LncRNA DLG1-AS1 Promotes Cancer Cell Proliferation in Triple Negative Breast Cancer by Downregulating miR-203

Abstract

Purpose: The oncogenic role of long non-coding RNA (lncRNA) DLG1-AS1 has been studied in cervical cancer, but its involvement in triple negative breast cancer (TNBC) is unknown. Here, we aimed to investigate the possible role and underlying mechanism of DLG1-AS1 in TNBC.

Methods: The differential expression of DLG1-AS1 and miR-203 in TNBC tissues and cells was determined using quantitative polymerase chain reaction assays. Correlations between DLG1-AS1 and miR-203 expression across TNBC tissues and non-tumor tissues were analyzed using Spearman rank correlation test. The effects of DLG1-AS1 and miR-203 overexpression, and DLG1-AS1 knockdown on the metastasis of BT-549 and MDA-MB-157 cells were evaluated using a transwell assay. The effects of DLG1-AS1 and miR-203 overexpression on the proliferation of BT-549 and MDA-MB-157 cells were evaluated using Cell Counting Kit-8 and cell colony formation assays.

Results: We found that DLG1-AS1 was upregulated whereas miR-203 was downregulated in tumor tissues of patients and in TNBC cells compared to the adjacent healthy tissues of patients with TNBC and in normal breast MCF-10A cells, respectively. Further, DLG1-AS1 and miR-203 were inversely correlated in tumor tissues. DLG1-AS1 overexpression mediated downregulation of miR-203, whereas miR-203 overexpression had no significant effects on DLG1-AS1 expression. DLG1-AS1 expression was increased, whereas miR-203 levels were decreased with advancing clinical stages. TNBC cell migration was promoted by DLG1-AS1 overexpression and inhibited by miR-203 overexpression or DLG1-AS1 knockdown. Moreover, TNBC cell proliferation was promoted by DLG1-AS1 overexpression and inhibited by miR-203 overexpression. Further, miR-203 overexpression reduced the effects of DLG1-AS1 overexpression.

Conclusion: These results indicate that DLG1-AS1 may promote cancer cell proliferation in TNBC by downregulating the tumor suppressor miR-203.

Keywords: Cell proliferation; Oncogene; RNA, long noncoding; Triple negative breast neoplasms.

Figure 1. DLG1-AS1 and miR-203 expression in TNBC tissues and corresponding OS.

The differential expression of (A) DLG1-AS1 and (B) miR-203 in TNBC was analyzed in both TNBC and non-tumor tissues from the 66 TNBC patients using qPCR assays. Data were compared between the 2 types of tissue by a paired t-test. PCR assays were repeated 3 times and mean values are presented. (C) Survival curve analysis showed that patients in the high DLG1-AS1 group showed significantly lower OS compared to that in patients from the low DLG1-AS1 group (p < 0.05). (D) Patients in the low miR-203 group showed significantly lower OS compared to patients in the high miR-203 group. TNBC = triple negative breast cancer; qPCR = quantitative polymerase chain reaction; OS = overall survival. ^*p < 0.001.

Figure 2. Inverse correlation of DLG1-AS1 and miR-203 in TNBC tissues and cells.

The correlations between DLG1-AS1 and miR-203 expression across TNBC tissues (A) and non-tumor (B) tissues were analyzed using the Spearman rank correlation test. qRT-PCR analysis of DLG1-AS1 levels was performed in normal human breast cells MCF-10A and in BT549, MDA-MB-157, MDA-MB-231, MDA-MB-468, BT20, and CAL51 TNBC cells. qRT-PCR = quantitative reverse transcription-polymerase chain reaction; TNBC = triple negative breast cancer. ^*p < 0.05.

Figure 3. miR-203 downregulation by DLG1-AS1 overexpression in TNBC cells.

BT-549 and MDA-MB-157 cells were transfected with DLG1-AS1 expression vector or miR-203 mimic to further analyze the interactions between DLG1-AS1 and miR-203. (A) Overexpression of DLG1-AS1 and miR-203 was confirmed by qPCR at 48 hours post-transfection. (B)The effects of DLG1-AS1 overexpression on miR-203 and (C) the effects of miR-203 overexpression on DLG1-AS1 were also analyzed by qPCR at 48 hours post-transfection. The experiments were repeated 3 times and mean values are presented. TNBC = triple negative breast cancer; qPCR = quantitative polymerase chain reaction; C = control; NC = negative control. ^*Compared to C, p < 0.05; ^†compared to NC, p < 0.05.

Figure 4. Association of DLG1-AS1 and miR-203 expression levels with clinical stages.

The 66 TNBC patients enrolled in this study included 12, 18, 19, and 15 cases at clinical stage I, II, III, and IV, respectively. (A) DLG1-AS1 and (B) miR-203 expression levels were compared among the 4 clinical stages using ANOVA (one-way) and Tukey test. TNBC = triple negative breast cancer; ANOVA = analysis of variance. ^*Stage I compared to stage II, p < 0.05; ^†stage II compared to stage III, p < 0.05; ^‡stage III compared to stage IV, p < 0.05.

Figure 5. Effects of DLG1-AS1 and miR-203 upregulation and DLG1-AS1 downregulation on BT549 and MDA-MB-157 cell migration.

A transwell assay was performed to study the effects of upregulated DLG1-AS1 and miR-203, and downregulated DLG1-AS1 on TNBC cell migration. NC = negative control; sh = short hairpin. ^*Compared to NC, p < 0.05; ^†compared to NC miRNA, p-value; ^‡compared to shNC, p < 0.05.

Figure 6. Promotion of TNBC cell proliferation by DLG1-AS1 through miR-203.

(A) The effects of DLG1-AS1 and miR-203 overexpression on the proliferation of BT-549 and MDA-MB-157 cells were examined using the CCK-8 cell proliferation assay. pCDH-CMV-DLG1-AS1 and pCDH-miR-203 expression vectors, were used to generate the TNBC cells with stable overexpression of DLG1-AS1 and miR-203. (B) The mock group included cells infected with the pCDH-CMV control. The RNA expression levels are shown. (C) Cell colony formation assays using stable overexpressing cells were carried out to study the long-term effect of upregulated DLG1-AS1 and miR203 on cell proliferation. Experiments were repeated 3 times and mean values are presented. ^*Compared to the pcDNA3.1 group, p < 0.05; ^†compared to NC miRNA; ^‡compared to pcDNA3.1, p < 0.05; ^§miR203 group compared to the NC miRNA group, p < 0.05; ^∥DLG1-AS1 compared to the mock group; ^¶compared to mock, p < 0.05.