Cell lines and cell culture
Human breast cancer cell lines (HCC1806, MDA-MB-231, MCF-7, ZR-75-1, BT549) and normal breast cell lines (MCF-10A) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured as monolayers in RPMI-1640 culture medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin and maintained in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37°C.
Reagents and antibodies
Antibodies against SHMT2(A1215),GAPDH(A19056),β-actin(AC026), VEGF(A12303), PEDF(A3475), were obtained from ABclonal Technology Co.,Ltd. (ABclonal,wuhan,China). Antibodies against JNK(AF6318), P38(AF6456), ERK(BF8004), P-JNK(Thr183+Tyr185)(AF3318), P-P38(Thr180/Tyr182) (AF4001), P-ERK(Thr202/Tyr204)(AF1015), cytochrome-c (AF0146), PARP(DF7198), Cleaved-PARP (Asp214) (AF7023), Cleaved-Caspase 3 (Asp175)(AF7022), Cleaved-Caspase 9 (Asp353) (AF5240), BAX(AF0120), Bcl-2 (AF6139), Goat Anti-Rabbit IgG (H+L) HRP (S0001) and Goat Anti-Mouse IgG (H+L) HRP (S0002) were purchased from Affinity Biosciences (Affbiotech, Jiangshu,China).ERK MAPK inhibitor FR180204 (HY-12275), p38 MAPK inhibitor SB202190 (HY-10295) were purchased from the Med Chem Express(MCE).
Preparation of shRNA or expression plasmid
The SHMT2 shRNA (HSH109479) and the overexpression plasmid of SHNT2 (p-SHMT2) (T3093) were purchased from GeneCopoeia, Inc. (Guangzhou, China). The sequence of the human SHMT2-specific shRNA was 5’-TCTGAACAACAAGTACTCGG-3’ and 5’-TCTCAGGATCACTGTCCGAC-3’, and the scramble shRNA was 5’-GGC TCC GAACGGGTCACGATT-3’. For the in vitro delivery of the shRNA and plasmid into tumor cells, the sequences were first encapsulated into Lipofectamine 3000 (L3000150, Thermo Scientific,) that had validated by many analyses by dissolving in Opti-MEM. The knockdown efficiency was validated by western blot.
A total of 2 × 105 HCC1806 and ZR-75-1 cells were seeded into each well of a six-well tissue culture plate (Nunc). The next day (when the cells were 70-80% confluent), the culture medium was aspirated, and the cell monolayer was washed with prewarmed sterile phosphate buffered saline (PBS). The cells were transfected with the shRNA or plasmid at the indicated dose using Lipofectamine 3000 (L3000150) (Thermo Scientific) . The cells were harvested after 48 h of transfection, and western blot analyses or other experiments were performed.
Western blot analysis
The proteins in cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA) and electrophoretically transferred to a PVDF membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The western blots were probed with specific antibodies, and the protein bands were detected using enhanced chemiluminescence.
The tissue array consisted of 140 formalin-fixed, paraffin-embedded (FFPE) breast cancers and corresponding adjacent normal tissues. These tissue samples were previously obtained with informed consent from patients having no anticancer treatment prior to tumor resection. The tissue specimens were histologically examined and classified according to the 2004 World Health Organization classification system . Detailed clinical and pathologic information, including the clinical and pathologic tumor-node-metastasis (TNM) stage, overall survival (OS) duration, and time to recurrence, was available for all cases. The pathological TNM status of all of the breast cancers was assessed according to the criteria of the seventh edition of the American Joint Committee on Cancer.
Immunohistochemistry was conducted using Envisionþ Kit/HRP (DakoCytomation). Briefly, slides were immersed in Target Retrieval Solution (pH 9; DakoCytomation) and boiled at 108°C for 15 min in an autoclave for antigen retrieval. The anti-SHMT2 antibody was added to each slide after blocking of the endogenous peroxidase and proteins, and the sections were incubated with HRP-labeled anti-rabbit IgG as the secondary antibody. The substrate-chromogen was added, and the specimens were counterstained with hematoxylin. A negative control was obtained by replacing the primary antibody with normal rabbit IgG.
To evaluate the immunohistochemical staining, two independent observers blinded to the clinicopathologic information performed scoring using light microscopy (magnification 20×). The intensity of SHMT2 staining was semiquantitatively evaluated using the following criteria: strongly positive (scored 2+), dark-brown staining in more than 50% of the tumor cells, completely obscuring the nucleus; weakly positive (scored 1+), any lesser degree of brown staining appreciable in the tumor cell nucleus; absent (scored 0), no appreciable staining in the tumor cells. Cases were accepted as strongly positive if 2 investigators independently defined them as such.
Cells plated in 96-well plates (1000 cells/well) were transfected with control or specific shRNA. 48 h later, 10ul MTT reagent (5 mg/ml) was added in single well and treated for 2.0 h. Then MTT was discarded and 150ul Dimethyl Sulphoxide (DMSO) was added in single well. Finally, the OD value at 490 nm was measured.
Colony formation assay
Cells were transfected with SHMT2 shRNA or plasmid for 24 h, trypsinized, and resuspended as single cells. The cells (1 × 103/ml) were then mixed in 21 ml of 1640 culture miadiumr containing 5% FBS. The cultures were maintained in a 37°C/5% CO2 incubator for 12-14 days. The cell clones were then washed three times with phosphate-buffered saline (PBS), fixed in methanol for 10 minutes, and stained with Crystal Violet for 10 minutes at room temperature. The dye was washed off, and the colonies that contained more than 50 cells were counted.
RNA was extracted from the vector group and the SHMT2 OVER ZR-75-1cells using the RNeasy Kit (Qiagen) according to the manufacturer’s protocol. RNA quality control was performed using an Agilent Bioanalyzer. RNA-seq libraries were generated using Illumina TruSeq mRNA stranded kits following Illumina protocols. Libraries were quantitated using an Agilent bioanalyzer, and the pooled libraries were sequenced with an Illumina HiSeq 4000 system using Illumina reagents and protocols.Abundance quantifications were imported into R software, and gene expression matrix was constructed using R Bioconductor package tximport (41). Count values summarized by tximport were analyzed using the DESeq2 algorithm. Differential expression was defined at a threshold of FDR = 0.05 and absolute log fold change > 1.
HCC1806 cells were inoculated in 6-well plates at a certain density and starved overnight (12 h). After cell adherence, HCC1806 cells were transfected with SHMT2 shRNA. At 48 h after transfection, the cells were harvested by trypsinization and fixed in 70% cold ethanol for 30 minutes, then stained with 5 μl Annexin V-FITC and 5 μl PI (propidium iodide) using an Annexin V-FITC/PI-staining kit (Genechem, Shanghai, China). The cells were placed at room temperature for 20 min in the dark and then analyzed using flow cytometry (Beckman Coulter). Apoptosis was calculated in terms of the FITC-positive cells. The raw data were analyzed using Multicycle for Windows (Beckman Coulter).
The treated Cells were seeded in 6 well Culture plate cover with glass slide(24mmX24mm) at a density of 1 × 105 cells per well. After 48 h, the cells were washed with PBS, fixed with 4% paraformaldehyde solution, and permeabilized with 0.1% Triton X-100. The cells were incubated with a rabbit anti-cytochrome-c antibody and then incubated with a Goat anti-Rabbit IgG (H+L), Alexa Fluor 568 (A-11036,Thermo Scientific). The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI), and the cells were examined under a fluorescence microscope.
The concentrations of VEGF and PEDF were determined by ELISA
HCC1806 and ZR-75-1 cells were seeded in 6-well plates and treated with SHMT2 shRNA or overexpression plasmids for 48 hours. The VEGF and PEDF levels in the culture supernatant were quantified using a VEGF Immunoassay Kit (ml064281) (Enzyme-linked Biotechnology, Shanghai, Chnia) and a Chemikine PEDF ELISA Kit (ab213815) (Abcam, Shanghai, Chnia) according to the manufacturer’s protocols.
All animal maintenance and operational procedures were carried in accordance with the animal licence protocol approval by Animal Care and Ethics Committee of Sun-Yat Sen University. Female nude mice (Balb/c) aged 5 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were randomized for xenograft tumor growth assays. For xenograft tumor formation, the mice were randomly divided into two groups, sh-control(KD-NC), sh-shmt2(KD-SHMT2). 8 × 106 HCC1806 cells with stable knockdown of SHMT2 gene or control shRNA were respectively suspended in 100ul PBS and injected subcutaneously into the right flank of each mouse. The tumor volume was measured after two weeks of injection. The tumor volume was calculated as V= (width2 ×length)/2 and the data was recorded every three days for four weeks. Mice were sacrificed and tumors were taken from mice for weighting and photographing. Partial tissues were transferred to liquid nitrogen immediately, and partial were fixed in formalin for IHC assay.
Immunohistochemistry staining assay in xenograft tumor
The examined cancer tissues were fixed in 4% paraformaldehyde, washed with PBS three times, transferred to 70% ethanol, cut into small pieces and then embedded in paraffin in accordance with standard procedures. After being dewaxed and antigen retrieval, the tissue sections were stained using SP kit (SP-9000, ZSGB-BIO, China) according to its instructions. Briefly, the sections were blocked with blocking reagents, and then were exposed overnight at 4 ℃ to antibodies against SHMT2, KI67, P-P38, or P-ERK. The slides were washed with PBS and incubated with anti-mouse/rabbit biotin antibodies for 1 h. After washing, the slides were added with HRP-conjugated streptavidin, developed with HRP substrate(DAB), and counterstained with hematoxylin. In the end, the paraffin sections were dehydrated by using Gradient alcohol, sealed by means of neutral balsam (Solarbio) and photographed with microscope.
All results were presented as the mean ± SE. A Student t-test was performed to compare the two independent groups of data. A statistical analysis was performed using the SPSS statistical software package (standard version 18.0; SPSS, Chicago, IL). Survival curves were calculated using the Kaplan Meier method. The log-rank test was used to analyze overall survival (OS) time between different expression of SHMT2 group in BRCA.