Cell culture and treatments
Ovcar3 (ATCC No: HTB-161), SKOV3 (ATCC No: HTB-77) and A2780 (ECACC No: 93112519) were purchased from American Type Culture Collection (ATCC) and proceeded to their authentication by STRS analysis. The cell lines were cultured in RPMI-1640 (HyClone, Logan, UT, USA) or Dulbecco’s Modified Eagle’s Medium (HyClone) media supplemented with 10% fetal bovine serum (FBS, HyClone) and incubated in 5% CO2 at 37°C. For 3-methyladenine (3-MA, MCE, HY-19312) treatment, cells were incubated with or without 1 mM 3-MA for 24, 48 and 72 hours.
Stable cell line establishment
The expression plasmid of human SOX30 was constructed by synthesis, subcloned into pIRES2-EGFP vector (Invitrogen Preservation, Carlsbad, CA, USA) and verified by direct sequencing as previously described[14]. The plasmids were transfected into cells using ViaFect Transfection Reagent (Promega). The stably transfected cells were screened under G418 (Calbiochem, La Jolla, CA, USA) for 2 weeks. Cell clones were obtained by the cylinder method. Stable overexpression of SOX30 in the cell lines was confirmed by western blotting.
Xenografts in mice
All procedures with animals were conducted in accordance with the National Institute of Health guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Chongqing Medical University. To create the xenograft tumor model, 5-week-old male nude mice were randomly divided into control group (empty vector, n = 8) and SOX30 overexpressing group (n = 8). Ovcar3 cells stably transfected with empty vector and SOX30 in PBS (8x106 cells/mouse) were subcutaneously injected into the left and right flanks of 6-week-old male Balb/c nude mice, respectively or into the only left flanks of 6-week-old Balb/c nude mice for tumorigenicity assay. At 6 weeks after subcutaneous injection, all injected nude mice were sacrificed, and the tumors were excised, measured and weighted.
Cell proliferation and colony formation assay
The cell proliferation was measured by MTS assays. Briefly, Ovcar3, SKOV3 and A2780 cells were plated at 4 × 103 cells per well on 96-well plates. The cells on 96-well plates were transfected with SOX30 or vector control plasmids after culturing overnight. Cell proliferation was evaluated among 5 days by determining the cell number with a Cell Proliferation Reagent MTS (Promega). For colony-formation assay, cells were seeded in 6-well plates at a density of 200 cells per well and were grown for 10–14 days. Colonies were fixed with 4% paraformaldehyde and then stained with crystal violet for 30 min at room temperature. The colonies were captured and manually counted. Each experiment was repeated at least three times independently.
Western blot analysis
Cells were harvested and lysed using RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors (MCE, USA). Protein concentration was quantified using BCA Protein Assay Kit (Beyotime, Shanghai, China), separated by using 8–10% SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with QuickBlock (P0260, Beyotime, Shanghai, China) for 30 min at room temperature, and then incubated with anti-SOX30 (1:1000, ab71033, Abcam), anti-LC3 (1:1000, L7543, Sigma-Aldrich) or anti-β-Actin (1:5000, ab8227, Abcam) overnight at 4°C on a shaker. The next day, the membranes were then incubated with HRP-conjugated secondary anti-rabbit antibody (1:2000, Beyotime, Shanghai, China) or anti-mouse antibody (1:2000, Beyotime, Shanghai, China) and visualized by Tanon-5200 Chemiluminescence Imager with ECL western blotting substrate (Millipore).
Cell apoptosis assay
Ovcar3, SKOV3 and A2780 cells were plated in 6-well plates at 3×105 cells per well, and transfected with SOX30 or vector control plasmids after culturing overnight. The transfected cells were harvested at 48h post-transfection. The cell apoptosis was determined by dual staining with Annexin V-APC (APC) and 7-amino-actinomycin D (7-AAD) (KeyGEN, Nanjing, China) using the FACS Calibur System. Briefly, cells were harvested and washed with phosphate-buffered saline twice. The washed cells were re-suspended in 500 µl binding buffer. Then, APC (5 µl) and 7-ADD (5 µl) were added into the re-suspended cells one after another and mixed gently. The cells were incubated for 15 min in the dark and were analyzed using the FACS Calibur System. The relative proportion of Annexin V-positive cells was determined as apoptotic cells using ModFit software and counted. The assays were carried out in triplicate for three times.
GFP-LC3 punctate assay
Cells were transfected with plasmids expressing GFP-LC3 (#22405, Addgene). GFP-LC3-expressing cells were fixed in 4% paraformaldehyde for 15 min and washed with PBS. Finally, the cover slides were then placed on the glass slide and sealed with anti-fluorescence quencher prior to observed by confocal microscopy (LSM510, Carl Zeiss). Cells representing five or more intense GFP-LC3 puncta with no nuclear GFP-LC3 were considered autophagic, whereas those with a mostly diffuse distribution of GFP-LC3 in the cytoplasm and nucleus were classified as non-autophagic.
Receiver operating characteristic curve analysis
By analyzing the expression of SOX30 in tumor and normal tissues, the diagnostic value of SOX30 was explored in ovarian cancer. The x-axis and y-axis are the specificity and sensitivity of diagnosis, respectively. The area under the receiver operating characteristic (ROC) curve (AUC) is calculated to evaluate the predictive ability of SOX30 for discriminating between normal and tumor tissues.
Correlation analysis
The clinical information and gene expression data of TCGA ovarian cancer cohort were obtained from the TCGA Data Portal (https://tcgadata.nci.nih.gov/tcga/) to investigate the genomic and gene expression profiling of SOX30, as well as the correlation of SOX30 expression with autophagy-related gene expression in ovarian cancer. The expression of genes was quantified as FPKM (fragments per kilobase million), which are provided by TCGA consortium. Processing of the data was according to the guidelines of this database.
For the copy number variation (CNV) profile, we download the level 3 CNV dataset of ovarian cancer in the SNP6.0 microarray from TCGA and analyzed the dataset with GISTIC2.0 software as described previously[15]. We procured the CNV number of each corresponding sample by examining the distribution of log2 ratios to identify peaks associated with copy number states. Final copy number variations were interpreted qualitatively as deleted, unchanged or amplified.
For DNA methylation profiling analysis, we used TCGA DNA methylation data (Illumina Infinium HumanMethylation450 platform) to perform methylation measurements. SOX30 gene expression values from ovarian cancer tissues were also extracted. Pearson’s correlation between SOX30 gene expression levels and methylation of its CpG islands was evaluated.
Enrichment analyses
SOX30 co-expressed genes were obtained from Linked Omics database (http://www.linkedomics.org/login.php), an online platform that allows researchers to analyze over 30 cancer-related cubes. We used the ovarian cancer cohorts, selected TCGA ovarian cancer as the search and target datasets in the HiSeq RNA platform, and used pearson correlation test as the statistical method. SOX30 co-expressed genes were explored using the Link Finder module, and the findings were presented graphically in volcano maps and heat maps. The SOX30 co-expressed genes were further subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using DAVID database (https://david.ncifcrf.gov/) with standard settings. GO and KEGG enrichment analysis was implemented by the clusterProfiler R package.
Statistical analysis
The data were presented as mean ± SE. Student’s t tests were used to determine statistical significance of differences between groups. Pearson’s correlation coefficient was used for correlation analysis. All tests were two-sided, and P values less than 0.05 was considered significant. Graphs were generated in GraphPad Prism software version 8.0 (GraphPad, La Jolla, CA, USA).