A total of 68 female patients with TNBC were enrolled in the Heilongjiang Farms and Land General Hospital (Haerbin, China) between April 2016 and July 2018. The inclusion criteria were as follows: i) Patients diagnosed by histopathological biopsies; ii) patients with complete medical records; and iii) patients who were willing to participate in the study. The exclusion criteria were as follows: i) Patients with other types of malignancies; ii) patients with chronic disease; and iii) patients who were treated within three months prior to admission. All patients received breast biopsies and tumor tissue as well as healthy adjacent tissue samples were obtained from each patient. The age of patients ranged between 32 and 66 years (median, 49 years; mean ± standard deviation, 48.6±5.7 years). The patients included 13 cases at stage I, 22 cases at stage II, 16 cases at stage III and 17 cases at stage IV. Tumor size was determined by performing magnetic resonance or X-ray imaging. Patients were divided into groups according to tumor size: i) 0–2 cm (n=19); ii) 2–5 cm (n=30); and iii) >5 cm (n=19). Patients were divided into groups according to the absence or presence of metastasis: i) Non-metastasis group (n=30); and ii) metastasis group (n=38). The Ethics Committee of Heilongjiang Farms & Land General Hospital approved the present study prior to enrolment of patients. All patients signed informed consent.

The present study included two TNBC cell lines, BT-549 and HCC70. The two cell lines were purchased from the American Type Culture Collection (ATCC). Cells were cultured using RPMI-1640 medium (ATCC) supplemented with 10% fetal bovine serum, 1% Penicillin and Streptomycin (all from Sangon Biotech Co., Ltd.) at 37°C with 5% CO₂.

In order to detect the expression of lncRNA DRHC and lncRNA HOTAIR in both in vitro cultivated TNBC cells and tissue samples from TNBC patients, the RNeasy Mini kit (Qiagen GmbH) was used to extract total RNA, according to the manufacturer's instructions. The Applied Biosystems™ High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to perform reverse transcription (25°C for 5 min, 55°C for 20 min and 75°C for 10 min). The qScript One-Step RT-qPCR kit (Quantabio) was used to perform qPCR, according to the manufacturer's protocol. All qPCR reactions were performed using an ABI 7500 system. lncRNA DRHC, lncRNA HOTAIR and GAPDH endogenous control primer pairs were designed and synthesized by Shanghai GenePharma Co., Ltd. The following primer sequences were used: lncRNA DRHC forward, 5′-GCTTAGAAATTTCTTCCACCTG-3′; lncRNA DRHC reverse, 5′-CTGCCCTAGCGATTTGTGAA-3′; lncRNA HOTAIR forward, 5′-CAGTGGGGAACTCTGACTCG-3′; lncRNA HOTAIR reverse, 5′-GTGCCTGGTGCTCTCTTACC-3′; GAPDH forward, 5′-GTCTCCTCTGACTTCAA-3′; and GAPDH reverse, 5′-ACCACCCTGTTGCTGTA-3′. The following thermocycling conditions were used: 95°C for 2 min, followed by 40 cycles of 95°C for 10 sec and 55°C for 30 sec. The expression of lncRNA DRHC and lncRNA HOTAIR was normalized to GAPDH endogenous control using the 2^−ΔΔCq method (12). The sample with the lowest expression level was set to ‘1’ and all other samples were normalized to this sample.

Vectors expressing lncRNA DRHC (11) or lncRNA HOTAIR (NR_047517.1) as well as the empty pcDNA3.1 vector were purchased from Sangon Biotech Co., Ltd. BT-549 and HCC70 cell lines were cultured overnight to reach 70–80% confluence. Vectors (10 nM) were transfected into cells using Lipofectamine^® 2000 (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.). Cells treated with Lipofectamine 2000 but without vectors were used as control (C) cells. Cells transfected with empty vectors were used as the negative control (NC) cells. Cells were collected 24 h following transfection for subsequent experimentation. The transfection efficacy was determined using RT-qPCR.

Cell proliferation was assessed using transfected cells with lncRNA DRHC or lncRNA HOTAIR overexpression rates of 200%, as determined by RT-qPCR. Briefly, cells were harvested and 0.1 ml cell suspension (4×10⁴ cells/ml) in RPMI-1640 medium was added into each well of a 96-well plate. A total of 10 µl Cell Counting Kit-8 (CCK-8, Sigma-Aldrich; Merck KGaA) solution was added to each well following 24, 48, 72 and 96 h of incubation (37°C, 5% CO₂). The cells were cultured for an additional 4 h and the optical density values were measured at a wavelength of 450 nm.

All experiments in this study were repeated three times and the data are presented as the mean ± standard deviation (SD). All statistical analyses were performed using the GraphPad Prism software (version 6.0; GraphPad Software, Inc.). Comparison of the expression levels of lncRNA DRHC or lncRNA HOTAIR between tumor tissues and adjacent healthy tissues were performed using the paired t-test. Comparisons among three groups were performed by the one-way analysis of variance followed by the Tukey's test. Correlations between the expression levels of lncRNA DRHC and lncRNA HOTAIR in tumor tissues and adjacent healthy tissues were analyzed using Pearson's correlation coefficient. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of lncRNA DRHC and lncRNA HOTAIR in tumor and adjacent healthy tissues obtained from 68 patients with TNBC were measured using RT-qPCR. Results revealed that lncRNA DRHC was significantly downregulated (Fig. 1A), while lncRNA HOTAIR was significantly upregulated (Fig. 1B) in tumor tissues compared with adjacent healthy tissues (P<0.05).

Correlations between the expression levels of lncRNA DRHC and lncRNA HOTAIR in tumor and adjacent healthy tissues were analyzed using Pearson's correlation coefficient. As shown in Fig. 2, the expression levels of lncRNA DRHC and lncRNA HOTAIR were negatively correlated in tumor tissues (P<0.0001; r, −0.86; Fig. 2A) but not in adjacent healthy tissues (P=0.21; r, −0.16; Fig. 2B).

In vitro overexpression experiments were performed to further investigate the interactions between lncRNA DRHC and lncRNA HOTAIR. As shown in Fig. 3A, overexpression of lncRNA DRHC in the TNBC cell lines BT-549 and HCC70 decreased the expression level of lncRNA HOTAIR (P<0.05). However, lncRNA HOTAIR overexpression did not significantly affect the expression level of lncRNA DRHC (Fig. 3B).

As shown in Fig. 4A, the expression levels of lncRNA DRHC in tumor tissues decreased significantly as the tumor diameter increased (P<0.05). However, no significant difference in the expression level of lncRNA DRHC was found between the metastasis and non-metastasis groups (Fig. 4B).

The correlation between DRHC and tumor size indicated the possible involvement of DRHC in the proliferation of TNBC cells. Therefore, the CCK-8 assay was performed to investigate the role of DRHC in the proliferation of TNBC cell lines. Compared with the C and NC groups, the overexpression of lncRNA DRHC inhibited while the overexpression of lncRNA HOTAIR promoted the proliferation of TNBC cell lines (P<0.05; Fig. 5). In addition, lncRNA HOTAIR overexpression attenuated the inhibitory effects of lncRNA DRHC overexpression on cancer cell proliferation (P<0.05; Fig. 5).