Forty-six cervical cancer patients who underwent surgical treatment in Xinjiang Uygur Autonomous region Maternal and Child Health 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-33 A, 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 trypsinization. We selected C-33 A and Hela cells as auxiliary research objects since their large expression differences.

C-33 A and HeLa cells (at the logarithmic growth stage) were trypsinized by 0.25% trypsin and transited into 6-well plates (5 × 106 per well). After cell growth was stable, they were transfected with the 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 according to the instructions of FuGENE® HD Transfection Reagent (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. The 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-Aldrich, St. Louis, Missouri, USA). The reverse transcription reaction (for FBXL19-AS1 and COL1A1) 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. For the cDNA synthesis of miR-193a-5p, a commercially purchased HyperScript III miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Cat.No.R601, NovaBio, Shanghai, China) was used, and all procedures were performed according to the instructions of the manufacturer. The fluorescence quantitative PCR reaction system was 25 µL, containing 500 ng cDNA template, 250 nmol L−1 reverse and forward primers, and 12.5 µL of 2×SYBR Green PCR Master mixture (Solarbio, Beijing, China). β-actin 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 β-actin: forward 5′-CTCCATCCTGGCCTCGCTGT-3′, reverse 5′-CCAAGGAGTAAGACCCCTGG-3′; primers for U6, forward 5′-TCTTCGTCATCACATATACTAAAAT-3′, reverse 5′-CTCTTCACGAATTTTCGTGTCAT-3′. The reaction tube was placed into the MX3000P Real-time PCR reaction instrument (Agilent, California, USA), and the reaction conditions were: 94 °C, 55 °C, 72 °C, 45 cycles, with fluorescence signal monitoring. The 2(−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 β-actin)-in the control group (Ct target gene − Ct β-actin).

C-33 A 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 as 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 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-h incubation, the absorbance (A) value of each well was evaluated and recorded with Multiskan FC Microplate Reader (Thermo Fisher, MA, USA) at the wavelength of 450 nm.

We applied the Total Protein Extraction [Cat. no. C006225-0050, Sangon Biotech (Shanghai) Co., Ltd.] to extract the total proteins of different cell groups. BCA method (Beyotime, Shanghai, China) was applied for protein concentration SDS-PAGE electrophoresis was taken to isolate the total protein with 50 µg total proteins per pore. After 2-h electrophoresis, the proteins were wet transferred to PVDF membranes, sealed with 5% skim milk powder (1 h) 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 (39,952, 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, Abcam, USA) (1 h). The protein bands were exposed by Chemistar™ High-sig ECL Western Blotting Substrate (TANON SCIENCE & TECHONLOGY CO., Shanghai, China). The experiment was repeated three times.

The C-33 A and HeLa CC cells were seeded into the Transwell chambers (2.5 × 104 cells per 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-Aldrich, St. Louis, Missouri, USA) was laid inside the chamber to simulate extracellular matrix (final concentration 2 mg mL−1, 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-h 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 1 × PBS 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.

All luciferase reporter vectors (FBXL19-AS1-MT, FBXL19-AS1-WT, COL1A1-MT, COL1A1-WT) were constructed by Promega (Madison, WI, USA). C-33 A 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-33 A 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.

For evaluating the role of FBXL19-AS1 on the growth of CC cells, 5 × 106 C-33 A cells transfected FBXL19-AS1 overexpression plasmids or the negative control (NC) were subcutaneously injected into the right side of Balb/c nude mice (6-week-old, 22–25 g). During the next four days of incubation, the tumor volume was measured every week and calculated (the volume calculating formula: 0.5 × length × width2). At the 28th day, the mice got anaesthesia (Phenobarbital sodium, 50 mg kg−1) and sacrificed. Then the formed tumors were weighted and subjected to immunohistochemistry for detecting the expression of E-cadherin, Vimentin and Snail referred to [24]. All nude mice were fed in a specific pathogen free (SPF) environment free for food and water (12-dark and 12-night in a cycle, 25 ℃, 50% humidity). All animal experiments were conducted following national and international guidelines and approved by the Animal Ethic Committee of Xinjiang Uygur Autonomous region Maternal and Child Health Hospital.

We applied SPSS17.0 statistical software (SPSS Inc., Chicago, IL, USA) to analyze the results. Measurement data were presented as mean ± standard deviation (x ± sd). We employed the t-test for the mean of two sample groups and the one-way analysis of variance (ANOVA) followed by Tukey post hoc test for the analysis of multiple samples. Pearson’s Correlation Analysis analyzed the correlation between the FBXL19-AS1 and miR-193a-5p level in CC tissues. Chi square test was used for analyzing the relationship of FBXL19-AS1 with the clinicopathological features of CC patients. p < 0.05 was considered to be statistically valuable.