PBX3-activated DLG1-AS1 can promote the proliferation, invasion, and migration of TNBC cells by sponging miR-16-5p

Abstract

DLG1-AS1 and PBX3 have been identified as acting as an oncogene in cervical cancer. However, they have not been well explored in triple-negative breast cancer (TNBC). As TNBC is one of the malignancies causing increasing death throughout the world, this study aimed to probe into the regulatory relationship between DLG1-AS1 and PBX3 in TNBC cells. In this study, real-time quantitative PCR (qRT-PCR) and western blot experiments were conducted to investigate the RNA and protein levels of genes of interest in TNBC cells. Functional experiments were implemented, such as 5-ethynyl-2'-deoxyuridine (EdU), transwell, and wound healing assays, to assess the changes in TNBC cell phenotype. Chromatin immunoprecipitation, luciferase reporter, RNA binding protein immunoprecipitation, and RNA pull-down assays were conducted to investigate the binding relationships among subject genes. The results show that DLG1-AS1 and PBX3 displayed high expression in TNBC cells, and PBX3 worked as the transcriptional activator of DLG1-AS1. Also, DLG1-AS1 served as an oncogene in TNBC cells and as a sponge for miR-16-5p to up-regulate JARID2. Meanwhile, JARID2 and PBX3 exerted oncogenic effects on TNBC cell growth. In conclusion, PBX3-activated DLG1-AS1 can promote the proliferation, invasion, and migration of TNBC cells by sponging miR-16-5p and elevating JARID2 expression.

Keywords: DLG1-AS1; JARID2; PBX3; miR-16-5p; triple-negative breast cancer.

Graphical abstract

Figure 1

DLG1-AS1 and PBX3 are highly expressed in TNBC cells and PBX3 acts as the transcriptional activator of DLG1-AS1 (A and B) The expression levels of DLG1-AS1 (A) as well as PBX3 (B) were examined by qRT-PCR, and the protein level of PBX3 was detected through western blot assay in normal cells (MCF-10A) and TNBC cells (BT-549, MDA-MB-231, and MDA-MB-468). (C) The efficiency of PBX3 overexpression and knockdown was tested by qRT-PCR and western blot assays in MDA-MB-231 and MDA-MB-468 cells. (D) qRT-PCR was used to investigate the effect on the expression of DLG1-AS1 caused by inhibited and overexpressed PBX3. (E and F) The DNA motif of PBX3 (E) and the binding site between the DLG1-AS1 promoter and PBX3 (F) are presented with the data recorded from JASPAR. (G) ChIP assay was used to prove that PBX3 can bind to DLG1-AS1. (H) The activity of the binding site of PBX3 was examined by luciferase reporter assays. ∗p < 0.05, ∗∗p < 0.01. The data were measured using mean ± SD.

Figure 2

DLG1-AS1 acts as an oncogene in TNBC cells and as a sponge for miR-16-5p (A) The interference efficiency of DLG1-AS1 was tested by qRT-PCR. (B) EdU experiments were implemented to test the influence of inhibited DLG1-AS1 (sh-DLG1-AS1#1 and sh-DLG1-AS1#2) on the proliferation of TNBC cells (MDA-MB-231 and MDA-MB-468). (C) Transwell assays were conducted to study the invasion situation of MDA-MB-231 and MDA-MB-468 cells affected by sh-DLG1-AS1#1 or sh-DLG1-AS1#2. (D) The migration situation of MDA-MB-231 and MDA-MB-468 cells transfected with sh-DLG1-AS1#1 or sh-DLG1-AS1#2 was evaluated by wound healing experiments. (E and F) The location of DLG1-AS1 in MDA-MB-231 and MDA-MB-468 cells was tested by FISH (E) and subcellular fraction assays (F). (G) Potential miRNAs were dug out by testing their expression in TNBC cells (BT-549, MDA-MB-231, and MDA-MB-468) through qRT-PCR experiments. (H) The binding site of DLG1-AS1 and miR-16-5p is presented according to starBase. (I) The overexpression efficiency of miR-16-5p was tested by qRT-PCR. (J and K) The binding relationship of DLG1-AS1 and miR-16-5p was examined by luciferase reporter assays (J) and RNA pull-down assays (K). ∗∗p < 0.01. The data were measured using mean ± SD.

Figure 3

JARID2 acts as an oncogene in TNBC cells and its expression is regulated by DLG1-AS1 and miR-16-5p (A) A Venn diagram was drawn to display the candidate mRNAs of miR-16-5p according to starBase. (B) The expression of mRNAs in TNBC cells (BT-549, MDA-MB-231, and MDA-MB-468) was investigated by qRT-PCR to find the cells with highly expressed mRNA. (C) The binding site between miR-16-5p and JARID2 is presented according to starBase. (D) The binding relationship between miR-16-5p and JARID2 was proven by luciferase reporter assays. (E) qRT-PCR and western blot were implemented to investigate the influence of reduced DLG1-AS1 and overexpressed miR-16-5p on the expression and protein level of JARID2. (F) The interference efficiency of JARID2 was investigated by qRT-PCR and western blot assays in MDA-MB-231 and MDA-MB-468 cells. (G–I) The proliferation, invasion, and migration situation of MDA-MB-231 and MDA-MB-468 cells was tested to investigate the influence of JARID2 depletion on TNBC cells by EdU (G), transwell (H), and wound healing (I) assays. ∗∗p < 0.01. The data were measured using mean ± SD.

Figure 4

The malignant behaviors of TNBC cells are affected through the DLG1-AS1/JARID2 axis (A) The overexpression efficiency of JARID2 was tested by qRT-PCR and western blot assays in MDA-MB-231 and MDA-MB-468 cells. (B–D) EdU (B), transwell (C), and wound healing (D) assays were implemented in different groups (sh-NC, sh-DLG1-AS1#1, and sh-DLG1-AS1#1+pcDNA3.1-JARID2) to explore the effect of DLG1-AS1 and JARID2 on TNBC cell proliferation, invasion, and migration. ∗∗p < 0.01. The data were measured using mean ± SD.