Research subjects
Our study included 65 patients with CSCC and 58 healthy females who were admitted by Wenzhou Medical University from May 2016 to May 2018. All CSCC patients were diagnosed by histopathological examinations. Among the 65 patients, 40 were HPV positive and 25 were HPV negative. Inclusion criteria of CSCC patients: 1) first time diagnosis; 2) no history of malignancy; 3) no therapy initiated before admission. Exclusion criteria: 1) with other clinical disorders besides CSCC; 2) patients transferred from other hospitals; 3) received any treatments within 3 months before admission. Age of the 65 patients with CSCC ranged from 30 to 61 years, with a mean age of 45.0 ± 4.2 year. Fifty-eight healthy females were enrolled in the physical health center of Wenzhou Medical University, and physical health parameters of them were all within normal range. Age of the 58 healthy females ranged from 32 to 64 years, and the mean age was 45.9 ± 4.3 year. This study was approved by Wenzhou Medical University Ethics Committee. All patients and healthy females signed informed consent.
Specimens and cell lines
Fasting blood (5 ml) was extracted from each participant before therapies. Blood was centrifuged for 12 min at 1200 g in EDTA tubes to prepare plasma samples. C33A (HPV negative) and SiHa (HPV positive) cell lines were used in this study to perform in vitro experiments. These 2 cell lines were bought from ATCC (USA) and were cultivated in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS) in a 37 °C and 5% CO2 incubator.
Real-time quantitative reverse transcription PCR (RT-PCR)
To detect the expression of GATA6-AS, total RNAs were extracted using Trizol reagent (Invitrogen, USA), SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) was used to perform reverse transcriptions and Applied Biosystems™ PowerUp™ SYBR™ Green Master Mix was used to prepare all PCR reaction systems with 18S rRNA as endogenous control.
To detect the expression of miR-205, miRNAs were extracted using miRNeasy Kit (Qiagen). MiRNA reverse transcriptions were performed using TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems), and PCR reaction systems were prepared using TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific). U6 was used as the endogenous control. 2-∆∆CT method was used to perform all data normalizations.
Transient transfection
Scrambled negative control miRNA and MISSION® microRNA Mimic hsa-miR-205 were purchased from Sigma-Aldrich (St. Louis, MO, USA). GATA6-AS full-length genomic DNA was inserted into the pcDNA3.1 vector to construct GATA6-AS expression vector. Lipofectamine 2000 reagent (Invitrogen, USA) was used to perform transient transfection with vectors and miRNAs at doses of 10 nM and 40 nM, respectively. Cells with no transfection were used as control cells. Cells transfected with negative control miRNA or empty vectors were used as negative control cells. Cells were collected at 24 h after transfection to perform subsequent proliferation and apoptosis assays.
Cell proliferation assay
C33A and SiHa cells were collected at 24 h after transfection to perform in vitro cell proliferation assay. Eagle’s Minimum Essential Medium containing 10% FBS was used to prepare single-cell suspension and the cell concentration was 4 × 104 cells/ml. After that, cells were cultivated in a 96-well plate with 0.1 ml cell suspension per well, followed by the addition of 10uL CCK-8 (Sigma-Aldrich) to each well every 24 h until 96 h. After that, cells were cultivated for additional 4 h, and 10 uL DMSO was added. After that, OD values (450 nm) were measured.
Cell apoptosis assay
C33A and SiHa cells were collected at 24 h after transfection to perform in vitro cell apoptosis assay. Non-serum Eagle’s Minimum Essential Medium was used to prepare single-cell suspension, and the cell density was 6 × 104 cells/ml. Cells were then cultivated in a 6-well plate with 2 ml per well. Cell culture was performed for 36 h and cells were then subjected to 0.25% trypsin digestion. Following staining with Annexin V-FITC and propidium iodide (PI), apoptotic cells were detected by flow cytometry.
Statistical analysis
Each experiment was performed 3 times (biological replicates). Data were processed using GraphPad Prism 6 software. Comparisons between CSCC patients and healthy controls or between HPV-positive and HPV-negative patients were performed by unpaired t-test. Comparisons among different cell treatment groups were performed by ANOVA (one-way) and Tukey test. Linear regression was performed to analyze the correlation between plasma levels of miR-205 and GATA6-AS. Diagnostic values of plasma GATA6-AS for CSCC were evaluated by performing ROC curve analysis with CSCC patients as true positive cases and healthy females as true negative cases. Difference with p < 0.05 was considered as statistically significant.
