Sample acquisition and Cell culture
CC tissue samples were collected from Yantai Affiliated Hospital of Binzhou Medical College, 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 line (HcerEpic) were purchased from the BNBIO (Beijing, China). HeLa, C-33A, SW756 and CaSki cells were cultured under 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), 100 μg/ml streptomycin. HcerEpic cells were cultured under 5% CO2 at 37 °C in MEM medium (E600024; Sangon, Shanghai, China) with 10% fetal bovine serum, 100 μg/ml streptomycin.
The small interfering RNAs of circ_0084927 (si-circ_0084927) and CDK2 (si-CDK2), and a negative control siRNA (si-NC) were synthesized by GenePharma (Shanghai, China). miR-1179 control, miR-1179 negative control, miR-1179 mimic (for luciferase reporter gene assay) and miR-1179 inhibitor were purchased from RiboBio Co., Ltd. (Guangzhou, China). 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) through lipofectamine transfection method for 20 minutes. Cells were incubated for 2 days at 37 °C, then analyzed by qRT-PCR.
Subcellular location using a nuclei-cytoplasm fractionation method
The separation of nuclear and cytoplasmic fractions was conducted prior to nuclear and cytoplasmic RNA isolation using the PARIS kit (AM1921; Thermo Fisher Scientific, Waltham, Mass., USA). The isolated RNA products in nuclei and cytoplasm were analyzed by qRT-PCR. The expression of circ_0084927 and ESRP1 mRNA was detected in nuclei and cytoplasm. GAPDH was used as a reference control for cytoplasmic expression, and U2 was used as a reference control for nuclear expression.
We first used Trizol reagents (15596026; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the instructions to isolate and detect total RNA from the tissue samples and cell lines. The obtained RNA was then reverse transcribed into cDNA. MiR-1179 was reverse transcribed following the protocol of mirVana™ qRT-PCR miRNA Detection Kit (AM1558; Invitrogen™; Thermo Fisher Scientific, Inc., Waltham, MA, USA). 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 used to perform qRT-PCR. The qPCR products were validated using agarose gel electrophoresis method. The data were analyzed with 2-ΔΔCt method. GAPDH was used as the internal control of circ_0084927 and CDK2 mRNA. 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). circ_0084927 wild type, circ_0084927 mutant, CDK2 3′UTR mutant and CDK2 wild-type were inserted into the Dual-Luciferase miRNA target expression vector (pGL4) to construct luciferase reporter plasmid. HeLa and C-33A cells were 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 lysed to collect 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 cell culture was digested using trypsin and collected after trypsin treatment. The cells were lysed by RIP lysis buffer. Cell lysates were 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 as a negative control. The mixture was then incubated with Proteinase K. The immunoprecipitated RNA was isolated and analysed 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 besides of the dual luciferase reporter gene assay. Firstly, Biotinylated double-stranded RNA of miR-1179 (Bio-miR-1179) and biotinylated negative control RNA (Bio-NC) were designed by GenePharma (Shanghai, China). 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 one day, resuspended in 1 mL lysis buffer, and incubated for 20 min on ice. The lysate was centrifuged at 12000×g for 15 min, and 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 for 2 hours incubation at 4°C. The pulled-down CDK2 mRNA in the bio-miR-1179 or bio-NC group was detected by RT-qPCR.
After the transfected cells were trypsinized, 100 μl of the transfected cell suspension was seeded into a 96-well plate (2x103 cells/well). The plate after cell seeding is placed in a 37 °C incubator for the appropriate time (24 hours, 48 hours, and 72 hours). Then 10 μl of CCK-8 solution was added to each well according to the procedures in the manual 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 detection kit was used to detect cell proliferation ability. Anti-BrdU antibodies were used to detect 5-bromo 2'-deoxyuridine (BrdU) incorporated into cell DNA during cell proliferation. Briefly, 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 permeabilized with 0.5% Triton (R) X-100 for 10 minutes. Mouse anti-IgG and anti-BrdU antibodies (diluted at 1:50) were incubated with the cells overnight at 4 °C. Subsequently, cells were washed with PBST and incubated with HRP-conjugated secondary antibodies (A24494; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 1:500 in PBS at room temperature in the dark. 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 96-well plates (4414133; Thermo Fisher Scientific, Inc., Waltham, MA, USA) previously coated with 10 μg/ml type I collagen (C7661, Sigma-Aldrich, USA), the cells were deprived from serum for at least 8 h. After 30 minutes to 1-hour 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 treated with MTT for an additional 2 h at 37 °C. Finally, the MTT-treated cells were treated with 100 µl DMSO. The absorbance at 570 nm was assessed at 570 nm using a microplate reader (Benchmark, Bio-Rad, U.S.A.).
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 cleavage from the peptide substrate DEVD-pNA may reflect the cell apoptosis level. Briefly, the transfected Hela and C-33A cells (1x105) in different groups were 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. determined by measuring OD405 of the released pNA using a microplate reader (Benchmark, Bio-Rad, U.S.A.).
Western blot analysis
Quantitative analysis was performed after total 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 for equilibrium, and then transferred to a polyvinylidene fluoride membrane. Primary antibodies were diluted at a ratio of 1:1000. The membrane was incubated with diluted primary antibodies against CDK2 (D220395; Rabbit-Human; Sangon; Shanghai, China) and β-actin (SAB5500001; Rabbit-Human; Sigma-Aldrich, China) for 2 hours. The hybrid membrane was then blocked with 5% skimmed milk and incubated at 4 °C overnight. The membrane was subsequently incubated with diluted secondary antibodies (A32731; Goat-Rabbit; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 2 hours. Finally, a hypersensitive ECL chemiluminescence kit (C510043; Sangon; Shanghai, China) was used to detect protein 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 resuspended once in pre-chilled 1xPBS, then were diluted to 1x105 cells/ml in 1x Annexin binding buffer. 100 µl of cell suspension (10,000 cells) was used in every assay. Briefly, the transfected HeLa and C-33A cells were resuspended and treated with absolute ethanol for 30 minutes. Cells were then incubated with RNase for 30 minutes to remove RNA to eliminate the influence of the binding between PI and RNA. Cells were then 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 DNA of cells. The stained cells were put into a flow cytometer, and the proportion of cells in each phase of the cell cycle was obtained from the linked BD FACSuite software.
All means and standard deviations were calculated based on three independent experiments using Microsoft Excel. GraphPad Prism 8.0 (GraphPad Prism, Inc., La Jolla, CA, USA) was used to produce the diagrams. One-factor analysis of variance (ANOVA) test was used for statistical analysis between multiple groups. Student's t test was used for statistical analysis of two groups. In terms of gene expression in tissue samples, we used Wilcoxon test for the CC tissue samples and matched adjacent healthy tissue samples comparison. P< 0.05 was considered to be statistically significant, and P< 0.01 was considered be extremely significant.