Forty-six cervical cancer patients who underwent surgical treatment in our hospital from October 2014 to October 2015 were selected. No radiotherapy or chemotherapy or other anti-tumor treatment was given to the patients before surgical treatment. The patients had an average age of 52 years, with the youngest aged 32 and the oldest 69 years. All subjects in this study have signed informed consent, and the research ethics committee of Xinjiang Uygur Autonomous region Maternal and Child Health Hospital approved the study.
Human healthy cervical cells (HUCEC) and human cervical cancer cells (HeLa, Caski, C-33A, AV3) were purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured in an RPMI1640 (Thermo Fisher Scientific, MA, USA) medium containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, MA, USA) and 1% penicillin/streptomycin (Invitrogen, CA, USA) at 37 ℃ with 5% CO2 volume fraction. During the logarithmic growth phase, 0.25% trypsin (Thermo Fisher HyClone, Utah, USA) was used for cell passage. We selected C-33A and Hela cells as auxiliary research objects since their large expression differences.
Cells, taken from the logarithmic growth stage, were trypsinized and transited into 6-well plates (5 × 106 per well). After cell growth was stable, they were transfected. Over-expressed FBXL19-AS1 plasmids, small inference RNA targeting FBXL19-AS1 (Si-FBXL19-AS1) or COL1A1 (si-COL1A1), miR-193a-5p mimics, miR-193a-5p inhibitor (anti-miR193a-5p and their negative controls were transfected into C-33A and HeLa respectively according to the instructions of FuGENE®HDTransfectionReagent (Roche, Shanghai, China). At 37 ℃ with 5% CO2, the cells were cultured in an incubator. After 24 h of transfection, the medium was changed with fresh compete medium and the cells were kept incubated for another 48 h. Total RNA of the cells was extracted for real-time fluorescent quantitative PCR (RT-PCR) to detect the transfection efficiency. After successful transfection, they were used in subsequent experiments.
TRIzol method was performed to extract total RNA from CC tissues and cells routinely, and the extracted total RNA was used to remove genomic DNA by deoxyribonuclease I (Sigma). The reverse transcription reaction was carried out according to the operation procedure of the reverse transcription kit (Thermo, Shanghai, China), and the reaction conditions were: 70 ℃, 10 min; 5 min on ice; 42 ℃, 60 min; 95 ℃, 5 min; 0 ℃, 5 min. The fluorescence quantitative PCR reaction system was 25 µL, containing 500 ng cDNA template, 250 nmol/L reverse and forward primers, and 12.5 µL of 2 × SYBR Green PCR Master mixture (Solarbio). GAPDH and U6 were used as the endogenous control. Primers for FBXL19-AS1: forward 5'-CCCATTGTCCCCATTTTGCA-3 ', reverse 5'-AGGCAGCAGGAATCAGTCTT-3'; miR-193a-5p primers: forward 5'-CAGTGCAGGGTCCGAGGT-3 ', reverse 5'-AACAATTGGGTCTTTGCGGGC-3'; Primers for COL1A1: forward 5'-CCTGGATGCCATCAAAGTCT-3 ', reverse 5'- AATCCATCGGTCATGCTCTC-3'. Primers for GHPAD: forward 5'-TGGTTGAGCACAGGGTACTT-3 ', reverse 5'-CCAAGGAGTAAGACCCCTGG-3'. The reaction tube was placed into the MX3000P Real-time PCR reaction instrument, and the reaction conditions were: 94 meters, 55 meters, 72 meter, 45 cycles, with fluorescence signal monitoring. The 22(−ΔΔCt) value indicates the relative expression of genes, and Ct value represents the number of amplification cycles passed when the fluorescence signal of the amplified product reached the set threshold during the PCR amplification process. Δ Δ process sample under test (Ct target gene - Ct GAPDH) - in the control group (Ct target gene - Ct GAPDH).
C-33A and HeLa CC cells in the exponential growth phase were taken and made into a single-cell suspension. After being counted, cell density was adjusted (1000 cells per well). We inoculated them in 96-well plates (6 replicates in each group, 6 well plates in total) and cultured for 12 h, 24 h, 48 h, 72 h and 96 h. After adherent culture, with the addition of 90 µL medium and 10 µL CCK-8 solution (Beyotime, Shanghai, China) into the samples, blank control wells containing only CCK-8 solution and medium were set. After a 2-hour incubation, the absorbance (A) value of each well was evaluated and recorded with a Microplate Reader at the wavelength of 450 nm.
We applied the protein extraction kit to extract the total proteins of different cell groups. BCA method was applied for protein concentration SDS-PAGE electrophoresis was taken to isolate the total protein with 50 µg total proteins per pore. After 2-hour electrophoresis, the proteins were wet transferred to PVDF membranes, sealed with 5% skim milk powder (1 hour) and incubated with primary antibodies of Anti-Bax antibody (ab32503, 1:1000, abcam, USA), Anti-Bcl2 antibody (ab59348, 1:1000, abcam, USA), Anti-Cleaved Caspase3 antibody (ab2302, 1:1000, abcam, USA), Anti-E Cadherin antibody (ab40772, 1:1000, abcam, USA), Anti-Vimentin antibody (ab92547, 1:1000, abcam, USA), Anti-SNAIL antibody (ab53519, 1:1000, abcam, USA), Anti-COL1A1 antibody(39952, Cell Signaling Technology, USA), Anti-beta Actin antibody(ab8227, 1:1000, abcam, USA) overnight at 4 ℃. The next morning, the membranes were rinsed with TBST and incubated at 37 ℃ with the addition of horseradish peroxidase-labeled Goat Anti-rabbit (ab6721, 1:300, USA) (1 hour). The protein bands were exposed by Chemistar™ High-sig ECL Western Blotting Substrate. The experiment was repeated three times.
The C-33A and HeLa CC cells were seeded into the Transwell chambers (2.5 × 104 cells/well) 24 h after transfection. The cells in the upper chambers were resuspended in a serum-free medium and the lower chambers were added with medium containing 20% serum. For invasion experiments, a layer of Matrigel (Sigma) was laid inside the chamber to simulate extracellular matrix (final concentration 2 mg/ml, diluted with serum-free medium, 40 µL each chamber, 37 ℃, 30 min to 1 h until gelation), while for migration experiments, no Matrigel was added to the chamber. After 24-hour cell migration and invasion, the cells were removed, secured with a mixture of formaldehyde and acetic acid for 15 min, rinsed with PBS, stained with crystal violet, and washed with 1XPBS after staining. Finally, the cells in the chamber were wiped off with cotton swabs, and the quantity of those cells invaded and migrated were counted under the microscope.
Dual luciferase activity experiment.
All luciferase reporter vectors (FBXL19-AS1-MT, FBXL19-AS1-WT, COL1A1-MT, COL1A1-WT) were constructed by Promega (Madison, WI, USA). C-33A cells (4.5 × 104) were inoculated in a 48-well plate and cultured till 70% confluence. Then, we used lipofectamine 2000 to co-transfect those luciferase reporter vectors with miR-193a-5p mimics or negative control in C-33A cells. Forty-eight hours after transfection, the luciferase activity was detected following manufacturer's instructions of the dual luciferase reporter assay system (Promega, Wisconsin, USA). All experiments were made in triplicate and repeated three times.
We applied SPSS17.0 statistical software (SPSS Inc., Chicago, IL, USA) to analyze the statistics. Measurement data were presented as mean ± standard deviation (x ± s). We employed the t-test for the mean of two sample groups and the one-way analysis of variance for the mean of multiple samples. P < 0.05 was considered to be statistically valuable.