Background
Cervical cancer (CC) is the second most frequent malignancies among females worldwide, with about 500,000 new cases diagnosed every year. Although significant advances have been made to protect women against CC (such as HPV vaccines), the prognosis and survival rates of CC patients at advanced stages are extremely poor [
1‐
4]. The molecular mechanism underlying CC carcinogenesis remains unclear. Therefore, the underlying mechanism and novel biomarkers for CC are urgently needed.
Circular RNAs (circRNAs) are a class of novel noncoding RNAs, characterized by a covalently closed continuous loop without any 50 to 30 polarity or a polyadenylated tail [
5,
6]. Increasing studies demonstrated that circRNAs regulate gene expression acting as competing endogenous RNA (ceRNAs), also known as microRNAs (miRNAs) sponges, which sequester miRNAs to terminate the regulation of their target genes [
7‐
11]. Besides, circRNAs play a key role in various biological processes, such as cell proliferation and metastasis [
12], and act as potential biomarkers in many diseases including cancers [
13‐
21]. But the biological or pathological functions of circRNAs in particular cancer remain largely obscure. Further investigation of circRNAs will enable us to better understand the tumorigenesis and improve the diagnosis and therapies of cancers.
Here, we aimed to identify a novel circRNA hsa_circ_0000069 that is clinically relevant to CC and to investigate its role in CC pathogenesis. Using bioinformatics analysis, hsa_circ_0000069 was highly expressed in CC cells and tissues compared with matched normal groups. We firstly found that hsa_circ_0000069 was upregulated in CC, and this high expression promoted the proliferation, migration, and invasion of CC. Mechanically, hsa_circ_0000069 could bind to and sponge miR-873-5p, consequently upregulating the TUSC3 expression and promoting tumor progression.
Methods
Clinical samples
A total of 50 pairs of CC tissues and para-tumor tissues were obtained from the Department of Gynecology, Affiliated Hospital of Nantong University. All specimens were immediately frozen in − 80 °C liquid nitrogen until RNA extraction. The study was approved by the ethical committee of the Affiliated Hospital of Nantong University. Informed consent was obtained from all patients.
Cell culture and transfection
The normal human cervical epithelial cell line End1/E6E7, and human CC cell lines including SiHa, C-4I, HeLa and C-33A were purchased from the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, USA), and cultured at 37 °C in a humidified 5% CO
2 incubator. And small interfering RNAs (siRNAs), miR-873-5p mimics, inhibitors, and their NC negative controls were purchased by Shanghai Biotend Biotechnology Co, Ltd (Shanghai, China). For TUSC3 overexpression, the full-length sequence of TUSC3 was cloned into pcDNA3.1 (Invitrogen, CA, USA) plasmid to generate pcDNA3.1-TUSC3 (Additional file
1: Fig. S1). For transfection, Lipofectamine 2000 (Invitrogen, USA) was used according to the manufacturer’s protocol.
The siRNA sequences for transfection were following
hsa_circ_0000069-siRNA-#1, 5′-CTACTTCAGGCACAGGTCT-3′;
hsa_circ_0000069-siRNA-#2: 5′-CTTCAGGCACAGGTCTTC-3′;
scramble-siRNA, 5′-GGACUCUCGGAUUGUAAGAUU-3′.
CircInteractome
A predicted binding site of miR-873-5p within hsa_circ_0000069 by bioinformatic analysis using the CircInteractome database (
https://circinteractome.nia.nih.gov/) as standard procedures [
22], which based on the Targetscan algorithm, was an online software to predict the binding sites of circRNAs and miRNAs. The data provided in CircInteractome are predicted based on sequence matches.
Transwell assay
Transwell assay was conducted for the detection of cell migration and invasion. After 48 h of transfection, 1 × 105 cells in 200 μl of serum-free medium were placed in the upper chamber (8.0 μm pore size; Corning, USA; Catalog number 3422) with a porous membrane with Matrigel solution (BD, USA) for invasion assay, while the lower chamber was inserted into a 12-well filled with 600 μl medium added with 10% FBS. After 24 h of incubation at 37 °C, noninvasive cells were removed from the upper surface of the membrane with cotton swabs, and invasive cells on the lower membrane surface were fixed with 4% formaldehyde and stained with 0.1% crystal violet (Beyotime, China). Five random 200 × visual fields per well were photographed and calculated under a Nikon Inverted Research Microscope Eclipse Ti microscope. Cell migration assay was simultaneously conducted as above, except for the chambers without Matrigel.
Cell counting kit-8 (CCK-8) assay
2 × 103 cells were seeded into 96-well plates and incubated for 0, 24, 48 and 72 h, respectively. Then, 10 μl CCK-8 solution (Dojindo, Japan) was added and incubated in the dark at 37 °C for another 1 h. The absorbance was detected using the microplate reader (Synergy H4 Hybrid Reader, BioTek, USA) at a wavelength of 450 nm at indicated time points using the microplate reader (Synergy H4 Hybrid Reader, BioTek, Winooski, USA). Each data point is the mean ± SD. of three independent experiments.
A total of 500 cells were seeded into 6-well plates. After the cells were grown for 2 weeks, and then fixed with 4% paraformaldehyde, and stained with 0.4% crystal violet (Beyotime, China) for 30 min, and colonies were counted under the microscope.
In vivo xenograft experiments
BALB/c nude female mice aged 6 weeks were to perform xenograft experiments. All animal protocols were approved by the Institutional Animal Care and Use Committee at the Affiliated Hospital of Nantong University. In brief, 1 × 107 SiHa and HeLa cells transfected with the indicated siRNA using the in vivo transfection reagent, JetPEI (Polyplus Transfection, Illkirk, France) were subcutaneously injected into the flank. Mice were monitored daily, and caliper measurements began once tumors became visible. The tumor volume was measured every 7 days via calipers, which were calculated using the following formula: Tumor volume (mm3) = (height) × (width)2/2. After 35 days, mice were sacrificed, and tumors were dissected and weighed. Tumor tissues were collected and snap frozen in liquid nitrogen and stored at − 80 °C for subsequent analyses.
Luciferase reporter assays
5 × 104 cells were seeded in 24-well plates the day before transfection. Then cells were transfected with Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s instructions. After 48 h of transfection, luciferase activities were analyzed using the Dual-Luciferase Reporter Assay System (Promega, USA).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the collected tumor samples and cells using TRIzol reagent (Invitrogen, USA) following the instruction of the manufacturer. Gene expression was analyzed with SYBR Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA, Catalog number 4309155) in an ABI 7900 Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific, Inc. USA), and GAPDH was served as an internal control. The relative mRNA expression was calculated using the 2−ΔΔCt method. The primers for qRT-PCR were as follows:
GAPDH F: 5′-AAGGTGAAGGTCGGAGTCA-3′;
R: 5′-GGAAGATGGTGATGGGATTT-3′;
hsa_circ_0000069 F: 5′-CTACTTCAGGCACAGGTCTTC-3′;
R: 5′-CTGACTCACTGGATGAGGACT3′;
miR-873-5p F: 5′-GCATGGCAGTGGTTTTACCCTA -3′;
R: 5′-ATCCAGTGCAGGGTCCGAGG -3′;
TUSC3 F: 5′-GAACGGATGTTCATATTCGGGT-3′
R: 5′-CGCTTAAAGCAAACCTCCAACAA-3′;
U6 F: 5′-CTCGCTTCGGCAGCACA-3′;
R: 5′-AACGCTTCACGAATTTGCGT-3′.
Cellular nucleo-cytoplasmic fractionation
Cells were fractionated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher, USA) following the manufacturer’s protocol. CC Cells (5 × 106/sample) were re-suspended in buffer C (20 mM Tris–HCl pH 7.5, 75 mM NaCl, 5 mM MgCl2, 0.5% p/w sodium deoxycholate, 0.2% Triton, 1 mM DTT, 0.5% glycerol) added protease inhibitor cocktail (Sigma, USA) and 1 U/μL RNase inhibitor (Thermo Scientific, USA). After centrifugation, supernatants were collected (cytoplasmic lysates). Then pelleted nuclei were washed extensively and carefully (4 × 200 μL) with 1 × PBS. Pelleted nuclei were resuspended in buffer N (10 mM Tris–HCl pH 8, 25 mM NaCl, 5 mM MgCl2, 1% p/w sodium deoxycholate, 1% Triton, 0.2% SDS, 1 mM DTT) added protease inhibitors and RNase inhibitors, and consequently sonicated. RNA from each portion was isolated as above.
Pull-down assay
hsa_circ_0000069 and Negative Control (NC) were biotinylated to be bio- hsa_circ_0000069, and bio-NC by GenePharma Company (Shanghai, China). Next, they were transfected into SiHa and Hela cells for 48 h, cells were collected and incubated with Dynabeads M-280 Streptavidin (Invitrogen, USA) for 10 min. After cells were washed with buffer, the bound RNAs were quantified and analyzed by qRT-PCR.
Western blot analysis
When transfection finished, SiHa and Hela cells were lysed in RIPA buffer (Beyotime, China). Total protein concentration was determined with the BCA Protein Assay kit (Beyotime, China). Next, proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then incubated with primary antibodies (TUSC3, ab77600, dilution 1:1000; PCNA, ab92552, dilution 1:2000; Ki67, ab92742, dilution 1:1000; Cyclin D, ab226977, dilution 1:1000; CDK1, ab32094, dilution 1:2000; E-cadherin, ab15148, dilution 1:500; N-cadherin, ab18203, dilution 1:1000; MMP9, ab76003, dilution 1:1000; GAPDH, ab181602, dilution 1:10000) at 4 °C overnight. The secondary antibodies were selected according to each primary antibody’s instructions. All these antibodies were purchased from Abcam (Cambridge, UK). An Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore) was used for detection. The protein bands were quantified with the ImageJ software (USA).
Statistical analysis
Statistical analysis was conducted using Microsoft Office Excel 2016. The significance of difference was evaluated with Student’s t test in two groups. One-way ANOVA was used in more than two groups and different times points. P values less than 0.05 were considered significant (*P < 0.05; **P < 0.01). The data present the mean ± SD. of three independent biological experiments.
Discussion
CircRNAs are a type of endogenous RNA that regulates gene expression at the post-transcriptional or transcriptional level through sponging miRNAs expression and their target genes [
5‐
11]. Emerging evidence demonstrated that the circRNAs act as an oncogene in various cancers, affecting the proliferation and invasion capability of cancers [
12‐
21]. Thus, CircRNAs can serve as a biomarker for cancers. Cervical cancer (CC) is the second leading cause of cancer deaths in females worldwide. Due to the lack of effective therapies, the overall prognosis and survival rate of CC patients is very low [
1‐
4]. Therefore, it is urgent to explore the exact mechanism and identify novel biomarkers to develop novel therapeutic strategies for CC.
MiR-873-5p was recently identified as a tumor suppressor, which directly repressing TUSC3 and inhibiting the TUSC3/AKT pathway in cancers, thus regulating cancer cell proliferation, colony formation, and invasion [
24‐
27]. Tumor suppressor candidate 3 (TUSC3) was reported to be upregulated and correlated with tumor progression and prognosis, which could be used to predict prognosis in cancer patients [
25,
27,
28]. It has been reported that TUSC3 accelerates cancer proliferation and induces epithelial-mesenchymal transition by upregulating claudin-1 in non-small-cell lung cancer cells [
27]; Besides, TUSC3 plays an oncogenic role in non-small cell lung cancer and participates in hedgehog signaling pathway [
29]; TUSC3 also regulates the proliferation, migration and invasion of breast cancer cells via SOX2/miR-181a-5p, miR-30e-5p/TUSC3 axis [
30]. Thus, TUSC regulates multiple malignant processes of cancer development including tumor proliferation, migration and invasion.
In our study, we firstly analyzed the biofunction of hsa_circ_0000069 and the clinical relevance in CC progression. With the array analysis, we found that hsa_circ_0000069 was obviously upregulated in CC cells and tissues, and negatively associated with the lymph node metastasis and survival rate of CC patients, suggesting that hsa_circ_0000069 may act as an oncogene in CC. To validate our hypothesis, we analyzed the function of hsa_circ_0000069 in CC cell proliferation, migration, and invasion. As expected, the knockdown of hsa_circ_0000069 robustly inhibited CC cell proliferation, migration, and invasion. More importantly, we found that hsa_circ_0000069 can directly bind to and inhibit miR-873-5p function in CC. The knockdown of miR-873-5p promotes CC progression, indicating that hsa_circ_0000069 may promote CC development through sponging miR-873-5p. Moreover, miR-873-5p can bind to and inhibit the TUSC3 function. We also found that the knockdown of hsa_circ_0000069 inhibited both mRNA and protein levels of TUSC3, while the miR-873-5p inhibitor rescued the inhibitory effect on TUSC3 expression, as well as overexpression of TUSC3 can restore the proliferation, migration, and invasion defects resulted from hsa_circ_0000069 deficiency. Overall, our results confirm for the first time that a new circRNA hsa_circ_0000069 regulates TUSC3 expression through miR-873-5p. This indicates that hsa_circ_0000069 plays a key role in the development of CC, through the function of TUSC3 as regulated by miR-873-5p. The exact regulatory mechanism of this axis in tumorigenesis and its important function in other cancer types still need our further study. It will be interesting to explore whether hsa_circ_0000069 can also regulate the tumor apoptosis or tumor microenvironment through this mechanism.
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