Human sample and clinicopathological data collection
This study was received the approval of the Ethics Committee of Shanxi Medical University (Ethical code: 2019220221). Frozen tissues specimens were collected 35 patients with ovarian cancer without any therapy before surgery at the Department of Gynaecology and Obstetrics from Tumor Hospital of Shanxi (China). The average age of the patients was 53.9 years (24-69 years). In formed written consent was obtained from all patients enrolled.
Cell culture and transfections
This human ovarian cancer cell line SKOV3 was purchased from the American Type Culture Collection (ATCC) and cells were cultured in RPMI medium 1640 (Gibco, Thermo Fisher Scientifc, Inc.) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, USA) and 1% penicillin/streptomycin (Invitrogen, Waltham, MA) in a stable humidified atmosphere with 5% CO2 at 37℃. siRNA targeting MTHFD2 (si-MTHFD2) and siRNA negative control (si-NC) were constructed and purchased from GenePharma (Shanghai, China).
Oncomine database verification
The Oncomine (www.oncomine.org) platform is a public bioinformatics network database including 715 independent datasets and 86, 733 samples which has become an industry-standard tool cited in more than 1,100 peer-reviewed journal articles [22]. MTHFD2 gene expression levels in malignancies were analyzed, selecting the cancer type as ovarian cancer. Paired Student’s t-test was performed to compare group means. A fold-change of at least 2 with a P-value < 0.001 was used as clinically significant, as previously described.
Gene expression profiling interactive analysis
Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/) was performed to explore the data of tumor/normal differential expression analysis, profiling according to cancer types or pathological stages, patient survival analysis, similar gene detection, correlation analysis and dimensionality reduction analysis from TCGA and GTEx databases with bioinformatics tools CIBERSORT, EPIC, and quan Tlseq [26]. In GEPIA, the expression of MTHFD2 and MOB1A in various human cancers and adjacent normal tissues was obtained, furthermore the expression of MTHFD2 in ovarian cancer and corresponding normal tissues was analyzed.
University of California Santa Cruz (UCSC)Cancer Genomics Browser Analysis
UCSC Xena platform (http://xenabrowseer.net), included 758 cases of ovarian cancer with genomic and clinical, was utilized to access TCGA ovarian cancer data [6]. The relationship between the mRNA expression of MTHFD2 and MOB1A was conducted thorugh TCGA-Ovarian Cancer.
cBioportal database analysis
cBioPortal (http://chioportal.org) was utilized to analyze MTHFD2 alterations observed in ovarian serous cystadenocarcinoma (TCGA, Nature 2011, n=489) [2, 5]. By analyzing various types of mutations, putative copy number variations, and co-expression data, the tab OncoPrint displayed an overview of the genetic changes in each sample as gene mutations and heat maps of MTHFD2 expression.
UALCAN analysis
UALCAN (http://ualcan.path.uab.edu), an an open public web resource, is utilized to analyze the mRNA expression of potential genes in various tumor subtypes, including age, gender, tumor stages, and other clinicopathological features [3]. In our study, UALCAN was pertormed to access the MTHFD2 expression levels in primary ovarian carcinoma tissues and its association with multiple clinical and pathological parameters.
LinkedOmics Analysis
The LinkFinder module of LinkedOmics (http://www.linkedomics.org/login.php), included multi-omics data from all 32 TCGA Cancer types and 10 Clinical Proteomics Tumor Analysis Consortium (CPTAC) cacner cohorts, is utilized to search for attributes that are associated with a query attribute, such as mRNA or protein expression signatures of genomic alterations, candidate biomarkers of clinical attributes, and candidate target genes of transcriptional factors, microRNAs, or protein kinases [28]. In our study, LinkFinder module was performed to identify differentially expressed genes related to MTHFD2 in the TCGA OV section. To derive biological insights from the association results, the LinkInterpreter module performs enrichment analysis based on Gene Ontology, biological pathways, network modules, among other functional categories.
Kaplan Meier plotter
The Kaplan Meier plotter (http://kmplot.com/analysis/) is capable to evaluate the role of 2,190 genes on survival in ovarian cancer from the databases containing GEO, EGA, and TCGA [15]. We accessed Kaplan Meier plotter to analyze the prognostic value of MTHFD2 in ovarian cancer. The patient samples were split into two groups by median expression to analyze the overall survival (OS) with hazard ratios (HRs) with 95% confidence intervals and log-rank p-values.
TIMER Database Analysis
Tumor Immune Estimation Resource (TIMER, https://cistrome.shinyapps.io/timer/) is a website for comprehensive analysis of the tumor-infiltrating immune cells and gene expression in different types of cancers [12]. We evaluated the expression of MTHFD2 in ovarian cancer in relation to tumor purity and the abundance of immune infiltrating cells including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. The levels of gene expression were expressed as Log2 RSEM.
TISIDB Analysis
TISIDB database (http://cis.Hku.hk/TISIDB) is a websit for comprehensive analyzing tumor and immune cell interactions which integrates into various heterogeneous data types for each gene [23]. We access this platform to analyze the relationship between MTHDF2 expression and tumor-infiltrating lymphocytes.
Quantitative RT-PCR (qRT-PCR)
Total RNA of tissues and cells was extracted with TRIzol reagent (Invtrogen, Carlsbad, CA, USA) and the concentration of total RNA was measured with Nano-DropTM Spectrophotometers (Thermo). Appropriate amounts of total RNA were reverse transcribed using the Prime Script™ RT Master Mix kit (Thermo Fisher Scientic, Carlsbad, CA, USA). Real-Time PCR was performed with the SYBR® Premix Ex Taq Kit (TaKaRa, Otsu, Shiga, Japan) and primers (listed below) using CFX96 (Bio-Rad company, Shanghai, China). The following PCR primer sequences as follows: MTHFD2, 5′-GATCCTGGTTGGCGAGAATCC-3′ (forward) and 5′-TCTGGAAGAGG CAACTGAACA-3′ (reverse), MOB1A, 5′-CAGCAGCCGCTCTTCTAAAAC-3′ (forward) and 5′-CCTCAGGCAACATAACAGCTTG-3′ (reverse), GAPDH, 5′-ACCACAGTCCATGCCATCA C-3′ (forward); and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse). The method of 2−ΔΔCt was performed to calculate the relative mRNA levels of target genes.
Western blot
Total protein from ovarian carcinoma cell lines was extracted using lysis buffer (KeyGEN BioTECH, China) containing phosphatase and protease inhibitors (KeyGEN BioTECH, China). Protein concentration was detected using BCA protein assay kit (KeyGEN BioTECH, China). Equal amount of protein (10 μg) was loaded onto 10% SDS-PAGE (PG112, Epizyme Biotech, China) and then transferred to PVDF membranes (Millipore, USA). All membranes were then blocked in TBST for 1 hour with 5% BSA at room temperature and followed by primary antibodies at 4℃ overnight. Then membranes were incubated at room temperature for 2 hours with HRP-conjugated secondary antibodies (701051, Zen Bioscience, China; ZB-2301, ZSGB-BIO, China). Protein signals were visualized with a chemiluminescence kit (Millipore, USA) and an Image system (Bio-Rad, USA). GAPDH was used as the internal control. Primary antibodies were as follows: MTHFD2 (ab151447, 1:3000, Abcam, Cambridge, MA, USA), MOB1A (ab236969, 1:3000, Abcam, Cambridge, MA, USA) and GAPDH (cat.no. T0004; 1:5000; Affinity Biosciences).
CCK-8 detection of viability
Cell Counting Kit-8 was accessed to evaluate the cell viability. Cells were seeded into 96-well plates at 5×103 cells per well and subjected in 100 μl serum-free medium for 24, 48, 72, and 96h. Then, 10 μl CCK-8 solution (CCK-8, ATgene, Taiwan, China) was added to each well for 2 hours and the absorbance (OD) values was detected at 450 nm by microplate reader (BioTek, Epoch, VT). All tests were repeated eight times, and each experiment was experimented at least three replicate.
Cell migration and invasion ability assay
Invasion and migration abilities of ovarian cancer cells transfected with si-MTHFD2 and si-NC were determined using transwell migration and invasion assay. 600 μl DEME/F12 supplemented with 10% FBS was added to lower chambers, followed by Transwell chamber (Millipore, Burlington, MA, USA) seeded with 5×104 cells. For cell invasion assay, 100 μg/ml Matrigel (BD Biosciences, Boston, MA, USA) was added to the upper layer of chamber. After incubating for 24h, the cells in the upper chamber were removed. Cells were fixed with 600μl methanol for 10 min. The chamber was stained with 0.25% cfystal violet (Solarbio) for 15 min. Finally, an inverted microscope (Eclipse Ti2; Nikon Corporation) was performed to photograph and calculate the invading and migratory cells in three random fields.
Statistical analysis
GraphPad Prism 7.0 software was used to statistical analysis. Student’s t-test was performed to compare the differences between two groups. One-way ANOVA was performed to compare multiple groups. Overall survival was presented as Kaplan-Meier survival curves and the statistical comparisons were calculated by Log-rank test. P-value < 0.05 was set as the threshold.