Sample acquisition and cell culture
CC tissue samples were collected from Yantai Affiliated Hospital of Binzhou Medical College, China, and the study protocols were approved by the Ethics Committee of Yantai Affiliated Hospital of Binzhou Medical College. CC cell lines (HeLa, CaSki, SW756 and C-33A) and normal cervical epithelial cell lines (HcerEpic) were purchased from the Beijing Beina Chuanglian Biotechnology Research Institute (BNBIO.com). HeLa, C-33A, SW756, and CaSki cells were cultured in 5% CO2 at 37 °C in RPMI-1640 medium (E600028; Sangon, Shanghai, China) with 10% fetal bovine serum (16140071; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in addition to 100 μg/ml streptomycin. HcerEpic cells were cultured in 5% CO2 at 37 °C in MEM medium (E600024; Sangon, Shanghai, China) with 10% fetal bovine serum and 100 μg/ml streptomycin. The characteristics of the patients are shown in Table 1, and the representative histopathological examination results are illustrated in Supplementary Figure 1.
H&E staining
Tissue sections were deparaffinized twice using xylene treatment (10 min each time), and they were re-hydrated by decreasing the alcohol concentration. After washing the tissue sections in distilled water for 1 hour, they were stained by hematoxylin solution for 8 minutes and by eosin for 3 minutes. After that, the tissue sections were dipped in 0.2% saturated lithium carbonate solution for 30 seconds. The eosin solution was then used to stain the tissue sections for 1 minute after washing the sections in running tap water. Finally, the H&E staining images were photographed with the Nikon TE2000-U inverted microscope (Japan).
Cell transfection
The small interfering RNAs of circ_0084927 (si-circ_0084927) and CDK2 (si-CDK2), as well as the negative control siRNA (si-NC), were synthesized by GenePharma (Shanghai, China). Some items were purchased from RiboBio Co., Ltd. (Guangzhou, China), such as miR-1179 control, miR-1179 negative control, miR-1179 mimic (for luciferase reporter gene assay) and miR-1179 inhibitor. HeLa and C-33A cells were transfected with si-NC, miR-1179 inhibitor, si-circ_0084927, si-CDK2, miR-1179 inhibitor plus si-circ_0084927 or miR-1179 inhibitor plus si-CDK2 via Lipofectamine ™ 2000 (11668019; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and via lipofectamine transfection method for 20 minutes. After the cells were incubated for 2 days at 37 °C, they were analyzed by qRT-PCR.
Subcellular location using a nuclei-cytoplasm fractionation method
Before the nuclear and cytoplasmic RNA isolation, nuclear and cytoplasmic fractions were separated using the PARIS Kit (AM1921; Thermo Fisher Scientific, Waltham, Mass., USA). The isolated RNA products in nuclei and cytoplasm were analyzed by qRT-PCR. Then, the expression of circ_0084927 and ESRP1 mRNA was detected in the nuclei and cytoplasm. GAPDH and U2 were subsequently employed as a reference control for cytoplasmic expression and nuclear expression, respectively.
qRT-PCR
The trizol reagents (15596026; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were first used, according to the instruction manual, to isolate and detect total RNA from the tissue samples and cell lines. The obtained RNA was then reverse-transcribed into cDNA. Then, miR-1179 was reverse-transcribed using the protocol of mirVana™ qRT-PCR miRNA Detection Kit (AM1558; Invitrogen™; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The reverse-transcription CDK2 mRNA and circ_0084927 was conducted with SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (; Thermo Fisher Scientific, Inc., Waltham, MA, USA). StepOnePlus Real-Time PCR System (4376600; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was later used to perform qRT-PCR. The qPCR products were then validated using the agarose gel electrophoresis method. Next, the data were analyzed with the 2-ΔΔCt method. GAPDH was then utilized as the internal control of circ_0084927 and CDK2 mRNA, while U6 was used as the internal control of miR-1179
Luciferase reporter gene assay
Oligonucleotides comprising the circ_0084927 mutant (the sequence containing the miR-1179 binding site was mutated to GAUACGA) or the CDK2 mRNA 3'UTR mutant (the sequence containing the miR-1179 binding site was mutated to GAUACGA) were synthesized by GenePharma (Shanghai, China). Inserted into the dual-luciferase miRNA target expression vector (pGL4) were wild-type circ_0084927, circ_0084927 mutant, CDK2 3′UTR mutant, and wild-type CDK2 3’UTR. This insertion was performed to construct luciferase reporter plasmid. HeLa and C-33A cells were also co-transfected with luciferase porter plasmid and miR-1179 mimic. After 48 hours of incubation, the culture medium was removed to collect the cells. The collected cells were then lysed to obtain cell lysates. The luciferase activity was measured by Pierce Renilla-Firefly Luciferase Dual Assay Kit (16185; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the protocol.
RNA immunoprecipitation (RIP) assay
The Hela and C-33A cells transfected with miR-1179 mimic were cultured to an appropriate density. The cultured cells were digested using trypsin and were collected after trypsin treatment. The cells were lysed using RIP lysis buffer. The cell lysates were then incubated with RIP buffer containing magnetic beads coupled with anti-Argonaute2 (MA5-23515; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or IgG for 1 hour, with IgG serving as a negative control. The mixture was subsequently incubated with Proteinase K. After that, the immunoprecipitated RNA was isolated and analyzed using qRT-PCR.
RNA pull-down assay
RNA pull-down assay was conducted to further validate the regulatory binding relationship between miR-1179 and CDK2 mRNA. The biotinylated double-stranded RNA of miR-1179 (Bio-miR-1179) and biotinylated negative control RNA (Bio-NC) were designed by GenePharma (Shanghai, China). Whereas the sense sequence of bio-miR-1179 was 5’- AAGCAUUCUUUCAUUGGUUGG-biotin-3’, the antisense sequence of bio-miR-1179 was 5’-CCAACCAAUGAAAGAAUGCUU-3’. 1×105 Hela and C-33A cells were cultured in 6-well plates for 1 day, resuspended in 1 mL lysis buffer, and incubated in ice for 20 min. The lysate was centrifuged at 12000×g for 15 min before the supernatant was collected. The mixture of bio-miR-1179 or bio-NC and streptavidin-coated magnetic beads (Invitrogen, USA) was added to the supernatant and incubated at 4°C for 2 hours. The pulled-down CDK2 mRNA in the bio-miR-1179 or bio-NC group was detected by qRT-PCR.
CCK-8 assay
After the transfected cells underwent trypsinization, 100 μl of the transfected cell suspension was seeded into a 96-well plate (2x103 cells/well). The plate was then placed in a 37 °C incubator for some hours (24 hours, 48 hours, and 72 hours). Then, 10 μl of CCK-8 solution was added to each well, based on the manual guidelines of CCK-8. After the cells were incubated with CCK-8 for 2 hours, the absorbance was measured at 450 nm.
BrdU incorporation ELISA assay (A colorimetric BrdU assay)
BrdU cell proliferation assay kit was used to detect cell-proliferation ability. Anti-BrdU antibodies were used to detect 5-bromo 2'-deoxyuridine (BrdU), which was incorporated into the cell DNA during cell proliferation. Then, a trypsin-treated suspension containing 104 cells was added to each well of a 24-well plate. The culture medium was changed every 6 hours. After the cells were cultured for 24 hours, 10 μm BrdU (E607203; Sangon, Shanghai, China) was added, and the culturing was continued for 4 hours to allow the proliferating cells to incorporate BrdU into their DNA. The cultured cells were then fixed using the fixing solution and were permeabilized with 0.5% Triton (R) X-100 for 10 minutes. Mouse anti-IgG and anti-BrdU antibodies (diluted at 1:50) were then incubated with the cells overnight at 4 °C. Subsequently, cells were washed with PBST and incubated in the dark with HRP-conjugated secondary antibodies (A24494; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 1:500 in PBS at room temperature. In the end, the absorbance at 450 nm was proportional to the amount of BrdU incorporated into the cell, which directly reflected cell proliferation.
Cell-matrix adhesion assay
Before the cell suspension (10,000 cells/well) was seeded in the 96-well plates (4414133; Thermo Fisher Scientific, Inc., Waltham, MA, USA) that were previously coated with 10 μg/ml type I collagen (C7661, Sigma-Aldrich, USA), the cells were deprived of serum for at least 8 h. After 30 or 60 minutes adherence at 37˚C in a 5% CO2 atmosphere, the wells were washed with PBS for at least three times to remove the non-adherent cells. The remaining cells were then treated with MTT for two more hours at 37 °C. Finally, the MTT-treated cells were treated with 100 µl DMSO. The absorbance recorded using a microplate reader (Benchmark, Bio-Rad, USA) was 570 nm.
Assays for caspase 3 activation
Caspase-3 is an active cell-apoptosis protease and an early indicator of the onset of apoptosis. Colorimetric detection at 405 nm of p-nitroaniline (pNA), after the cleavage from the peptide substrate DEVD-pNA, may reflect the cell apoptosis level. The transfected Hela and C-33A cells (1x105) in different groups were briefly harvested and lysed in 50 ml of ice-cold cell lysis buffer. Cell lysates were centrifuged at 10,000 g for 10 min to obtain the supernatant. Then, 50 μl of 2x Reaction Buffer/DTT Mix and 5 μl of 1 mM DEVD-pNA (substrate for caspase-3) from caspase 3 colorimetric assay kit (630217, Takara Biomedical Technology (Beijing) Co., Ltd., China) were added to the cell lysates. The absorbance was determined by measuring OD405 of the released pNA using a microplate reader (Benchmark, Bio-Rad, USA).
Western blot analysis
Quantitative analysis was performed after all the protein was extracted with RIPA lysis buffer (C500005, Sangon; Shanghai, China) from Hela and C-33A cells in different groups. An equal amount of protein was separated by 10% SDS-PAGE. The gel was immersed in a transfer buffer to achieve equilibrium before transferring it to a polyvinylidene fluoride membrane. Primary antibodies were diluted at a ratio of 1:1000. The membrane was later incubated for 2 hours with diluted primary antibodies against CDK2 (D220395; Rabbit-Human; Sangon; Shanghai, China) and β-actin (SAB5500001; Rabbit-Human; Sigma-Aldrich, China). Following that, the hybrid membrane was blocked with 5% skimmed milk and incubated at 4 °C overnight. Next, the membrane was incubated for 2 hours with diluted secondary antibodies (A32731; Goat-Rabbit; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Finally, a hypersensitive ECL chemiluminescence kit (C510043; Sangon; Shanghai, China) was used to detect proteins according to the reagent instructions. The intensity of the protein bands was read using ImageJ software.
Cell cycle by flow cytometry
The transfected HeLa and C-33A cells were re-suspended once in pre-chilled 1xPBS and were subsequently diluted to 1x105 cells/ml in 1x Annexin binding buffer. In every assay, 100 µl of cell suspension (10,000 cells) was used. The transfected HeLa and C-33A cells were then re-suspended and treated with pure ethanol for 30 minutes. After that, cells were incubated with RNase for 30 minutes not only to remove RNA but also to eliminate the influence of the binding between PI and RNA. Cells were subsequently stained with the red-fluorescent stain, PI (V13242; Thermo Fisher Scientific, Inc., Waltham, MA, USA), in a dark room at room temperature to allow PI to bind to the DNA of the cells. The stained cells were finally put into a flow cytometer before the proportion of cells in each phase of the cell cycle was obtained from the linked BD FACSuite software.
Statistical analysis
With Microsoft Excel, all the means and standard deviations were calculated based on three independent experiments. GraphPad Prism 8.0 (GraphPad Prism, Inc., La Jolla, CA, USA) was used to produce the diagrams. One-factor analysis of variance (ANOVA) test and Student's t-test were used for the statistical analysis between multiple groups and for the statistical analysis of two groups, respectively. In terms of the gene expression in tissue samples, we used the Wilcoxon test for the CC tissue samples and matched adjacent healthy tissue samples for comparison. P<0.05 was considered to be statistically significant, while P< 0.01 was considered to be extremely significant.