tumor (Aran et al., 2017). Herein, the samples at 5cm from BCa tumor margin
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were obtained as AN tissues which were then confirmed by three
histopathologists. All patients did not receive any preoperative treatment.
Tumor and AN specimen from resection surgery were instantly frozen at
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liquid nitrogen and then preserved at -80oC freezer until use. Written
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informed consents were obtained from all participants. This research was
of Sichuan University.
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2.2 Cell lines
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approved and supervised by the ethics committee of West China Hospital
Human normal bladder cell SV‑ HUC‑ 1 and human BCa cell lines (T24,
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UMUC3, 5637, J82) were acquired commercially from Shanghai Institute of Cell Biology (Shanghai, China) and kept under 5% CO2 at 37oC. All
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cells were incubated routinely in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco), 100 IU/mL penicillin and 100 IU/mL streptomycin. 2.3 Quantitative real-time PCR (qRT-PCR) TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was
employed to extract total RNA and whole procedure was conducted following the user guide. RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA) was applied for reverse-transcription from total RNA to cDNA. SYBR Green PCR Master Mix (Takara) was employed for quantitative PCR on Step-One Plus System (Applied Biosystems, Foster City, CA, USA). GAPDH was an internal control for lncRNA and
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mRNA, while U6 was an endogenous reference for miRNA. The relative quantification method (2-ΔΔCT) was applied for determination of quantitative variation.
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2.4 Transfection
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TMPO-AS1 or EBF1-specific short hairpin RNAs (shRNAs) and negative control (sh-NC) were constructed by RiboBio (Guangzhou,
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China). The whole length of EBF1 was sub-cloned into pcDNA3.1 vector
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so that EBF1 was overexpressed. Empty vector acted as the negative control for pcDNA3.1/EBF1. To overexpress miR-98-5p, miR-98-5p
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construct were synthesized by GeneCopoecia (Guangzhou, China) with NC construct as negative control. Transfection was performed in UMUC3
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and J82 cells using Lipofectamine2000 kit (Invitrogen, Carlsbad, CA, USA) for 48 h. 2.5 CCK-8 assay Transfected UMUC3 and J82 cells were reaped and put into 96-well plates (2 × 103 cells/well), followed by cultivation for 0, 24, 48, 72 or 96
h. Later, 10 μL of Cell Counting Kit 8 reagent (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added and then cells were cultivated at 37oC. After 4 h of incubation, cell viability was estimated via examining the absorbance at 450 nm with a microplate reader. And then cell viability was estimated. 2.6 Colony formation assay
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Plate clone formation assay was applied to analyze the proliferative ability of indicated cells. UMUC3 and J82 cells (500 cells per well) treated with indicated transfection plasmids were harvested and planted
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into 6-well plates. The plates were cultured at 37oC for 14 days. Then,
2.7 TUNEL staining
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counting and recording manually.
re
colonies were subjected to 0.1% crystal violet in methanol, followed by
na
Transfected cells were fixed with 1% formaldehyde and permeabilized with Triton X-100. Then, cells were treated with dUTP-end labeling from
ur
Clontech (Mountain View, CA, USA). After nucleus staining with DAPI, cells were observed by fluorescence microscope (NIKON, Tokyo, Japan).
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Red represented apoptotic cells, blue represented the nuclei of total cells. The rate of apoptosis was calculated as TUNEL positive cells/total cells by Image pro-plus. 2.8 Wound-healing assay UMUC3 and J82 cells were first seeded in 96-well plates all night at 37oC.
The wound was scratched by sterile pipette tip. Subsequently, cells were washed in phosphate-buffered saline (PBS) and detected after 36 h. The change of scratch and the percentage of wound healing were calculated by Image J. 2.9 Transwell invasion assay 48 h post-transfection, cells were put into the top chamber of inserts with
ro of
24 well plates and 8-μm pore polycarbonate filters (Corning Incorporated, Corning, NY, USA) coated with Matrigel membrane (BD Biosciences,
Franklin Lakes, NJ, USA). The invaded cells were then dyed in crystal
-p
violet and visualized through microscope (magnification × 200). Finally,
re
stained cells were manually counted in five random fields and an average number was calculated using Image J software.
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2.10 Bioinformatics analysis
na
To predict the upstream transcription factor of TMPO-AS1, we used UCSC (http://genome.ucsc.edu/) for searching the potential transcription
ur
factor that could bind to TMPO-AS1 promoter and JASPAR (http://jaspar.genereg.net/) for predicting binding sites. To predict the
Jo
TMPO-AS1-binding
miRNAs,
we
applied
starBase
(http://starbase.sysu.edu.cn/index.php), resulting in a series of miRNAs that could interact with TMPO-AS1. 2.11 Luciferase reporter assay The wild-type or mutation of EBF1 binding sites to TMPO-AS1 promoter
was separately cloned to pGL3 luciferase reporter vector (Promega Corporation, Madison, WI, USA). J82 and UMUC3 cells in 96-well plates were co-transfected with above vectors and sh-EBF1#1/2 or sh-NC for 48 h. The reporter vectors pmirGLO-TMPO-AS1-WT and pmirGLO-EBF1-WT were generated via sub-cloning the predictive interacting sequences of miR-98-5p in the sequence of TMPO-AS1 or
ro of
EBF1 into pmirGLO Luciferase miRNA Target Expression Vector
(Promega). Their mutant vectors were formed using the point mutations
of miR-98-5p seed region binding sites. These reporter vectors were
-p
transfected into cells with indicated transfection plasmids for 48 h. After
re
transfection, the Luciferase Reporter Assay System (Promega) was applied. Relative luciferase activity was normalized to the Renilla
na
2.12 ChIP assay
lP
luciferase internal control.
ChIP assay was conducted utilizing Magna ChIP and EZ-Magna ChIP Kit
ur
(Millipore) following the manufacturer’s instruction. Briefly, UMUC3 and J82 cells were fixed by formaldehyde and then treated with glycine to
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form DNA-protein cross-links. Afterwards, cells were lysed in lysis Buffer and then sonicated to break the DNA chains into small fragments and then antibodies against EBF1 and control normal immunoglobulin G (IgG)
(Millipore,
Bedford,
MA,
USA)
were
utilized
for
immunoprecipitation with the sonicated DNA. At length, precipitated
chromatin DNA was analyzed with qRT-PCR. 2.13 Nucleus-cytoplasm separation A PARIS™ kit (Thermo Fisher Scientific) was used for total RNA isolation. To separate nuclear and cytoplasmic fraction, RNAs from J82 and UMUC3 cells were isolated with Cytoplasmic and Nuclear RNA Purification Kit (Norgenbiotek, ON, Canada). RNA extracted from
ro of
nuclear or cytoplasmic fractions was reversely transcribed into cDNA and then analyzed through qRT-PCR as above described. GAPDH and U6 RNA were tested as the control for cytoplasmic RNA and nuclear RNA.
-p
2.14 RIP assay
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Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit from Millipore was applied for this experiment based on the guidebook. Briefly,
lP
cells were lysed in a complete RIP buffer, and then cell extraction was
na
cultured with magnetic beads bounded with anti-Ago2 (required for RISC) or anti-IgG antibody which was taken as control (Millipore). The
ur
retrieved RNA was assayed via qRT-PCR. 2.15 RNA pull-down
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Pierce™ RNA 3' End Desthiobiotinylation Kit (Thermo 20163) was used to label the biotinylation for RNAs. To test the interaction between miR-98-5p and TMPO-AS1 or EBF1, cells were transfected with 50 nM biotinylated miR-98-5p-WT or miR-98-5p-Mut (Bio-miR-98-5p-WT or Bio-miR-98-5p-Mut). 48 hours later, cells were harvested and subjected
to PBS washing. Afterwards, cells were lysed using a specific lysis buffer (Ambion, Austin, Texas, USA) for about 10 minutes. Then, M-280 streptavidin magnetic beads (S3762, Sigma, USA) were applied to incubate the lysate. Next, lysate was subjected to pre-coating of RNase-free BSA and yeast tRNA (TRNABAK-RO, Sigma, USA) to prevent non-specific binding of RNA and protein complexes, and
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followed by cultivation for 3 h at 4°C. Afterwards, these beads were washed by lysis buffer and low salt buffer. After purification of bound RNAs with Trizol, RT-qPCR was applied to determine RNA expression.
-p
2.16 Statistical analysis
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The data were obtained and then processed by SPSS 19.0 statistical software. Later, the processed data was imported to GraphPad Prism 6.0
lP
to generate graph. Overall survival of BCa patients was plotted by
na
Kaplan-Meier method. Pearson’s correlation method was used for gene expression correlation. Continuous variables from three replications were
ur
exhibited as mean ± SD. Significance of differences between groups was evaluated by Student’s t test or one-way ANOVA, with the threshold of
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p-value less than 0.05.
3. Results 3.1 TMPO-AS1 predicts poor prognosis in BCa In order to determine the biological role of TMPO-AS1 in BCa, qRT-PCR
was utilized to examine TMPO-AS1 expression in two types of tissues. The results displayed that TMPO-AS1 expression was remarkably higher in tumor tissues (Fig. 1A). Then, TMPO-AS1 expression in BCa cell lines (T24, UMUC3, 5637, J82) was also examined, and SV-HUC-1 cell was treated as control. Data from qRT-PCR uncovered high expression of TMPO-AS1 was in BCa cells (Fig. 1B). According to clinical analysis
ro of
shown in Table 1, there was a close association between TMPO-AS1 and
tumor size, tumor stage and tumor grade. Further, the analysis of survival
rate unveiled that BCa patients with high level of TMPO-AS1 presented a
-p
worse prognosis than those with low level of TMPO-AS1 (Fig. 1C).
re
Besides, proportional hazards method analysis indicated an independent prognostic significance of TMPO-AS1 expression (Table 2). In a word,
lP
TMPO-AS1 predicts poor prognosis in BCa.
na
3.2 TMPO-AS1 drives cell growth, migration and invasion in BCa In order to understand the function of TMPO-AS1 in BCa cell growth,
ur
loss-of-function assays were applied. Firstly, shRNAs targeting TMPO-AS1 (sh-TMPO-AS1#1/2) and negative control (sh-NC) were
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transfected into the UMUC3 and J82 cells. The decreased expression of TMPO-AS1 in the above cells was examined by qRT-PCR (Fig. 2A). The effect of TMPO-AS1 on the proliferation of UMUC3 and J82 cells was then analyzed by CCK-8 and colony formation assays. Results showed that TMPO-AS1 silencing notably suppressed BCa cell proliferative
ability (Fig. 2B-C). Next, data from TUNEL assay indicated that TMPO-AS1 depletion induced an increase in apoptosis rate (Fig. 2D). Later, wound healing assay demonstrated a reduced migration rate of BCa cells after knockdown of TMPO-AS1 (Fig. 2E). Subsequently, transwell assay was conducted to further evaluate the influence of TMPO-AS1 on the invasion. As expected, the number of invaded cells was notably
ro of
reduced after the transfection of TMPO-AS1-specific shRNAs (Fig. 2F). Conclusively, TMPO-AS1 functions in BCa by promoting cell proliferation, migration and invasion.
-p
3.3 EBF1 protein binds to TMPO-AS1 promoter
re
Based on bioinformatics analysis, we found that EBF1 might be an upstream gene of TMPO-AS1, and EBF1 has been reported as a
lP
transcription factor (Belarbi et al., 2018). Hence, we hypothesized that
na
EBF1 might promote the transcription of TMPO-AS1 in BCa. To verify our hypothesis, EBF1 expression in BCa was firstly detected. As expected,
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EBF1 expression was quite high in BCa tissues and cells (Fig. 3A-B). Besides, we found that there was a positive relationship between
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TMPO-AS1 and EBF1 through Pearson’s correlation analysis (Fig. 3C). Further, we investigated the effect of TMPO-AS1 on EBF1 expression, the results depicted that EBF1 expression was significantly diminished in sh-TMPO-AS1-transfected BCa cells (Fig. 3D). Later, we knocked down EBF1 expression for the follow-up experiments. The results displayed
that EBF1 expression was markedly reduced with the transfection of sh-EBF1#1/2 (Fig. 3E). Subsequently, data from luciferase reporter assay revealed the repressive impact of EBF1 knockdown on the transcriptional activity of TMPO-AS1 promoter (Fig. 3F). According to ChIP assay, only the -1500~-2000 bp (upstream of the initiation start site) of TMPO-AS1 promoter region was revealed to be significantly enriched in EBF1
ro of
antibody group (Fig. 3G). Then, the binding sequences between
TMPO-AS1 promoter and EBF1 protein were predicted by JASPAR and UCSC database. As shown in Fig. 3H, the predicted binding sites were at
-p
-1636~1646 bp upstream transcription start site (TSS). Besides, data from
re
luciferase reporter assay suggested that the luciferase activity of wide type TMPO-AS1 promoter was evidently decreased upon EBF1
lP
knockdown, while no significant change was observed in the group of
na
mutant TMPO-AS1 promoter (Fig. 3I). All the data confirmed that EBF1 protein binds to TMPO-AS1 promoter and acts as a transcription
ur
activator.
3.4 TMPO-AS1 sponges miR-98-5p in BCa
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To examine the potential mechanism by which TMPO-AS1 functioned in BCa, we firstly performed nucleus-cytoplasm separation assay, and observed that TMPO-AS1 was mainly distributed in cytoplasm, suggesting the regulation of TMPO-AS1 at post-transcriptional level (Fig. 4A). Increasing studies have manifested that lncRNA regulates cancer
progression through serving as a competitive endogenous RNA (ceRNA) (Cheng et al., 2017). Thus, starBase was used to search miRNAs that potentially bind with TMPO-AS1. Ten candidates (let-7i-5p, miR-370-5p, miR-4500,
let-7g-5p,
miR-670-3p,
miR-873-3p,
miR-98-5p,
miR-3150b-3p, miR-4784 and miR-383-5p) were screened out and subjected to RNA pull down assay. It showed that miR-98-5p was
ro of
markedly enriched in the TMPO-AS1 probe compared to other miRNAs
(Fig. 4B). Therefore, miR-98-5p was used to conduct the following
experiments. As illustrated in Fig. 4C, a considerably decreased
-p
expression of miR-98-5p was found in BCa tissues and miR-98-5p
re
expression was negatively associated with TMPO-AS1 expression. Moreover, miR-98-5p was down-regulated in BCa cells (Fig. 4D). In
lP
addition, miR-98-5p expression was signally elevated by silenced
na
TMPO-AS1 (Fig. 4E). Furthermore, starBase predicted the binding sites of miR-98-5p on TMPO-AS1 (Fig. 4F). After miR-98-5p expression was
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increased by miR-98-5p construct in BCa cells (Fig. 4G), the luciferase activity of pmirGLO-TMPO-AS1-WT was evidently restrained, but the
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luciferase activity of pmirGLO-TMPO-AS1-Mut was not affected (Fig. 4H). Lastly, data from RNA pull down assay displayed that TMPO-AS1 exceedingly enriched by bio-miR-98-5p-WT compared with bio-NC and bio-miR-98-5p-Mut, further verifying the direct combination between TMPO-AS1 and miR-98-5p (Fig. 4I). In brief, TMPO-AS1 sponges
miR-98-5p in BCa. 3.5 MiR-98-5p targets EBF1 mRNA To further support the ceRNA mechanism of TMPO-AS1, we applied RIP assay and observed that TMPO-AS1, miR-98-5p and EBF1 mRNA bound to RISC complex in BCa cells (Fig. 5A). The binding site of miR-98-5p on EBF1 mRNA was presented in Fig. 5B. Additionally, TMPO-AS1
shown
in
Fig.
5D,
the
decreased
ro of
expression was up-regulated for the subsequent assays (Fig. 5C). As luciferase
activity
of
pmirGLO-EBF1-WT caused by miR-98-5p upregulation was restored by
-p
the transfection of pcDNA3.1/TMPO-AS1, while no notable changes
re
were found in that of pmirGLO-EBF1-Mut. Results of RNA pull down assay further unveiled the combination between miR-98-5p and EBF1
lP
mRNA (Fig. 5E). Later, the negative expression association between
na
miR-98-5p and EBF1 was unveiled by Pearson’s correlation analysis (Fig. 5F). To further confirm this, qRT-PCR was utilized and data revealed that
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overexpressing miR-98-5p significantly down-regulated the expression of EBF1 (Fig. S1A). Then, EBF1 expression in UMUC3 and J82 cells was
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up-regulated for further study by transfecting pcDNA3.1/EBF1 (Fig. S1B). Results of colony formation presented that EBF1 overexpression rescued the inhibitive effect of up-regulated miR-98-5p on the proliferative capacity of BCa cells (Fig. S1C). Through TUNEL assay, we observed that the induced apoptosis caused by miR-98-5p overexpression
was reserved by transfecting pcDNA3.1/EBF1 (Fig. S1D). Furthermore, transwell assay indicated that overexpressed EBF1 recovered miR-98-5p overexpression-mediated repressive function on BCa cell invasion (Fig. S1E). All these findings indicated that miR-98-5p targets EBF1 mRNA. 3.6
TMPO-AS1/miR-98-5p/EBF1
axis
promotes
BCa
cell
role
of
proliferation, migration and invasion last,
we
planned
to
investigate
the
ro of
At
TMPO-AS1/miR-98-5p/EBF1 axis in the progression of BCa. And rescue
experiments were designed to reveal the effect of the above axis. Data
-p
from CCK-8 and colony formation manifested that EBF1 overexpression
re
counteracted the repressed proliferation in TMPO-AS1-silenced cells (Fig. 6A-B). According to TUNEL assay, it was observed that TMPO-AS1
lP
silencing-mediated promotion on cell apoptosis was recovered by
na
overexpression of EBF1 (Fig. 6C). Additionally, wound healing assay suggested that the suppressed migration caused by knockdown of
ur
TMPO-AS1 was rescued by the transfection of pcDNA3.1/EBF1 (Fig. 6D). According to transwell assay, it was discovered that up-regulated
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EBF1 reversed the restraining effect of silenced TMPO-AS1 on cell migration (Fig. 6E). Taken together, lncRNA TMPO-AS1 sponges miR-98-5p to liberate EBF1 mRNA, and thus increases EBF1 protein and enhances TMPO-AS1 transcription. This TMPO-AS1/miR-98-5p/EBF1 feedback loop promotes BCa cell progression (Fig. 7).
4. Discussion In recent years, accumulating evidence has implied that lncRNAs modulate gene expression in multiple biological processes, including chromatin modification, transcriptional and posttranscriptional regulation (Hon et al., 2017; Meng et al., 2015). For instance, HOTAIR and Xist
ro of
restrain gene regulation via interaction with PRC2 (Fu et al., 2017;
Yildirim et al., 2013). Additionally, increasing studies have indicated that lncRNA regulates cancer progression by acting as a ceRNA (Cheng et al., For
example,
LINC00460
functions
as
-p
2017).
a
ceRNA
in
re
nasopharyngeal carcinoma by sponging miR-149-5p and up-regulating IL6 to facilitate the tumorigenesis of nasopharyngeal carcinoma (Kong et
lP
al., 2018). LncRNA MYOSLID functions as a ceRNA in gastric cancer
na
and boosts cancer progression by targeting miR-29c-3p/MCL-1 axis (Han et al., 2019). Existing reports have verified the oncogenic role that
ur
TMPO-AS1 paly in prostate cancer (Huang et al., 2018), whereas its role in BCa has not been identified. Our research displayed that TMPO-AS1
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expression was up-regulated in BCa tissues and cells, and it predicted poor prognosis. Furthermore, TMPO-AS1 was validated to boost cell proliferation, migration, invasion and restrained cell apoptosis in BCa cells. All the above results confirmed that BCa progression was promoted by TMPO-AS1.
EBF transcription factor 1 (EBF1) has been reported as a transcription factor in a variety of diseases. For example, EBF1 has been certific ated to enhance the proliferation and hamper the apoptosis of bone marrow CD34+ cells in myelodysplastic syndrome via negatively regulating mitogen-activated protein kinase (Hou et al., 2018). Further, as a transcription factor, EBF1 protein has been revealed to transcriptionally
ro of
activate the expression of Aryl hydrocarbon receptor (AHR) to impair
human B lymphopoiesis (Li et al., 2017). This research revealed that
EBF1 was overexpressed in BCa tissues and cells. In addition, the
-p
expression of EBF1 was positively correlated with that of TMPO-AS1.
re
Importantly, EBF1 combined with TMPO-AS1 promoter to promote TMPO-AS1 transcription. To sum up, EBF1 functioned as a transcription
lP
factor in BCa and transcriptionally activated the transcription of
na
TMPO-AS1.
MicroRNAs (miRNAs) are a groups of small RNAs that cannot code
ur
proteins whereas bind to 3′ UTR of target genes to regulate target gene expression in human cancers (Stark et al., 2005; Tian et al., 2012). For
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instance, miR-675 binds to RB1 and negatively modifies its expression to promote glioma cell proliferation (Zheng et al., 2017). MiR-381 down-regulates the expression of SOX4 to suppress cell migration and invasion in gastric cancer (Zhang, M. et al., 2017). Interestingly, miRNAs are also sponged by lncRNAs and targets mRNAs. As reported, CRNDE
sponges miR-384 and up-regulates IRS1 to enhance cell proliferation and metastasis in pancreatic cancer (Wang et al., 2017). PCAT-1 sponges miR-129-5p to upregulate HMGB1 to facilitate the invasion and metastasis of hepatocellular carcinoma cells (Zhang, D. et al., 2017). MiR-98-5p has been validated as an anti-tumor gene in many cancers, such as pancreatic ductal adenocarcinoma (Fu et al., 2018), ovarian
ro of
cancer (Yang et al., 2018), hepatocellular carcinoma (Jiang et al., 2017) and so on. However, the role of miR-98-5p has not been studied in BCa.
In present study, miR-98-5p was sponged by TMPO-AS1, and showed a
-p
negative expression association with TMPO-AS1. Furthermore, EBF1
re
mRNA was uncovered as a target gene of miR-98-5p and present a reverse expression relationship with miR-98-5p. In line with rescue
lP
assays, overexpressed EBF1 could recover the promotion caused by
na
silenced TMPO-AS1 on BCa cell growth. In conclusion, this study unveiled that TMPO-AS1 is a tumor facilitator
ur
in the development of BCa and transcriptionally activated by EBF1 protein. Meanwhile, TMPO-AS1 contributes to BCa cell growth by
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sponging miR-98-5p and upregulating EBF1. This discovery indicated that TMPO-AS1/miR-98-5p/EBF1 positive feedback loop might be helpful for the treatment of patients suffered from BCa.
Availability of data
Not applicable.
Consent for publication The publication of this worked has received the permission of all authors.
Author’s contribution
ro of
Hua Luo: Conceptualization, Methodology, Software; Lu Yang: Investigation, Resources;
Chen Liu: Visualization, Supervision, Data curation;
re
Qiang Dong: Writing - review & editing;
-p
Xiaobo Wang: Formal analysis, Writing - original draft;
Liangren Liu: Project administration, Validation;
na
lP
Qiang Wei: Funding acquisition.
Conflicts of interests
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None exist. Funding
Jo
The project of Sichuan Deyang science and technology bureau (2017SZ097-1)
Acknowledgement
Jo
ur
na
lP
re
-p
ro of
We appreciate all participants involved in this experiment.
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Figure legends Figure 1 TMPO-AS1 predicts poor prognosis in BCa. (A) The expression of TMPO-AS1 in BCa tissues and paired non-tumor tissues. (B) TMPO-AS1 expression in BCa cell lines (T24, UMUC3, 5637, J82) and normal bladder cell line (SV-HUC-1). (C) The correlation between
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TMPO-AS1 expression and overall survival rate was assessed by
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Kaplan-Meier survival analysis (P = 0.013). **P < 0.01.
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Figure 2 TMPO-AS1 drives cell proliferation, migration and invasion, and inhibits cell apoptosis in BCa.
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(A) The transfection efficiency of sh-TMPO-AS1#1/#2 relative to sh-NC was showed. (B-C) The effect of TMPO-AS1 silencing on
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cell proliferation was unveiled by CCK-8 and colony formation. (D) TUNEL assay indicated the role of TMPO-AS1 knockdown in cell apoptosis. Green, apoptotic cells; blue, DAPI stained nuclei. Scale bar = 100μm. (E) Wound healing assay demonstrated the migration caused by knockdown of TMPO-AS1. Scale bar =
100μm. (F) The effect of TMPO-AS1 depletion on cell invasion was displayed through transwell assay. Scale bar = 50μm. **P <
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0.01.
Figure 3 EBF1 protein binds to TMPO-AS1 promoter. (A) EBF1 mRNA expression in BCa tissues and matched normal tissues. (B) EBF1 mRNA expression in BCa cell lines (T24, UMUC3, 5637, J82) and
normal cell line (SV-HUC-1). (C) Expression relation between TMPO-AS1 and EBF1 mRNA in BCa samples was showed by Pearson’s correlation analysis. (D) EBF1 mRNA expression in sh-TMPO-AS1#1/2 transfected cells was determined. (E) EBF1 mRNA expression was examined after transfecting with sh-EBF1#1/#2. sh-NC was used as negative control. (F) The luciferase reporter assay was conducted to
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determine the interaction between EBF1 mRNA and five section of
TMPO-AS1 promoter from -2000bp of TSS. (G) ChIP assay confirmed
the combination between EBF1 protein and TMPO-AS1 promoter. (H)
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The DNA motif of EBF1 and binding site between EBF1 and TMPO-AS1
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promoter was showed. (I) Luciferase reporter assay further verified that
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EBF1 mRNA interacted with TMPO-AS1 promoter. **P < 0.01.
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Figure 4 TMPO-AS1 sponges miR-98-5p in BCa. (A) The location of
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TMPO-AS1 was certified via nucleus-cytoplasm separation assay. (B) RNA pull down assay was employed to screen out the potential miRNAs interacted with TMPO-AS1 by using control probe or TMPO-AS1 probe to assess the relative enrichment of miRNAs. (C) The expression of miR-98-5p in BCa tissues and the association with TMPO-AS1 was
respectively determined by qRT-PCR and Pearson’s correlation analysis. (D) MiR-98-5p expression in BCa cell lines was evaluated by qRT-PCR. (E) MiR-98-5p expression in cells transfected with sh-TMPO-AS1 was displayed. (F) The binding sites between TMPO-AS1 and miR-98-5p were predicted by starBase. (G) The transfection efficiency of miR-98-5p construct was assessed. (H) Luciferase reporter assay confirmed the
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combination between TMPO-AS1 and miR-98-5p. (I) Interaction between TMPO-AS1 and miR-98-5p was confirmed by RNA pull down
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assay. *P < 0.05, **P < 0.01.
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Figure 5 MiR-98-5p targets EBF1 mRNA. (A) Ago2-RIP assay validated that TMPO-AS1, miR-98-5p and EBF1 mRNA were enriched in RISC. (B) The binding site between miR-98-5p and EBF1 mRNA was predicted. (C) TMPO-AS1 expression was tested in cells transfected with pcDNA3.1/TMPO-AS1. (D) The interaction between miR-98-5p and TMPO-AS1 (or EBF1) was implied with luciferase reporter assay. (E)
RNA pull down assay verified the interaction between miR-98-5p and EBF1 mRNA. (F) Negative expression association between miR-98-5p and EBF1 mRNA in BCa samples was showed by Pearson’s correlation
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analysis. **P < 0.01.
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Figure 6 TMPO-AS1/miR-98-5p/EBF1 axis promotes BCa cell proliferation, migration and invasion. (A-B) Cell proliferation was estimated via CCK-8 and colony formation. (C) TUNEL assay was conducted to estimate cell apoptosis. Green, apoptotic cells; blue, DAPI stained nuclei. Scale bar = 100μm. (D) Wound healing assay was employed to evaluate cell migration. Scale bar = 100μm. (E) Transwell
was utilized to analyze cell invasion. Scale bar = 50μm. *P < 0.05, **P <
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0.01.
Figure 7 Role of TMPO-AS1/miR-98-5p/EBF1 feedback loop in BCa was illustrated.
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Table1. Correlation between lncRNA TMPO-AS1 Expression and Clinical Features. (n=40) lncRNA TMPO-AS1
Variable
low
high
<60
12
15
≥60
8
5
Male
15
13
Female
5
7
Tumor size (cm) <4
13
3
≥4
7
17
15 5
5 15
Low
10
2
High
10
18
P-value
Age 0.501
Sex
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0.731
0.003**
Tumor stage
Tumor grade
0.004**
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<=T1 T2-T4
0.014*
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Low/high by the sample median. Pearson χ2 test. *P<0.05, **P<0.01 was considered to be statistically significant.
Table 2. Multivariate analysis of prognostic parameters in patients with BCa by Cox regression analysis Category
HR
Age
<60
0.796
CI(95%)
P-value
0.427-1.48
0.473
0.879
0.451-1.71
0.704
0.878
0.401-1.92
2.686
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Variable
≥60 Male
Sex
Tumor Size
<4
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Female
≥4 Tumor Stage
≤T1
Low
0.495
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Tumor Grade
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T2-T4
1.192-6.05
0.745
0.017*
0.232-1.05
0.069
1.27-6.121
0.011*
High Low
2.788
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TMPO-AS1
High
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Proportional hazards method analysis showed a positive, independent prognostic
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importance of TMPO-AS1 expression (P = 0.011*), in addition to the independent prognostic impact of Tumor Stage (P =0.017*). *P < 0.05 was considered statistically significant.