lncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

Abstract

Triple-negative breast cancer (TNBC) is a highly invasive subtype of breast cancer. This study investigated the molecular mechanism and influences of MIR503HG, miR-224-5p, and homeobox A9 (HOXA9) on TNBC cell growth and migration. Dual-luciferase reporter gene and RNA immunoprecipitation were performed to examine the regulation of MIR503HG, miR-224-5p, and HOXA9. Cell proliferation, apoptosis, migration, and invasion were evaluated by colony formation, flow cytometry, and Transwell assays. Finally, nude mice were employed to investigate the influence of MIR503HG on TNBC tumor growth. HOXA9 protein levels were detected by immunohistochemical staining. MIR503HG and HOXA9 expression were reduced in TNBC, while miR-224-5p was increased. Overexpression of MIR503HG or HOXA9 reduced the cell migration ability and proliferation and promoted apoptosis, and knockdown of MIR503HG or overexpression of miR-224-5p exhibited the opposite effects. Furthermore, MIR503HG promoted HOXA9 expression by inhibiting miR-224-5p. Overexpression of miR-224-5p reversed the effects of MIR503HG overexpression on TNBC cells, while overexpression of HOXA9 reversed the effect of MIR503HG knockdown. Additionally, an in vivo study proved that MIR503HG inhibited TNBC tumor growth via the miR-224-5p/HOXA9 axis. MIR503HG inhibited cell proliferation and promoted the apoptosis of TNBC cells via the miR-224-5p/HOXA9 axis, which may function as a novel target for the treatment of TNBC.

Keywords: HOXA9; MIR503HG; TNBC; lncRNA; miR-224-5p; triple-negative breast cancer.

Graphical abstract

Figure 1

Expression patterns of MIR503HG, miR-224-5p, and HOXA9 in TNBC cells and tissues (A–F) Relative expression levels of (A) MIR503HG, (B) miR-224-5p, and (C) HOXA9 in TNBC tissues and adjacent tissues, and relative expression levels of (D) MIR503HG, (E) miR-224-5p, and (F) HOXA9 in TNBC cells and normal human breast cells. (G–I) Correlations between MIR503HG, miR-224-5p, and HOXA9 in TNBC tissues. Data represent the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 2

After overexpression of MIR503HG, the expression of HOXA9 was increased by reducing the expression of miR-224-5p (A) Binding site of MIR503HG and miR-224-5p. (B) Detection of the expression levels of miR-224-5p in MDA-MB-231 and MDA-MB-453 cell lines after miR-224-5p mimics transfection by quantitative real-time PCR. (C) Dual-luciferase reporter for MIR503HG and miR-224-5p gene detection. (D) RIP measurement. (E) Binding site of miR-224-5p and HOXA9. (F) Dual-luciferase reporter for HOXA9 and miR-224-5p gene detection. (G) Detection of the expression levels of MIR503HG, miR-224-5p, and HOXA9 by quantitative real-time PCR after overexpression of MIR503HG in MDA-MB-231 and MDA-MB-453 cell lines. (H) detection of HOXA9 expression level in MDA-MB-231 and MDA-MB-453 cell lines via quantitative real-time PCR after miR-224-5p inhibitor transfection. Data represent the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3

Overexpression of MIR503HG inhibited TNBC cell migration and invasion by inhibiting the expression of miR-224-5p (A–C) Expression of MIR503HG, miR-224-5p, and HOXA9 was determined by quantitative real-time PCR. (D and E) Transwell assays were performed to detect cell migration. (F and G) Transwell assays were performed to detect cell invasion. Data represent the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

Overexpression of MIR503HG inhibited TNBC cell proliferation and promoted apoptosis by inhibiting the expression of miR-224-5p (A and B) Clonal formation was performed to detect proliferation. (C and D) Apoptosis was detected by flow cytometry. (E and F) Detection of the protein expression levels of HOXA9, Bcl-2, Bax, and cleaved caspase-3 by western blotting. Data represent the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5

Overexpression of HOXA9 reversed the effect of MIR503HG knockdown on cell migration and invasion (A–C) Expression of MIR503HG, miR-224-5p, and HOXA9 was determined by quantitative real-time PCR. (D) Quantitative real-time PCR detection of HOXA9 levels in cells after overexpression of HOXA9. (E and F) HOXA9 protein levels detected by western blotting. (G and H) Transwell assays were performed to detect cell migration. (I–J) Transwell assays were performed to detect cell invasion. Data represent the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6

Overexpression of HOXA9 reversed the effects of MIR503HG knockdown on cell proliferation and apoptosis (A and B) Clonal formation was performed to detect proliferation. (C and D) Apoptosis was detected by flow cytometry. (E and F) Protein expression levels of HOXA9, Bcl-2, Bax, and cleaved caspase-3 were detected by western blotting. Data represent the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 7

MIR503HG inhibited TNBC tumor growth by downregulating miR-224-5p expression to increase HOXA9 levels in the body (A) Tumors collected from mice. (B) Effect of MIR503HG overexpression on tumor volume in mice. (C) Effect of MIR503HG overexpression on tumor weight in mice. (D) Quantitative real-time PCR detection of MIR503HG, miR-224-5p, and HOXA9 expression levels in mouse tumor tissues after overexpression of MIR503HG. (E) Immunohistochemical detection of HOXA9. (F–G) Western blotting detection apoptosis-related proteins. Data represent the means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.