Cell lines and reagents
The human breast epithelium cell line MCF-10A was grown in DMED/HAM F12 1:1 mixed medium (Hyclone, Logan, UT, USA), which contains 5% horse serum (Gibco, Billings, MT, USA), hydrocortisone, cholera toxin, insulin (Sigma-Aldrich, St. Louis, MO, USA) and EFG (PeproTech, Cranbury, NJ, USA). The human BC cell lines SK-BR-3, BT-474 and MCF-7 were cultured in RPMI 1640, DMEM F12 or DMEM H21 (Hyclone), respectively, comprising 10% fetal bovine serum (Gibco). All cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in a humidified atmosphere with 5-% CO2 at 37 °C. MEK inhibitor U0126 and DMSO was purchased from Sigma-Aldrich. A final concentration of 50 μM (1:1000, v/v) U0216 or the same volume of DMSO was applied to treat the cells.
Patients and samples
The tissue samples of 210 female patients with BC who had received breast-conserving surgery or modified radical mastectomy during the period from January 2012 to December 2015 at China-Japan Union Hospital of Jilin University were enrolled in our study. The patients who received chemotherapy, radiotherapy, or endocrine therapy before surgery were excluded. The cases with recurrent or metastatic cancer at the time of diagnosis were also eliminated. The last date of follow-up was January 2020. Furthermore, a total of 40 normal or para-tumor tissues were acquired as controls. The study was approved by the Research Ethics Committee of the China-Japan Union Hospital of Jilin University and conducted with written informed consent from all patients.
Total RNA extraction was isolated using TRIzol reagent (Ambion, Austin, TX, USA). The reverse transcription was performed with a 5 × PrimeScript RT Master Mix for Real-Time kit (TaKaRa, Tokyo, Japan). qRT-PCR was done using an SYBR® Premix DimerEraser™ Perfect Real-Time kit (TaKaRa). All operating procedures followed the supplier’s instructions. RT-qPCR analyses were done with a Mastercycler (Eppendorf, Hamburg, Germany). The primer sequences were as follow: TGM2, F: 5′- CAG CAG GGC TTT ATC TAC CA-3′, R: 5′- GAT CCC ATC TTC AAA CTG CC-3′; LDHA, F: 5′- TTC CAG TGT GCC TGT ATG G-3′ R: 5′- TTA TCA GTC CCT AAA TCT GGG TG-3′; LDHB, F: 5′- ACA ATA AGA TCA CTG TAG TGG G-3′, R: 5′- CAT CAG CCA GAG ACT TTC C-3′; GAPDH, F: 5′-GAA GGC TGG GGC TCA TTT GCA GGG-3′, R: 5′-GGT GCA GGA GGC ATT GCT GAT GAT-3′. The relative fold-change was normalized to GAPDH and calculated by the 2-ΔΔCt method.
All the antibodies were obtained from Abcam (Cambridge, UK), except if stated otherwise. The cells were harvested in a lysis buffer and the proteins were extracted by RIPA buffer. The protein concentration was quantitated using the Bio-Rad (Hercules, CA, USA) method. Proteins (25 μg) were loaded into each well and separated on a 10% SDS-PAGE gel, and transferred to a nitrocellulose membrane. Blots were cut prior to hybridization with antibodies. After blocking with 5% fatty-free milk, the membranes were incubated with either TG2, p-MEK/MEK, p-ERK/ERK, LDHA/B, or GAPDH at a dilution of 1:1000 overnight. Anti-rabbit secondary antibodies were coupled to horseradish peroxidase (Proteintech, Wuhan, China). Immunoreactivity was detected using enhanced chemiluminescence (Beyotime, Shanghai, China) and visualized through autoradiography. Protein levels were normalized against GAPDH and analyzed.
Immunochemistry staining (IHC)
To identify the level of TG2 in BC tissues, IHC staining was performed. Briefly, 3 μm thick sections were deparaffinized, rehydrated and submerged into EDTA for antigen retrieval. All sections were then treated with hydrogen, heated, and incubated in bovine serum albumin. Subsequently, they were incubated with TG2 antibody (Abcam) overnight at 4 °C. Negative controls were employed with normal goat serum. The sections were washed and incubated with secondary antibody and incubated with streptavidin-horseradish peroxidase complex (Invitrogen, Waltham, MA, USA). Each section was immersed in 3-amino-9-ethyl carbazole followed by counterstaining with Mayer’s hematoxylin, dehydrated, and then mounted.
The plasma staining of TG2 was considered positive. The intensity of staining was categorized as follows: 0 (no staining), 1 (light yellow), 2 (yellow-brown), and 3 (brown). The percentage of positive cells were grouped as: 0% = 0, 1–10% = 1, 11–25% = 2, 25–50% = 3, and > 50% = 4. IHC intensity, with a range between 0 and 12, was calculated by the score of the staining indexes which equals the staining intensity × proportion of positive cells. The staining score of TG2 level was grouped as: 0, no level; 1–3, weak; 4–6, mild; and > 6, strong.
Establishment of TG2 overexpression and shRNA knockdown cell colonies
Empty vector (EV), wildtype TG2 for overexpression (OE), scramble control (SC), and shRNA targeting TG2 (knockdown, KD) were constructed into lenti-virus (Hanbio, Shanghai, China). BT-474 cells were infected with wildtype TG2 or EV. The cell line SK-BR-3 was infected with shRNA or SC. Concisely, 2 × 105 cells were incubated with lenti-virus and polybrene (Hanbio) for 8 h. Fresh growth medium was added, and stable cell lines were gained in selected medium containing puromycin. Prepared cells were then used for further analysis as described.
Cell proliferation assay
Cell proliferation was evaluated using a cell counting kit-8 (CCK-8, Meilunbio, Dalian, China). Cells were suspended into a 96-well plate in a 100 μL growth medium. After incubation for 24, 48, and 72 h, CCK-8 was added and further cultured for 4 h. The optical density (OD) value was obtained at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).
Glucose consumption and lactate production
The glucose/lactate assay kit (RSBio, Shanghai, China) was used to detect the levels of glucose and lactate in the cell culture medium following the manufacturer’s instructions. Briefly, 3 × 105 cells/well were seeded into 6-well plates and incubated for 2 days. The medium was then replaced with a fresh medium and incubated for another 2 days. The medium was collected, and the glucose or lactate level was measured at the end of the incubation. The difference between the concentration of glucose or lactate and the controls (0 h) was used to calculate the glucose or lactate content. Glucose consumption, as well as lactate production, was normalized by cell numbers .
Gene set enrichment analysis (GSEA)
The mRNA profile data were obtained from the TCGA database and included 1104 BC tissues and 114 normal tissues. The mRNA level of TG2 was divided into a low- or high-level group based on the median ratio. Briefly, gene sets (h.all.v6.2.symbols.gmt) were obtained from the Molecular Signatures Database and analyzed using GSEA v3.0. Normalized enrichment scores (NES) were obtained from 1000 site permutations with a p-value < 0.05 and false discovery rate (FDR) < 0.25 considered statistically significant.
Mice xenograft model
Six female nude mice (Huafukang Bioscience, Beijing, China) aged 4–5 weeks were randomly separated into two groups. SK-BR-3 cells (4 × 106, either SC or KD) were injected into the fourth mammary fat pad of each mouse. The tumor size was measured using calipers every 3 days for 1 month. At the end of the experimental period, the mice xenograft tumors were excised and weighed after euthanasia. Organs such as the brain, heart, lung, and liver were fixed in formalin and pathologically investigated for signs of toxicity and metastatic lesions. All procedures of animal handling were approved by the Laboratory Animal Care and Use Committee of Jilin University.
The statistical data were analyzed using SPSS v21.0 (IBM, Armonk, NY, USA). The Chi-square test as well as Spearman’ s correlation analysis was performed for analysis of the relationship between clinicopathological parameters and TG2 expression. Survival curves drawn by the Kaplan-Meier method were compared using the log-rank test. Univariate and multivariate Cox-regressive analyses were performed for further assessment. Student’s t-test or one-way ANOVA was adopted to investigate data among or between groups. A p-value < 0.05 signified statistical significance. All the in vitro experimentations are performed independently at least three times, and the data were expressed as mean ± standard deviation.