Research patients in this study were 69 TNBC patients (38 to 71 year; median age: 54 years) selected from the 302 TNBC patients who were admitted by Longgang District Central Hospital of Shenzhen during the time period between December 2015 and December 2018. Inclusion criteria:1) newly diagnosed TNBC patients; 2) therapies were not initiated before admission. Excluson criteria: 1) other clinical disorders were observed; 2) any treatments for any diseases perfromed within 3 months before admission; 3) previous history or family history of malignancies. The 69 TNBC patients included 13, 27, 16 and 13 cases at AJCC stage I-IV, respectively (table 1). All patients were negative for HER2, PR and ER. All patients were informed of the details of whole experimental procedure and signed informed consnet. Ethics Committee of Longgang District Central Hospital of Shenzhen approved this study.
Tissues and TNBC cells
Before any therapies were initiated, all patients were subjected to breast biopsy. During biopsy, paired fresh TNBC (cancer) and adjacent (within 3 cm around tumors) non-cancer tissues (weight 0.1 to 0.15 g) were collected from each patient. All TNBC and non-cancer tissues were confirmed by 3 experienced pathologists. This study included BT-549 and BT-20 TNBC cell lines to perform cell experiment. 10% fetal bovine serum (FBS) was added into RPMI-1640 Medium, and the mixture was used as the culture medium of BT-549 and BT-20 cells. Cells were cultivated under conditions of 37°C and 5% CO2.
Transient cell transfections
SEMA3B-AS1 (NCBI Accession: NR_110702.1) and CDK4 (NCBI Accession: CR542247.1) expression vectors were constructed using pcDNA3.1 vector, which was purchased from Sangon (Shanghai, China). Negative control miRNA and miR-545 mimic were bought from Sigma-Aldrich (USA). SEMA3B-AS1 siRNA and negative control siRNA were also designed and synthesized by Sangon. MiR-545 inhibitor and inhibitor negative control were synthesized by Sangon (Shanghai, China). 10 nM vector, 35 nM miRNA or 35 nM inhibitor were transfected into 106 BT-549 cells using Lipofectamine 2000 reagent (Invitrogen, USA). Transfection with empty vector, negative control miRNA or inhibitor negative control (NC) was used as negative control. Cells without any transfections were control (C) cells. Subsequent experiments were performed using cells collected at 24 h atfer trasnfection.
Tissues or cells were mixed with RiboZol™ RNA Extraction Reagent (VWR, USA) to extract total RNAs. After digestion with DNase I, RNA samples were subjected to reverse transcription using PrimeScript RT Reagent Kit (Takara, Japan). With cDNAs as template, qPCR mixtures were prepared using qScript One-Step RT-qPCR Kit (Quantabio, USA) to detect the mRNA expression of SEMA3B-AS1 and CDK4 with 18S rRNA and GAPDH as endogenous control, respectively.
Extractions of miRNAs from ground tissues and cells were performed using microRNA Purification Kit (Cat. 21300, Norgen Biotek Corp). QScript microRNA cDNA Synthesis Kit (Quantabio, Beverly, MA, USA) was used to perform miRNA reverse transcription, and qPCR mixtures were prepared using miScript SYBR Green PCR Kit (QIAGEN, Germany) to detect the expression of miR-545 with U6 as endogenous control.
Primer sequences were: 5'-CTCCAATATCTCAACCTCTC-3' (forward) and 5'-GGGCACGTTCACCAGACTCA-3' (reverse) for SEMA3B-AS1; 5'-AGTGTGAGAGTCCCCAATGG-3' (forward) and 5'-CCTTGATCTCCCGGTCAGTT-3' (reverse) for CDK4;
5'-CTACCACATCCAAGGAAGCA-3' (forward) and 5'-TTTTTCGTCACTACCTCCCCG-3' (reverse) for 18S rRNA; 5'-AGGTGAAGGTCGGAGTCAACG-3' (forward) and 5'-AGGGGTCATTGATGGCAACA-3' (reverse) for GAPDH. Forward primer sequence of miR-545 was 5’-TCAGCAAACATTTATTGTG-3’. U6 forward primer and universal reverse primer were from the kit. All PCR reactions were repeated 3 times, and data were normalized using 2−ΔΔCT method.
Measurement of cell proliferation ability
BT-549 and BT-20 cells were harvested at 24 h after transections. 1 ml RPMI-1640 Medium (10% FBS) was mixed with 3× 104 cells to prepare single cell suspensions with a final cell density of 3× 104 cells per ml. Cell suspensions were added into a 96-well plate with 0.1 ml cell suspension per well. Under the conditions of 5% CO2 and 37°C, cells were cultivated, and 10 µl CCK-8 solution (Sigma-Aldrich, USA) was added into each well every 24 h for 4 times. Following that, cells were cultivated for further 4 h and OD values were measured at 450 nM to reflect cell proliferation rate.
BT-549 cells were harvested at 24h after transections, and 105 cells were mixed with RIPA solution (Thermal Fisher Scientific) to extract total proteins. Following denaturing, electrophoresis was performed using SDS-PAGE gel (10%) to separate protein molecules with different molecular weights. After that, proteins were transferred to PVDF membranes, and blocking was performed for 2 h at 25°C in PBS containing 5% non-fat milk. PVDF membranes were then incubated with CDK4 (1: 1400, ab137675, Abcam) and GAPDH (1: 1400, ab9845, Abcam) rabbit polyclonal primary antibodies at 4°C overnight. Following that, membranes were further incubated with secondary antibody of IgG-HRP goat anti rabbit (1:1000, MBS435036, MyBioSource). Finally, signals were developed using ECL (Sigma-Aldrich, USA), and all data were processed by Image J v1.46 software.
Three biological replicates were included in each experiment, and mean values were calculated. Differences between TNBC and non-cancer tissues were analyzed by performing paired t test. Differences among different cell transfection groups were analyzed by performing one-way ANOVA and Tukey test. Correlations were analyzed by performing linear regression. p < 0.05 was statistically significant.