Bioinformatics analysis
The differential expression of the MTHFD2 gene in 31 kinds of tumors and adjacent tissues was analyzed by GEPIA online software (http://gepia.cancer-pku.cn), and the relationship between survival time and MTHFD2 expression in patients with bladder cancer was analyzed. UALCAN online software (http://ualcan.path.uab.edu) was used to analyze the expression of MTHFD2 in bladder cancer and adjacent tissues and the expression of MTHFD2 in tumor and adjacent tissues with different pathological stages and lymph node metastasis.
Patients and tissue samples
From March 2015 to January 2017, bladder cancer tissue and adjacent tissues were collected after transurethral resection of bladder tumor or radical cystectomy in The First Affiliated Hospital of Nanchang University. All specimens were confirmed by pathology and then detected by PCR and WB assays. All the patients in this study signed the informed consent form and were approved by the Ethics Committee of The First Affiliated Hospital of Nanchang University [No. (2021)51]. Tissue microarrays were purchased from Jiangxi Ke Rui Si Biotechnology Co., Ltd. (Jiangxi, China) (Supplementary Table S2). The main clinicopathological parameters and their patient numbers were shown in Supplementary Table S3.
Cell culture
Human bladder cancer cell lines, including UM-UC-3, T24, 5637, EJ, J82, BIU, and human bladder cell biochemistry Pillon (SV-HUC-1), were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology (Shanghai, China). T24 cells were cultured in high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; HyClone; GE Health care Life Sciences, Logan, UT, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin. UM-UC-3 cells were cultured in MEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were cultured at 37°C with 5% CO2.
Lentiviral construction
Lentivirus packaging was purchased from Hanheng Biological (Shanghai) Co., Ltd. T24 and UM-UC-3 bladder cancer cell lines were infected with lentiviral knockout MTHFD2 (HBLV-h-MTHFD2shRNA1/2/3-ZsGreen-PURO) or negative control (HBLV-ZsGreen-PURONC) constructs. The stably transfected cell lines were screened with puromycin.
Western blot analysis
Total proteins were extracted from cells or preserved bladder cancer tissues and adjacent tissues and were stored in a refrigerator at -80°C. Western blot analysis was performed as previously described[27]. The membranes were incubated with antibodies targeting β-actin (cat. no. 58169, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), MTHFD2 (cat. no. 98116, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-phospho-PI3K (cat. no. 17366, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-phospho-AKT (cat. no. 4060, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-PI3K (cat. no. 4257, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-AKT (cat. no. 4691, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), followed by incubation with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. ab6721, 1:3,000; Abcam) for 1.5 h at room temperature. ImageJ 1.45 software (NIH) was used to perform densitometric analysis of each band.
Immunohistochemistry
Tissue specimens were fixed with 4% neutral formaldehyde for less than 24 hours and then rehydrated in a graded alcohol series, transparent, paraffin embedded, and sliced; then, microwave antigen repair was performed for 15 min, hydrogen peroxide blocking and serum sealing. The primary antibody MTHFD2 (cat. no. ab151447, 1:200; Abcam) was incubated overnight at 4°C. The secondary antibody was incubated for 30 min and stained with DAB (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Slides were stained with hematoxylin and sealed with neutral gum.
The cytoplasmic staining results were evaluated based on the percentage of positive cells and the intensity of staining by two independent pathologists. The intensity of staining was scored as 0 (no staining), 1 (mild staining), or 2 (deep staining). The percentage of positive cells was given scores of 0 (no tumor cells stained), 1 (<10%), 2 (10%-50%), and 3 (>50%). The results were calculated as the intensity x proportion. A total score <2 was defined as negative expression, and >2 was defined as positive expression.
RNA extraction and reverse transcription-quantitative PCR (RT–PCR)
Total RNA of cells and tissues was extracted by TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.), the concentration and purity of the extracted RNA were judged by the value of OD260/OD280. cDNA synthesized and the RT–PCR experiment were conducted as previously described[27]. mRNA relative expression levels were evaluated using the 2-△△Ct method. The primer sequences were as follows: β-actin-forward: 5'-TCTCCCAAGTCCACACAGG-3'; β-actin-reverse: 5'-GGCACGAAGGCTCATCA-3'. MTHFD2-forward: 5'-CAGCAGATCAAGCAGGAAG-3'; MTHFD2-reverse: 5'-GCAGGATTCTCGCCAAC-3'. 2.
CCK-8 assay
A CCK-8 assay was performed to measure cell viability. Approximately 1*103 cells were seeded into 96-well plates and cultured for 24 hours. Then, 10 µl CCK-8 solution (Nanjing Keygen Biotech Co., Ltd., Nanjing, China) was added to each well at 0 h, 24 h, 48 h, and 72 h. After incubation for 2 hours, the absorbance at 450 nm was measured by a TECAN SPARK 10 M microplate reader (Tecan Group, Ltd., Männedorf, Switzerland).
Cell migration and invasion assays
A wound healing assay was performed to measure cell migration. Approximately 5*105 cells were seeded in 6-well plates. A wound was made with a 10 µl pipette tip. The area of the cell-free scratch was photographed using an Olympus CKX41 inverse light microscope (Olympus Corporation, Tokyo, Japan) at 0 h, 24 h, and 48 h.
Cell invasion abilities were determined using a Transwell assay as previously described[27]. Following incubation for 24 h, the cells were fixed using 4% formaldehyde, stained using crystal violet and photographed using an Olympus CKX41 inverse light microscope at 37°C for 30 min.
Flow cytometry
For apoptosis and cell cycle distribution detection, approximately 1x106 cells were collected and washed twice in pre-cooled PBS buffer, and then 7-ADD and Annexin V-APC binding solution were added according to the apoptosis kit (Lian Ke biology, China). PI staining solution (Mei Lun Biology, China) was added to detect the cell cycle by flow cytometry.
Colony formation assay
The appropriate cells (approximately 300 cells/well for T24 cells and approximately 400 cells/well for UM-UC-3 cells) were seeded in 6-well plates, and the cells were cultured in an incubator (37°C, 5% CO2) with 10% FBS medium for 10–14 days. The culture was terminated when the number of cells was more than 50 per colony under a microscope, or the colony was visible under the naked eye. The colonies were stained with crystal violet and photographed.
Tumor formation experiment in nude mice
Four-week-old female BALB/c nude mice were obtained from Hunan Si Lai Ke Jing Da Laboratory Animal Co., and fourteen mice were divided into two groups with 7 mice in each group and fed according to protocols approved by the Animal Care and Use Committees of Nanchang University [Institutional approval number for lab animal studies: SYXK (Gan) 2015-0001]. T24-control and T24-shRNA2 cells at a density of 1 × 108/ml were resuspended in 200 µL Matrigel and injected subcutaneously into the flanks of mice. The size of the tumor was recorded every 3–6 days, and the volume (V) = 1/2A*B2 (A is the long axis, B is the short axis). After 4 weeks of tumor formation, the nude mice were killed, and tumors were photographed, measured, weighed and immunohistochemically stained.
Construction of RNA sequencing libraries and RNA-sequencing (RNA-Seq)
RNA-Seq was performed by NovelBio Bio-Pharm Technology Co.,Ltd. (Shanghai, China). Total RNA was extracted from 3 pairs of T24-Control and T24-shRNA2 by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Agilent 2200 and kept at −80°C. The cDNA libraries were constructed for each RNA sample using the TruSeq Stranded mRNA Library Prep Kit (Illumina, Inc.) according to the manufacturer’s instructions. The libraries were quality controlled with Agilent 2200 and sequenced by HiSeq X(Illumina, San Diego, CA) on a 150 bp paired-end run. The clean reads were then aligned to the human genome (GRCh38, NCBI) using the Hisat2. HTseq was used to get gene counts and the RPKM method was used to determine the gene expression.
We applied the DESeq2 algorithm to filter the differentially expressed genes, after the significant analysis, P-value and FDR analysis were subjected to the following criteria: i) Fold Change>2 or < 0.5; ii), P-value<0.05, FDR<0.05. Gene ontology (GO) analysis was performed to facilitate elucidating the biological implications of the differentially expressed genes in the experiment. Fisher’s exact test was applied to identify the significant GO categories (P-value < 0.05). Pathway analysis was used to find out the significant pathway of the differentially expressed genes according to Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We turn to Fisher’s exact test to select the significant pathway, and the threshold of significance was defined by P-value< 0.05.
Statistical analysis
GraphPad Prism 8.00 software (La Jolla, CA, USA) was used for data analysis. The quantitative data are expressed as the mean ± standard deviation (x̄±S). Student's t-test or one-way analysis of variance (ANOVA) followed by post hoc Tukey's honest significant difference test were used for comparisons between groups. A P<0.05 was considered to indicate a statistically significant difference.