A total of 30 pairs of breast cancer and paracancerous tissue samples were obtained from the China-Japan Union Hospital (Changchun, China) between March 2018 and March 2019. The median age is 38 years (range, 28–45 years). The study was approved by the Ethics Committee of the China-Japan Union Hospital. The samples were obtained with signed informed consent from the patients or their family.

MCF-7 cells were gifted from Jilin University School of Pharmacy. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin G and 100 µg/ml streptomycin in an incubator at 37°C and 5% CO₂. (all from Invitrogen; Thermo Fisher Scientific, Inc.).

All materials for the SDS-PAGE were purchased from Bio-Rad Laboratories, Inc. The monoclonal antibody against β-actin (1,2000; cat. no. AAPR201-100) was purchased from Sigma-Aldrich; Merck KGaA. Rabbit polyclonal antibodies against TGF-β₁ (1:2,000; cat. no. RAB-0238) and TG2 (1:2,000; cat. no. CTA-DE056) were obtained from Cell Signaling Technology, Inc. The TG2 inhibitor (MDC) and the TGF-β₁ inhibitor (ITD) were purchased from Sigma-Aldrich; Merck KGaA.

The sections (3 µm) of breast cancer tissue were fixed in 4% paraformaldehyde followed by dehydration using a gradient ethanol series (80 and 95%). The section was subsequently stained with HE at 25°C (10 min). Images were captures on a fully automatic photomicrography device (magnification ×200; five field of views; Olympus PM-10AO; Olympus Corporation).

0.3% hydrogen peroxide formaldehyde solution was added to the paraffin sections and incubated at 37°C. The sections of breast cancer tissues and paracancerous tissues were washed with PBS and incubated with 10% bovine serum albumin (Thermo Fisher Scientific, Inc.) for 15 min. Rabbit anti-human TGF-β₁ and TG2 polyclonal antibodies (1:300) were added and incubated at 4°C for 12 h. The next day, color rendering was performed using 3,3′-diaminobenzidine and images were captures on a fully automatic photomicrography device (magnification ×200; five field of views; Olympus PM-10AO; Olympus Corporation).

MCF-7 cells were seeded in 96-well plates at 1×10³ per well, treated with 1 or 2 mg/l cisplatin (German Pharmaceutical Co., Ltd.) and incubated in an incubator at 37°C at 5% CO₂ for 24 h. Next, 10 µl MTT was added to each well and incubated for 30 min at 37°C. The absorbance was measured at 450 nm using a microplate reader.

The protein expression levels of TG2 and TGF-β₁ inMCF-7 cells were assessed by western blotting. Tissue samples were homogenized in a PIPA buffer (Qiagen, Inc.). Protein concentrations were determined using a bicinchoninic acid kit (Pierce; Thermo Fisher Scientific, Inc.). Protein (20 µg/lane) was separated using 12% SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with primary antibodies against TG2 (1:2,000), TGF-β₁ (1:2,000) and β-actin (1:2,000) at 4°C overnight. The membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (cat. no. ZB-2301; 1:2,000; Beijing Noble Technology Co., Ltd). TG2 and TGF-β₁ were detected using ECL development solution (Pierce; Thermo Fisher Scientific, Inc.). TG2 and TGF-β₁ expression levels were determined using Quantity One v4.6.2 software (Bio-Rad Laboratories, Inc.).

The fluorescence intensity of TG2 and TGF-β₁ in MCF-7 cells was assessed via immunofluorescence. MCF-7 cells (5×10⁴) were treated with cisplatin (1 mg/l), TGF-β₁ (150 µmol/l) and TG2 inhibitors (8.31 µmol/l), and incubated at 37°C and 5% CO₂ for 24 h. Rabbit monoclonal primary antibodies against TG2 (1:300) and TGF-β₁ (1:300) were added to the cells and incubated overnight at 4°C. Following overnight incubation with fluorescein-conjugated IgG (cat. no. ZB-2301; Beijing Noble Technology Co., Ltd.) antibody at 4°C. TG2 and TGF-β₁ expression levels were determined using Quantity One v4.6.2 software (Bio-Rad Laboratories, Inc.).

The crystal structure of TG2 [in complex with GTP; Protein Data Band (PDB) ID, 4PYG] and TGF-β₁ (in complex with scFv GC1009; PDB ID, 4KV5) were obtained from the PDB (http://www.rcsb.org/pdb). The protein files of TG2 and TGF-β₁ were prepared by removing water molecules and other ligands. Molecular docking studies and docking analysis were performed using the PatchDock server (http://bioinfo3d.cs.tau.ac.il/PatchDock/). Analysis and visualization of interactions in the docked complexes obtained by PatchDock server were analyzed by PyMOL (https://pymol.en.softonic.com/).

Quantitative data are presented as the mean ± standard deviation and were analyzed using SPSS 19.0 software (IBM Corp.). Student's t-test was used to compare two groups; one-way ANOVA followed by Dunnett's test was used to compare all treatment groups against an untreated control group. The χ² test was used to analyze the associations between protein expression and patient characteristics. P<0.05 was considered to indicate a statistically significant difference.

In the present study, tissues from 30 patients with breast cancer were evaluated using HE staining (Table I). These patients were recruited at the China-Japan Union Hospital of Jilin University. Clear structures and contours of the tissues were detected in paracancerous tissues (Fig. 1A). However, in the breast cancer samples, moderately heteromorphic and moderately differentiated cells were observed in the epithelial tissue (Fig. 1B). In addition, moderate atypia and interstitial fibrosis were observed in the breast cancer tissues (Fig. 1B).

In the present study, the expression levels of TG2 and TGF-β₁ were assessed via immunohistochemical staining. The results revealed that the expression levels of TG2 and TGF-β₁ in the cell membrane and cytoplasm in the paracancerous tissues were low. However, the expression levels of TG2 and TGF-β₁ were significantly higher in the breast cancer tissues compared with those in the paracancerous tissues (P<0.05; Fig. 2A-C). TG2 and TGF-β₁ were primarily expressed in the tumor and interstitial regions.

The viability of MCF-7 cells treated with cisplatin was analyzed by MTT assay. The results revealed that cell survival in untreated MCF-7 cells was lower compared with that in cells treated with 1 and 2 mg/l cisplatin (P<0.05; Fig. 3).

In the present study, immunofluorescence was used to determine the fluorescence intensity of TG2 and TGF-β₁ in MCF-7 cells. The results demonstrated that the fluorescence intensity of TG2 and TGF-β₁ decreased in cisplatin-treated MCF-7 cells compared with untreated MCF-7 cells (P<0.05; Fig. 4A-C).

In order to further understand and characterize the interactions between TG2 and TGF-β₁, the binding interaction of TG2 with TGF-β₁ was modeled by molecular docking. The present results found that there were ten sites that prove TGF-β₁ may be associated with TG2 (Table II; Fig. 5).

In the present study, the fluorescence intensity of TG2 and TGF-β₁ in MCF-7 cells treated with the inhibitors of TGF-β₁ and TG2 was assessed by immunofluorescence. The results demonstrated that the fluorescence intensity of TG2 and TGF-β₁ in MCF-7 cells treated with the TGF-β₁ inhibitor (8.31 µmol/l) and the TG2 inhibitor (150 µmol/l) was lower compared with that in untreated MCF-7 cells (P<0.05; Fig. 6). In addition, the fluorescence and protein levels of TG2 and TGF-β₁ in MCF-7 cells induced with TGF-β₁ were higher compared with that in untreated MCF-7 cells (P<0.001; Figs. 7 and 8). In Fig. 9, the expression levels of TG2 and TGF-β₁ in MCF-7 cells treated with ITD (TGF-β₁), Pt (cisplatin), and ITD plus Pt were lower compared with those in untreated MCF-7 cells (P<0.05 and P<0.001).