1 Introduction
Cervical cancer (CC) is a prevalent gynecological neoplasm with increasing incidence and mortality rates, particularly among younger individuals. It continues to be a highly perilous gynecological malignancy worldwide, particularly in developing nations, posing a significant threat to human well-being [
1]. Despite substantial progress in surgical interventions, chemotherapy, and radiotherapy, the 5-year survival rate for patients remains alarmingly low [
2]. The pathogenesis of CC is governed by a diverse array of molecules, and comprehending the molecular mechanisms involved is crucial.
Circular RNAs (circRNAs) possess covalent closed-loop structures [
3], which do not have 5ʹ-3ʹ polarity and polyadenylate termini. Moreover, the unique circular structure of circRNAs makes them resistant to RNase and relatively stable in tissues and cells. circRNAs are aberrantly expressed in cancer cells in a tissue-specific manner, making them promising biomarkers and therapeutic targets for tumors [
4‐
7]. Research has indicated that circRNAs are implicated in the carcinogenesis, including CC, and can function as ceRNAs by modulating the activity of miRNAs [
8,
9]. Previous investigations have identified multiple dysregulated circRNAs associated with tumorigenesis and metastasis in CC, such as circ_101996 [
10], circ_0018289 [
11], and circ_0000228 [
12]. circ_0006789 (circSLC25A43) is a circular RNA originating from the SLC25A43 gene and is known to exert a significant influence on tumor progression. Circ_0006789 was originally thought to be able to regulate the growth, migration and invasion of hepatocellular carcinoma cells by modulating the miR-1324/SOX 12 axis [
13], but no studies have yet explored circ_0006789's role in CC. Various miRNAs have been implicated in CC development, either as tumor suppressors or oncogenes[
14‐
16]. Notably, miR-615-5p has been implicated in ovarian cancer [
17]. HSF1 promotes tumor progression and tumorigenesis in cancer cells by promoting their survival and escaping programmed cell death [
18‐
20]. Also, it has been reported that HSF1 participates in the pathogenesis of CC [
21].
The objective of this study was to examine circ_0006789 expression and its biological role in CC. Our findings detected a significant upregulation of circ_0006789 in both CC tissues and cell lines. Additionally, we investigated the impact of circ_0006789 on CC development and demonstrated that circ_0006789 targets and sequesters HSF1 through miR-615-5p, thereby facilitating CC progression.
2 Materials and methods
2.1 Patients and tissue samples
CC tissues (n = 150), along with adjacent normal tissues, were collected from CC patients at The First Affiliated Hospital of Harbin Medical University between January 2021 and May 2023. Histopathology biopsy confirmation was required, followed by pathology confirmation after surgery, to determine whether a patient met the inclusion criteria. None of the subjects had undergone radiation, chemotherapy, or any treatments. The specimens were stored at − 80 °C. The clinical characteristics of the patients can be found in Table
1. Furthermore, all patients provided written informed consent. This study was endorsed by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University (No. 202010HS6).
Table 1
circ_0006789 expression and clinicopathological features
Age (years) |
≥ 45 | 35 | 45 | 0.1017 |
≤ 45 | 40 | 30 |
Lymph node metastasis |
Yes | 32 | 38 | 0.0623 |
No | 43 | 37 |
Tumor size (cm) |
< 4 | 44 | 26 | 0.0032* |
≥ 4 | 31 | 49 |
Clinical stage |
I/II | 56 | 34 | 0.0002* |
III/IV | 19 | 41 | |
2.2 Cell culture
Human CC cell lines (SiHa, HeLa, HCC94, and SW756) and normal cervical epithelial cells (H8) were supplied by the BeNa Culture Collection (Beijing, China). SiHa cells were cultured in DMEM (HyClone, UT, USA), while HeLa and H8 cells with RPMI 1640 medium (Thermo Fisher Scientific, MA, USA). All cells were cultured at 37 °C and 5% CO2 in a complete medium (Thermo Fisher Scientific) plus 10% FBS and 1% antibiotics.
2.3 Subcellular localization
RNA was extracted from the nucleus and cytoplasm of HeLa cells utilizing the PARIS™ Kit (Thermo Fisher Scientific). The presence of circ_0006789 was identified through PCR analysis in both nucleus and cytoplasm, with U6 and GAPDH as respective nuclear and cytoplasmic controls, respectively.
2.4 Fluorescence in situ hybridization (FISH)
FISH assay was conducted with the FISH kit (GenePharma, Shanghai, China). The Cy5- and farm-labeled probes exhibited specificity towards circ_0006789 and miR-615-5p, respectively. Nuclei were stained with 4ʹ,6-dimethyl-2-phenylindole, and images were captured with a Zeiss LSM880 NLO confocal microscope (Leica Microsystems, Germany).
2.5 Actinomycin D experiment
By adding 2 mg/ml actinomycin D (Sigma-Aldrich), HeLa and Siha cells were incubated at 37 °C for 4, 8, 12, and 24 h. Extracted RNA was collected using HiScript II first strand cDNA synthesis kit (Vazyme, Nanjing, China) to determine the stability of circ_0006789 using PCR.
2.6 RNAse R
The extraction of total RNA (2 μg) from HeLa and Siha cells was conducted using TRIzol reagent (Invitrogen). Subsequently, digestion was carried out at 37 °C for 10 min with 3 U/μg RNAse R (Epicentre, WI, USA). Following RNA purification with RNeasy MinElute Cleanup kit (Qiagen, Hilden, Germany), PCR was conducted to assess circ_0006789 and SLC25A43 levels.
2.7 Cell transfection
miR-615-5p mimic/inhibitor, si-circ_0006789, and negative controls were generated by RiboBio (Guangzhou, China). pc-HSF1, an HSF1 overexpression vector, was constructed using the pcDNA3.1 vector (Thermo Fisher Scientific). pcDNA3.1 (pc-NC) served as the negative control. HeLa and SiHa cells were seeded in 6-well plates at 3 × 105 cells/mL for 24 h, followed by transfection using Lipofectamine® 3000 (Invitrogen) and PCR verification.
2.8 PCR
Total RNA was isolated from using TRIzol reagents (Thermo Fisher Scientific). circ_0006789 and HSF1 were treated with PrimeScript™ RT Master Mix (Takara, Shiga, Japan), while miR-615-5p was detected with miRNA reverse transcription kit (TaKaRa). PCR was performed using the SYBR® Premix Ex TaqTM II kit (TaKaRa) on the StepOnePlus real-time PCR system (Thermo Fisher Scientific). GAPDH and U6 were internal parameters to calculate the relative gene expression by 2
−ΔΔCt method. The primers are listed in Table
2.
circ_0006789 | GACCCAGACCCTCTCCTTTC | CCGTTCCAGATTTTCTCCAGG |
SLC25A43 | CTGGAACCATCGTACAGGGG | CCCCTGGGCCTTCACTATCT |
HSF1 | CATGAGAATGAGGCTCTGTG | CTACGCTGAGGCACTTTTCA |
miR-615-5p | ATGCAGGGTCCGAGGTATTC | GGGGGTCCCCGGTGCT |
U6 | CTCGCTTCGGCAGCACA | AACGCTTCACGAATTTGCGT |
GAGPH | TGTGGGCATCAATGGATTT | ACACCATGTATTCCGGGTCAAT |
2.9 CCK-8 assay
HeLa cells were put into 96-well plates (2 × 103 cells/wells), on which each well was supplemented with 10 μL CCK-8 solution at 0, 24, 48, and 72 h (Dojindo, Kumamoto, Japan) and assayed for 2 h at 37 °C. Absorbance at 450 nm was read on a microplate reader (Bio-Rad, CA, USA).
2.10 Flow cytometry
Apoptosis was assessed using the annexin V-FITC apoptosis kit (Solarbio, Beijing, China). HeLa cells were seeded at 105 cells/well in a 12-well plate for 48 h. Following this, the cells were trypsinized, resuspended in a binding buffer, and subsequently incubated with 10 μL annexin V-FITC and PI for a duration of 10 min. Data quantification was done with flow cytometry (Agilent, Hangzhou, China), and apoptosis rate was calculated.
2.11 Transwell
Transwell chambers (Corning, MA, USA) were utilized for Transwell assays. For the cell migration assay, a suspension of 1 × 105 HeLa and SiHa cells in serum-free medium (150 µL) was transferred to the upper compartment. Following incubation at 37 °C for 24 h, the cells on the lower compartment were fixed with 95% ethanol and stained with 0.1% crystal violet for a duration of 25 min. The cells were enumerated utilizing a microscope manufactured by Olympus, Japan. A Matrigel layer was pre-covered in the Transwell chamber to perform invasion assay.
2.12 Dual luciferase reporter gene experiment
The CircInteractome (
https://circinteractome.nia.nih.gov/) and TargetScan (
https://www.targetscan.org/) biological information website predicted the potential binding sites of miR-615-5p with circ_0006789 or HSF1 3ʹUTR. circ_0006789 or HSF1 3'UTR sequences, containing either a wild-type or mutant binding sequence for miR-615-5p, were inserted into the pmirGLO vector (GenePharma). Following the Lipofectamine 3000 protocol, miR-615-5p mimic or miR-NC and the luciferase reporter were co-transfected into HeLa and SiHa cells for a duration of 48 h. Subsequently, the luciferase activity was evaluated using the luciferase reporter assay kit (Promega).
2.13 RIP
HeLa and SiHa cells were lysed with a complete RIP lysis buffer (Millipore, MA, USA). The supernatant was co-incubated with magnetic beads bound to Ago2 (ab32381, Abcam, UK) or IgG (02–6102, Invitrogen) at 4 °C for 6 h. After eluting the immunoprecipitate of the bound bead, circ_0006789, miR-615-5p, and HSF1 were measured by PCR.
2.14 Immunoblot
The isolation of total protein from tissues and cells was performed using RIPA lysis buffer (Beyotime, Shanghai, China), followed by quantification analysis using the BCA kit (Beyotime). Subsequently, protein samples (30 μg/lane) were separated through sodium dodecyl sulphate–polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene fluoride membrane (Millipore). Then, the membrane was sealed with 5% skim milk for 1 h, subjected to incubation with HSF1 (4356, CST), Vimentin (ab92547, Abcam), E-cadherin (14472, CST), N-Cadherin (13116, CST), and GAPDH (5174, CST) overnight and horseradish peroxidase-labeled IgG (ab124055, Abcam) for 1 h, and developed with ECL kits (Thermo Fisher Science) in a chemiluminescent imager (Image Quant LAS4000 mini, GE Healthcare, UK). The Image J software analyzed protein bands.
2.15 Xenotransplantation model
Five-week-old female BALB/c nude mice (Beijing Laboratory Animal Center, Beijing, China) were utilized to construct the xenotransplantation model. HeLa cells were transfected with circ_0006789 or si-NC (RiboBio, Guangzhou. China) and selected using purinomycin. Subsequently, each mouse was given a subcutaneous inoculation of stably transfected HeLa cells (5 × 106 cells) into the lateral abdomen (n = 6/group). The tumor volume was assessed weekly for a duration of 4 weeks. Volume (mm3) = length × width2/2. Under isoflurane anesthesia, the mice were euthanized after completing the experiment. The tumor samples were weighed, and then circ_0006789, miR-615-5p and HSF1 were detected by PCR and immunoblot. Animal treatment conformed to the approval by the Animal Management and Use Committee of The First Affiliated Hospital of Harbin Medical University (No. 202012B01).
2.16 Data analysis
All data were expressed as the mean ± standard deviation (SD) of more than 3 replicates and analyzed using GraphPad Prism 9.0 (GraphPad, CA, USA). One-way analysis of variance was employed for multiple-group differences, and Student’s t-test was used for comparing two-group differences. Chi-square test was utilized to evaluate the relationship between circ_0006789 expression and clinicopathological information. * P < 0.05 was considered statistically significant.
4 Discussion
CircRNA is considered a biomarker relevant to clinical trials and may contribute to poor prognosis in various tumors due to its stable structure.Studies have demonstrated the significant role of circRNA as a key regulator in CC [
22]. For instance, circ_0000228 facilitates the malignancy of CC through the miR-195-5p/Lysyl-oxidase-like protein 2 axis [
23], while circ_0000285 downregulation inhibits CC via the miR-197-3p-ELK1 axis [
24]. Additionally, circ_0003221 has been found to promote CC through the miR-758-3p/CPEB axis [
25]. The present study observed circ_0006789 up-regulation in both CC tissues and cell lines. Also, depleting circ_0006789 significantly hindered CC cells to proliferate, migrate, and invade, while concurrently enhancing apoptosis. These findings strongly imply that circ_0006789 possesses oncogenic properties and holds promise as a therapeutic target for CC treatment. However, the mechanism of action of CC treatment through circ_0006789 needs more exploration.
circRNA can function as a molecular sponge, exerting indirect control over gene expression through miRNA sequestration [
26]. A new mechanism by which non-coding RNA regulates biological processes is suggested by the ceRNA hypothesis. The interaction between miRNA and circRNA can regulate the malignancy and aggressiveness of cancer cells [
27,
28]. In this work, it is found that circ_0006789 was mainly distributed in the cytoplasm of HeLa cells, so it is speculated that circ_0006789 may have the function of ceRNA. miRNAs are an integral part of the ceRNA network and are characterized by their specific binding to the 3'UTR of target mRNAs. By blocking translation or inducing target degradation, miRNAs are involved in the regulation of gene expression at the post-transcriptional level. aberrant expression of miRNAs is also closely related to tumour development [
29]. miR-615-5p is a newly identified endogenous miRNA that has been associated with the development of several cancers [
30,
31]. In common with other studies, miR-615-5p was also in a dysregulated state in CC, and we found a trend of downregulation in CC tissues and cells. Meanwhile bioinformatics, dual luciferase reporter gene assay and RIP assay predicted and verified that miR-615-5p could specifically and directly bind to circ_0006789. Moreover, knocking down circ_0006789 in CC cells significantly increased miR-615-5p. Shortly, circ_0006789 participates in miR-615-5p's negative regulation in CC cells through ceRNA activity.
Multiple cellular responses related to tumorigenesis involve HSF1, including alterations in the tumor microenvironment, repairs to the genome, and other critical pathways [
32]. Generally, cancer cells exhibit elevated levels of HSF1 expression [
33]. Additionally, HSF1-associated non-coding RNAs have been identified in various cancer types. In the context of hepatocellular cancer cells, downregulating HSF1 reduces proliferative and anti-apoptotic abilities of tumor cells [
34]. This study confirmed HSF1 overexpression in CC. HSF1 was a target gene of miR-615-5p and could be positively regulated by circ_0006789. Overexpressing HSF1 partially offset the suppressive impact of circ_0006789 knockdown on CC cells. Furthermore, a mouse xenograft model was established to elucidate the in vivo anticancer effects of circ_0006789 knockdown on CC.
The findings of this study provide insights into the molecular mechanism underlying the dysregulation of circ_0006789 in CC and demonstrate that circ_0006789 exerts a carcinogenic influence on CC through the regulation of the miR-615-5p/HSF1 axis. This study provides a new mechanism to understand the pathogenesis of CC and suggests that circ_0006789 may be a target for CC treatment. However, this study has limitations in terms of cell lines, and further studies on the roles of circ_0006789 and miR-615-5p in CC in more cell lines are needed subsequently. In addition, although the mechanism of action of circ_0006789 in CC was analyzed in this study, its potential for clinical diagnosis, prognostic assessment and as a therapeutic target needs to be validated by large-scale clinical trials.
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