Background
Endometrial cancer (EC) is the sixth most common gynaecologic malignancy and threatens women’s health and life, increasing the disease risk and medical burden on society. In 2020, there were 417,000 new cases of endometrial cancer and 97,000 deaths from EC worldwide; its morbidity was 8.2% and mortality was 1.4% in Eastern Asia [
1]. The overall 5-year survival rate of patients with endometrial cancer is approximately 80% [
2,
3]. However, the clinical prognosis of advanced and specific subtypes of EC, such as high-grade EC and EC with papillary serous or clear cell histology, is extremely poor. Unexpected recurrence and poor outcomes with early-stage or well-differentiated endometrioid tumors do occur [
4]. Surgery, endocrine therapy, chemotherapy, radiotherapy, and immunotherapy are the principal treatments for endometrial cancer [
5]. Hence, it is pivotal to identify new molecular targets and molecular mechanisms to facilitate improved diagnosis and treatment of EC.
Epithelial-to-mesenchymal transition (EMT) is a process that is activated during cancer progression, causing cancer cells to acquire mesenchymal or stem cell properties that allow them to detach from the primary tumour site and invade surrounding tissues or vascular tissues to form distant metastases [
6,
7]. Loss of E-cadherin expression and acquisition of Vimentin expression are markers of EMT occurrence [
8]. Research has shown that EMT is critically associated with tumour metastasis in the development of tumorigenesis. LncRNA MAFG-AS1 mediates NFKB1-induced upregulation of IGF1 through miR-339-5p to promote EMT in ovarian cancer [
9]. In renal clear cell carcinoma, E2F1 modulates SREBP1 to induce cellular lipid accumulation and elevated lipase, thereby promoting tumour cell proliferation and metastasis [
10]. DLX6-AS1 contained in hepatocarcinoma exosomes modulates CXCL17 by binding competitively to miR-15a-5p and triggers M2 macrophage polarization, promoting EMT in hepatocarcinoma [
11]. The EMT process is primarily correlated with the Wnt signalling pathway, Notch signalling pathway, and Hedgehog signalling pathway, which are involved in tumour cell metastasis [
12]. SRA is engaged in EMT in EC cells by enhancing the expression of EIF4E-BP1 and activating the Wnt/β-catenin signalling pathway [
13]. The cytoplasmic polyadenylation element-binding protein (CPEB) family consists of sequence-specific RNA-binding proteins. CPEB1, CPEB2, CPEB3, and CPEB4 are four members of the CPEB family [
14]. The CPEB family is differentially expressed and activated in different tumours. CPEB4 has been identified as playing an important role in the proliferation of tumour cells and tumour development progression [
15‐
18]. Increasing evidence has shown that CPEB4 is involved in tumour invasion and metastasis. For example, CPEB4 can regulate tumour cell invasion and migration by regulating Vimentin expression in breast cancer; ZEB1-mediated EMT may be involved in CPEB4-promoted cell proliferation, invasion, and metastasis in gastric cancer [
19,
20]. However, the pathophysiological roles of CPEB4 in EC remain uninvestigated. Therefore, further studies are of paramount importance to determine the mechanism underlying CPEB4-driven EMT in EC.
Circular RNAs (circRNAs) are a type of closed-loop noncoding RNA without a 5′ cap or 3′ poly (A) tail [
21,
22]. They were initially regarded as the products of splicing errors, but they are now considered independent functional entities [
23,
24]. CircRNAs play important roles in many aspects of tumorigenesis, such as proliferation, metastasis, apoptosis, and autophagy [
25]. CircRNAs can also function as sponges for miRNAs that influence the mRNA translation process [
26]. The roles of circRNAs as microRNA (miRNA) sponges in EC are being gradually discovered. Circ_0002577, circ_PUM1, circTNFRSF21, circRNA WHSC1, circ_0002577, and circ_0067835 have been reported to influence the biological function of EC by sponging miRNAs [
27‐
32]. However, the roles of most circRNAs in EC remain uninvestigated.
In this study, bioinformatics analysis was performed to investigate the expression of circRNAs in EC tissue. The expression level of hsa_circ_0084927 (circESRP1), which is derived from the ESRP1 gene locus, was markedly upregulated in EC tissues. Up- or downregulation of circESRP1 correspondingly promoted or inhibited tumour cell proliferation and migration. CircESRP1 was demonstrated to sponge miR-874-3p, which degrades CPEB4 mRNA. In conclusion, circESRP1 regulates EMT and proliferation in EC by acting as a ceRNA to bind to miRNA-874-3p. CircESRP1 may serve as a novel biomarker for the diagnosis and prognosis of EC and may be a potential therapeutic target for EC.
Methods
Human tissues sample and databases
Human normal endometrial tissues (n = 10) and EC tissues (n = 19) were obtained from Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). The tissue specimens were all from patients undergoing surgery and were removed and stored in liquid nitrogen until proteins or RNAs were extracted. Patients read and signed an informed consent form before the surgery. Institutional Review Board of the Tongji Medical College, Huazhong University of Science and Technology approved this research.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA from cells or fresh tissues was extracted using TRizol (#9108, RNAiso Plus, Takara, Japan) according to the instructions. The cDNA was prepared by using PrimeScriptRT Reagent Kit (#RR037A, Takara, Japan). qRT-PCR was performed by the Biosystem StepOne Plus PCR System (ABI) with Real-time PCR kits (Takara, Japan). The RNA expression levels were calculated using GAPDH and U6 expression as internal references by the 2−ΔΔCT method. The sense and antisense primers of qRT-PCR were shown as follows: circESRP1 5′-GGAACGGAGAAGCTCTGGTTAG-3′ and 5′-GTAAACCTCGTGCCCTGACTAC-3′; linear ESRP1 5′-ACCAAGCCCTCCGACAGTTTA-3′ and 5′-ATCAGGTGAACCAGGGCAACA-3′; miR-874-3p 5′-CTGCCCTGGCCCGAGG-3′ and RT primer 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCGGTC-3′; miR-4694-5p 5′-CAAATGGACAGGATAACACCTATG-3′ and RT primer 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAAATA-3′; miR-4732-3p 5′-TGTCCTGTTCTGCCCCCAG-3′ and RT primer 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAGGGG-3′; miR antisense primers 5′-AGTGCAGGGTCCGAGGTATT-3′; CPEB4 5′-TGTGTGCTACGCTGGGATTGA-3′ and 5′-TGGCTTAACTTCCACCCGTTT-3′; U6 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH 5′-GGCAGAGATGATGACCCTTTT-3′ and 5′-AGATCCCTCCAAAATCAAGTGG-3′.
Cell culture
EC cell lines included RL95-2 and Ishikawa, which were purchased from the American Type Culture Collection (ATCC; Manassas, VA). Ishikawa cells and RL952 cells were cultured in DMEM/F12 medium (#11330032, Gibco) supplemented with 10% fetal bovine serum (#10099141, Gibco) and 1% streptomycin and penicillin (#PYG0016, Bosterbio, USA) in the 5% CO2, 37 °C incubator.
Transfection
Lentivirus vector containing short hairpin RNA (shRNA) and overexpression lentivirus were purchased from GenChem (Shanghai, China). Three shRNA sequences of circ0084927 were: sh-circ0084924-1: 5′-CTTAAAATTGCTGGTGCAGCA-3′; sh-circ0084927-2: 5′-AAAATTGCTGGTGCAGCAAGA-3′; sh-circ0084927-3: 5′-TGCTGGTGCAGCAAGATGGAA-3′; sh-negative control (sh-NC): 5′-TTCTCCGAACGTGTCACGT-3′. Lentiviral transfection of cells was performed when cell integration reached 20–30%. MiR-874-3p mimic/mimic control/inhibitor/inhibitor control were designed and purchased from RiboBio (Guangzhou, China). Transfection of miR-874-3p mimic, miR-874-3p mimic control, miR-874-3p inhibitor, and miR-874-3p inhibitor control was performed when EC cells were melted in 6-well plates with 60–70% conformation utilizing co-transfection reagents (Guangzhou, China). CPEB4 three small interfering RNAs (shRNA) and negative control shRNA were designed and synthesized by GenChem (Shanghai, China). The target sequences of CPEB4 were described as follows:
-
sh-CPEB4-1, 5′-TAGCCGATCCATTATCATC-3′;
-
sh-CPEB4-2, 5′-ATACTGTTCAACACATAAC-3′;
-
sh-CPEB4-3, 5′-ATATTATACCAAAGCGCAG-3′.
Cell counting kit-8 assay
Cell Counting Kit-8 assay (#34302, CCK8, Bimake, USA) was used to detect cell proliferation. Cells (4 × 103/well) were cultivated in 96-well plates, and the absorbance at 450 nm was detected at four-time points of 24 h, 48 h, 72 h, and 96 h according to the instructions. The absorbance was performed by using an automatic microplate reader (BioTek, VT, USA).
EdU assay
Cells (1 × 104 cells/well) were grown in 96-well plates and incubated at 37 °C, 5% CO2 thermostat for 24 h before EdU (5-ethynyl-2′-deoxyuridine) assay. EdU assay was measured using an EdU kit (#C0071S, Beyotime, shanghai) in accordance with the instructions. The EdU solution was prepared as a 1:1000 EdU medium. Add 50 µl/well of EdU medium to the pre-prepared 96-well plate and the cells were incubated at 37 °C for 2 h and then fixed using 4% paraformaldehyde for 15 min. Cells were incubated with 30 µl/well of click reaction solution for 30 min at room temperature and sheltered from light, and then Hochst33342 stained for 10 min. Cell proliferation was imaged using a 20× microscope, and 5 isolated fields were photographed for counting statistics.
Cells (1 × 103 cells/well) were seeded in 6-well plates and cultured at 37 °C with 5% CO2 for 3 weeks. Cells were fixed with 4% paraformaldehyde for 15 min and then stained with 0.1% crystal violet for 25 min. Clone number and size were used to assess the ability of cell clone formation, and five separate fields of view were used for enumeration.
Migration and invasion assays
Migration assays were performed in 24-well plates. 600 µl of medium containing 20% fetal bovine serum was added to the lower part of the transwell chamber (#3422, Corning, USA) and 200 µl of serum-free medium containing 10 × 104 cells was added to the upper part of the transwell chamber. Following 24 h of incubation at 37 °C, cells penetrated from the top of the chamber membrane (8 μm pore size) to the bottom. Cells penetrating the chamber membrane were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 30 min, followed by imaging under a 10× microscope, and four separate fields of view were taken for counting. In contrast to the cell migration assay, the cell invasion assay requires the addition of Matrigel (#356234, Corning, USA) to the upper part of the cell chamber before seeding the cells, and the same steps as the cell migration assay.
Wound healing assay
Cells were implanted in six-well plates and cultured for 24 h to reach 100% fusion. Cell wounds were made by a 10 µl pipette tip, and cells were rinsed clean by PBS. The cells were continued to be cultured in a serum-free medium, and the width of the wound was photographed at 0 h and 24 h under a 40× magnification microscope.
RNase R treatment and actinomycin D
Total RNA (2 μg) was incubated with or without 3 U/μg of RNase at 37 °C for 45 min. For the actinomycin D treatment assay, total RNA was obtained using 2 mg/μl of actinomycin D after treating the cells for 0 h, 4 h, 8 h, and 12 h. After actinomycin D and RNAase treatment, qRT-PCR was operated to evaluate variations in the expression of circESRP1 and ESRP1 mRNA.
Nuclear-cytoplasmic fraction
Cytoplasmic and nuclear RNA isolation was performed by utilizing the Cytoplasmic and nuclear RNA purification kit (#21000, Norgenbiotek, Canada). Cells were washed twice with PBS and 200 µl of pre-cooled Lysis buffer J was added and incubated on ice for 5 min. The cell lysate was centrifuged at 12,000 rpm for 10 min at 4 °C to separate the supernatant of the cytoplasmic fraction from the nuclear pellet. The cytoplasmic supernatant and the nuclear pellet were washed sequentially by vortexing using Buffer K and 100% ethanol and subsequently transferred to a centrifuge column for centrifugation. The RNA solution was obtained by washing the centrifuge column 3 times with wash solution A and then lysing the RNA with 50 µl of Elution Buffer E.
Fluorescence in situ hybridization
Cy3-labeled circ0084927, human U6, and human18S probes were designed and synthesized by RiboBioCo. Ltd. (Guangzhou, China). The FISH assay was performed using the RiBoTM Fluorescent In Situ Hybridization Kit (#C10910, RiboBio Co. Ltd, Guangzhou, China) according to the instructions. Cells (1 × 104/well) were replanted in 24-well plates and incubated in the incubator for 24 h before the FISH assay. Cells were treated with PBS 2 times and then fixed using 4% paraformaldehyde at room temperature for 10 min, followed by permeabilization with 0.5% Triton-X100 at 4 °C for 5 min. After 30 min of pre-hybridization at 37 °C, cells were hybridized with the fluorescent probe overnight at 37 °C sheltered from light. Thereafter, cells were rinsed 3 times/5 min using 4×/2×/1× SCC solution at 42 °C under light-protected conditions and then stained with DAPI. The cells were eventually observed utilizing confocal microscopyat 400× magnification.
RNA pulldown
The biotinylated-circ0084927 probe was synthesized by Sangon Biotech (Shanghai, China). The cells were harvested and lysed and subsequently centrifuged, and the lysate was collected. The cell lysates were incubated with a circ0084927 probe and an oligo probe for 2 h at room temperature. Then, the cell lysates incorporating the probes were incubated with the streptavidin magnetic beads (#HY-K0208, MedChemExpress, USA) for 1 h at room temperature. The RNA bound to the magnetic beads was then purified and withdrawn using RNeasy Mini Kit (Qiagen, German). Lastly, qRT-PCR experiments were performed.
RNA immunoprecipitation
RNA binding protein immunoprecipitation (RIP) assay was performed with the EZ-Magna RIP kit (#17-704, Millipore, Burlington, MA, USA) based on the guidelines. The purpose of RIP was to extract and identify the RNA bound to AGO2 protein. Cells (1 × 107) were added to the lysis solution, lysed on ice for 5 min, and then centrifuged, and the supernatant was extracted. After incubation with AGO2 antibody and IgG antibody for 30 min, the magnetic beads were combined with 100 μl of cell lysate supernatant, respectively, rotated 360° and incubated overnight at 4 °C. After washing the magnetic bead protein RNA complex, RNA was extracted and qRT-PCR was performed.
Dual-luciferase reporter assay
Fragments of wild-type and mutant circ0084927 and CPEB4 were constructed and inserted downstream of the reporter plasmid pRL-SV40 with firefly fluorescence (GenChem, Shanghai, China). Cells were seeded in 6-well plates, and when cell fusion reached 50%, the plasmids and miR-874-3p mimic or controls were transfected using lipo3000. After 48 h of incubation, firefly luciferase and renilla luciferase activities were detected using the Dual-Luciferase Reporter System kit (#E1910, Promega, USA).
Western Blot (WB) analysis
Total cellular proteins were extracted with RIPA lysate (#P0013B, Beyotime, Shanghai, China) and the proteins were measured quantitatively using the BCA protein assay kit (#G2026, Servicebio, Wuhan, China). Total cellular protein (30 µg) was pipetted into 10% or 12.5% SDS-PAGE gel for gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies overnight at 4 °C after 2 h of blocking at room temperature using 5% skim milk. Membranes were washed three times with TBST buffer and incubated with goat anti-rabbit or goat anti-generic secondary antibodies for 1 h at room temperature. Finally, imaging was performed using The ChemiDoc MP (Bio-Rad, USA). The primary antibodies were as follows: CPEB4 (1:1000, #25342-1-AP, Proteintech Group, INC., USA), CRCP (1:1000, #14348-1-A, ProteintechGroup, INC., USA), Vimentin (1:1000, #10366-1-AP, Proteintech Group, INC., USA), E-cadherin (1:1000, #20874-1-AP, Proteintech Group, INC., USA), GAPDH (1:20,000, #AC002, ABclonal, Wuhan, China); secondary antibodies are as follows: HRP Goat Anti-Rabbit IgG (1:8000, #AS014, ABclonal, Wuhan, China), HRP Goat Anti-Mouse IgG (1:8000, #AS003, ABclonal, Wuhan, China), China).
Immunohistochemistry
Paraffin sections of mouse subcutaneous graft tumor tissue were dewaxed and dehydrated, rehydrated, and repaired with citric acid. After peroxidase blocking, the tissue sections were incubated with primary antibody overnight at 4 °C. The primary antibodies were as follows: CPEB4 (1:2000, #25342-1-AP, Proteintech Group, INC., USA), Vimentin (1:2000, #10366-1-AP, Proteintech Group, INC., USA), E-cadherin (1:3000, #20874-1-AP, Proteintech Group, INC., USA). The sections were washed with PBS and incubated with secondary antibody for 30 min at 37 °C. The sections were finally blocked and observed under the microscope. According to the staining score, the staining intensity was categorized as negative (score = 0), weak (score = 1), moderate (score = 2), and strong (score = 3); the number of positively stained cells 0–5%, 5–25%, 26–50%, 51–75%, and 76–100% were scored as 0, 1, 2, 3, 4, respectively. The final staining score was the product of the staining intensity and the number of positively stained cells products.
Hematoxylin–eosin staining (HE staining)
Xylene I and II were used to dewax the sections for 10 min each. 100% (I and II), 90%, 80% and 70% alcohol were used to dehydrate the sections for 5 min each and rinsed under running water for 5 min × 3. Hematoxylin was utilized to stain the sections for 5 min and rinsed under running water. Acetic acid fractionation for 1 min, rinse slides with running water. Eosin staining for 1 min, rinsing the slides under running water. 70%, 80%, 90%, 100% alcohol for 10 s each, xylene for 1 min, dehydrating the slides. Slides were dried naturally and sealed with drops of neutral gum.
In vivo tumor xenografts
All animals in experiments were approved and supported by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. 6-week-old female BALB/c nude mice were purchased from SPF Biotechnology Co., Ltd. (Beijing, China), and Ishikawa cells (4 × 106 per mouse) were injected subcutaneously into the back of mice. Mice were housed for 28 days in a specific pathogen-free class of animal room. The sizes of the mice subcutaneous tumors were measured weekly. Tumor size was measured and mice were sacrificed, and tumors were removed and weighed. The tumor volume was calculated by the formula: a × b2, where a indicated the longest diameter of the tumor and b was the length of the diameter perpendicular to a.
Database
The differential circRNA data in EC were extracted from the analysis of EC tissue samples by the team of Yongchao Dou, which consisted of 95 cases of EC and 49 normal tissue samples [
4]. Subsequently, the differential circRNAs were performed using R software, where the parameters were set fold change ≥ 2 and p
adj ≤ 0.001.
Statistical analysis
The data in this study, which were from 3 independent replicate experiments, were analyzed by GraphPadPrism software (version 7.0), and the results are expressed as mean ± SD. The significance of differences between groups was assessed by paired two-tailed Student’s t-test or χ2 test. Correlation analysis between circESRP1 and miR-874-3p was evaluated by Spearman’s test. A p-value 0.05 was admitted as statistically significant.
Discussion
Although the 5-year survival rate of EC is 80%, its annual morbidity is rising and threatens women’s health [
31]. Accumulating evidence indicates that circRNAs are involved in tumorigenesis and tumour development and may potentially serve as tumour markers in the future [
34]. Based on bioinformatics analysis, we selected circESRP1, a 287 bp exonic circRNA (EcircRNA) containing three exons from its parental gene ESRP1, to elucidate its biological and potential mechanisms. CircESRP1 is a novel circRNA that exhibited the most significant differences in expression among all circRNAs. Our study investigated the function and mechanism of circESRP1 in EC for the first time. Utilizing qRT-PCR, we found that the RNA level of circESRP1 was higher in EC tissues than in adjacent tissues. However, the lower RNA level of miR-874-3p in EC, which has been reported to be associated with tumour progression, was verified by qRT-PCR. These results suggest that the differential expression of circESRP1 and miR-874-3p may be significant for the diagnosis of EC and may serve as biomarkers of EC. Current research indicates that dysregulation of circRNA expression may contribute to progressive, uncontrolled tumour cell growth and metastasis [
35,
36], not simply a nonfunctional product of pre-mRNA splicing. Functionally, silencing circESRP1 reduced the proliferation and invasion abilities of EC cells, and conversely, overexpression of circESRP1 had enhancing effects in vitro and in vivo. Therefore, our data suggested that circESRP1 may play a key role in pathogenesis and assessing the progression of EC, providing a basis for being a prospective therapeutic target in the future.
Regarding circRNAs containing miRNA response elements, a vast number of studies have reported that miRNA adsorption is the most pivotal pathway by which circRNAs perform their biological functions, which can further modulate downstream target genes, thus influencing malignant tumour progression, recurrence, and chemoresistance [
22]. For example, circSPARC upregulates JAK2 expression by sponging miR-485-3p, leading to accumulation of phosphorylated p-STAT, which in turn promotes CRC cell proliferation and metastasis [
37]; in addition, circPDE3B can induce EMT to regulate ESCC cell proliferation, migration, and invasion through the miR-4766-5p/LAMA1 axis [
38]. Through the experiment, circESRP1 was identified as a miRNA sponge to promote EC cell progression. Bioinformatics analysis predicted that circESRP1 interacts with miR-874-3p, and the anti-AGO2 RIP, RNA pulldown, and dual-luciferase reporter assays confirmed our speculation. Our study revealed that miR-874-3p was expressed at low levels in EC, and rescue experiments reconfirmed that circESRP1 exerted its oncogenic effect through miR-874-3p. In papillary thyroid carcinoma, miR-874-3p suppressed tumour cell migration and invasion by downregulating the expression of FAM84A and served the same function in colon cancer, [
39,
40] consistent with our findings that the miR-874-3p mimic inhibited EC cell metastasis and that the miR-874-3p inhibitor enhanced EC cell function. This study shows for the first time that circESRP1 and miR-874-3p interact and that circESRP1 promotes EC cell progression via miR-874-3p. These results also indicate that miR-874-3p plays an important role in the progression of EC and may be a potential therapeutic target.
In addition, bioinformatics analysis and validation experiments indicated that miR-874-3p binds to CPEB4, a member of the CPEB family. CPEB4 is intimately associated with the metastasis of a variety of tumours. For example, CPEB4 can promote breast cancer metastasis through upregulation of Vimentin [
19] and facilitate the migration and invasion of lung cancer cells through activation of the AKT pathway [
41]. Thus, CPEB4 could enhance the metastasis of tumour cells. In our study, we detected upregulation of CPEB4 in EC tissues. Knockdown of CPEB4 inhibited migration, invasion, and corresponding changes in EMT-related proteins in EC cells, whereas the rescue experiments suggested that cotransfection of the circESRP1 vector partially attenuated these inhibitory effects. CircESRP1 overexpression increased CPEB4 expression, while the miR-874-3p mimic partially abolished these promotive effects. These results suggest that circESRP1 acts as a sponge for miR-874-3p to regulate CPEB4 and promote EMT, metastasis, and proliferation in EC.
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