There were 68 cases of cervical squamous cell carcinoma patients included in this study. All the patients were treated with surgery between January 2015 and December 2016. None of the patients were treated with any prior anticancer therapies and were histopathologically confirmed as cervical squamous cell carcinoma. Ethics Committee of Guangzhou First People’s Hospital approved this study. All the patients were informed and consent was obtained by all the patients.
All methods were carried out in accordance with relevant guidelines and regulations.
RNA-seq data of CESC
RNA-seq data of CESC was downloaded from the TCGA database. The data was retrieved from the TCGA using perl software and analyzed using R software.
Cell line and cell culture
The human CESC cell lines (SiHa, Hela and C33A) and human keratinocytes cell line HacaT cells were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and were cultured in RMPI1640 or DMEM supplemented with 10% fetal bovine serum (FBS).
The short hairpin RNA (shRNA) targeting HOXC-AS3 and scrambled negative controls were purchased from GenePharma (Shanghai, China). The cell transfection was conducted using Lipofectamine 2000 Transfection Reagent (Invitrogen) for 24 h according to the manufacturer’s instructions.
To construct an overexpression plasmid, cDNA sequence of HOXC-AS3 or FBXL17 was subcloned into the pcDNA3.1 expression vector by GeneCoppeia (Guangzhou, China). C33A cells were infected with lentivirus in the presence of polybrene and selected with puromycin to establish stale cells
Nucleus and cytoplasm fraction isolation
Cell cytoplasmic and nuclear fractions were isolated using Nuclear/Cytosol Fraction Kit (Biovision, CA, USA) following the manufacturer’s instructions. Briefly, the cells were incubated with cytosolic extraction buffer for 15 mins. After centrifugation, the cytosol supernatants were harvested and the following nuclear fractions were incubated with nuclear extraction buffer. After centrifugation, the nuclear fraction supernatants were collected. And the nucleus and cytoplasm fractions were used for the real time quantitative PCR. GAPDH and U6 were used as endogenous controls for normalization.
Real time quantitative PCR (qRT-PCR)
Total RNA from tissues and cell lines were extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The concentration of total RNA was assessed using NanoDrop 2000 (Thermo). The cDNA was synthesized using Takara cDNA Synthesis Kit. The resulting cDNA was performed to qRT-PCR on an Applied Biosystems 7900HT Fast Real-Time. The relative mRNA levels of genes were calculated based on the Ct value and normalized to the internal control GAPDH or U6. The primers used for qRT-PCR were as follows: 5’-CGTGCGCTCTGTAAAGG-3’ (F) and
5’-AGCCTCGGTCAGTTTGG-3’ (R) for HOXC-AS3;
5’-ACTGGCGTGACCTTTGTTTAG-3’ (F) and 5’-CAGACATACTGCGACAATCAGAA-3’ (R) for FBXL17;
5’-AAACAGTAGTGGAGAATCAAGCC-3’ (F) and
5’-AGAACTCGAACCAGATCCAGAG-3’ (R) for E-cadherin; 5’-AGTCAGTCGGAAAGTGAGCAG-3’ (F) and
N-cad-R 5’-ACATCAGCTATCCGTTCCTTCT-3’ (R) for N-cadherin;
5’-TGTGCCTCAGATCAACTGCG-3’ (F) and
5’-GCATACACGTCAGTGCCATAAA-3’ (R) for Vimentin; 5’-CTCCTCCTGTTCGACAGTCAGC-3’ (F) and 5’-CCCAATACGACCAAATCCGTT-3’ (R) for GAPDH; 5’-TTATGGGTCCTAGCCTGAC-3’ (F) and 5’-CACTATTGCGGGCTGC-3’ (R) for U6.
Total protein was extracted from tissues and cell lines using RIPA solution as previously described. The resulting protein lysates were separated by 10% sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE). After being transfected to a PVDF membrane and blocked, the membrane was incubated with the first antibodies at 4℃ overnight, which including Anti-E-cadherin (AF0131), anti-N-cadherin (AF4039) and anti-Vimentin (AF7013). All the antibodies were purchased from Affinity Biosciences. After washing, the membrane was incubated with secondary antibodies. Then the results were developed using an enhanced chemiluminescence detection system.
The cell proliferation was assessed using Cell Counting Kit-8 assay. Indicated cells were seeded in a 96-well plate at a concentration of 5,000 cells per well and collected at different time point, including 0, 24, 48, 72 h. After being incubated with CCK8 reagent, the absorbance was measured using a microplate reader at a wavelength of 450 nm.
In vitro cell migration and invasion were investigated using a 24-well transwell inserts (8.0 µm, Corning). Briefly, for cell migration, 5×104 cells were suspended in 200µl serum free culture medium and placed in the upper chamber. For cell invasion, 5×104 cells were placed in the insert pre-coated with Matrigel. After incubation for 48h, the cells adhered to the lower surface of the membrane were stained with 0.1% crystal violet solution. The cells migrated or invaded in five randomly selected fields were calculated under a light microscope. Magnification, X100.
The potential targets of HOXC-AS3 were predicted using on a R software.
The correlation between HOXC-AS3 expression and FBXL17 expression was confirmed on an online tool GEPIA (http://gepia.cancer-pku.cn/).
The data were analyzed using GraphPad Prism 7 software. The comparison between two groups was calculated using student’s t test. The correlations between HOXC-AS3 expression and clinicopathological parameters were assessed using chi-square test. The correlation between HOXC-AS3 expression and overall survival was calculated using Kaplan-Meier survival curve with log-rank test. A two tailed P value < 0.05 was considered statistically significant.