ln RNA LINC01234 promotes triple-negative breast cancer progression through regulating the miR-429/SYNJ1 axis

Abstract

Emerging evidence has illustrated that long noncoding RNA 01234 (LINC01234) has played a pivotal role in the development and progression of human cancer. The regulatory role and underlying mechanisms of LINC01234 in triple-negative breast cancer (TNBC) remains unknown. In this study, we analyzed the expression level of LINC01234 in several breast cancer cell lines. CCK-8, EdU, flow cytometry analysis, wound healing assay, and transwell assay were carried out to investigate the effect of LINC01234 on tumor proliferation, apoptosis, and migration. Bioinformatic analysis and luciferase reporter assays were performed to confirm the molecular binding. We found that LINC01234 was dramatically upregulated in breast cancer cell lines, especially in TNBC. The loss and gain-of functional experiments revealed that LINC01234 significantly promoted proliferation, migration, and suppressed cell apoptosis of MDA-MB-231 cells in vitro and inhibited tumorigenesis in vivo. Mechanistic investigations demonstrated that LINC01234 might act as a competing endogenous RNA (ceRNA) for miR-429 to regulate the SYNJ1 expression. The effects of miR-429 and SYNJ1 in MDA-MB-231 cells were also analyzed. Our results revealed that the novel LINC01234/miR-429/SYNJ1 axis played a critical role in progression of TNBC cell line MDA-MB-231, and it may serve as a therapeutic target for TNBC.

Keywords: LINC01234; SYNJ1; miR-429; progression; triple-negative breast cancer.

Figure 1

Knockdown of LINC01234 inhibited TNBC cell proliferation and induces cell apoptosis in vitro and in vivo. A. qRT-PCR analysis of LINC01234 expression in MDA-MB-231 cells transfected with si-NC or si-LINC01234. B. qRT-PCR analysis of LINC01234 expression in MDA-MB-231 cells transfected with empty control or pcDNA-LINC01234. C and D. The effect of LINC01234 on cell proliferation was evaluated by CCK-8 assays. E and F. EdU assays assessed the effect of LINC01234 on cell proliferation. Scale bar: 200 μm (100 ×). G. FACS was performed to determine the apoptosis rate. H. qRT-PCR analysis of LINC01234 expression in MDA-MB-231 cells transfected with sh-RNA. I. Tumor volumes were measured once a week (N=5). J. Images of tumors of each group. K. Tumor weights were evaluated at the end of experiment. NC, negative control. EdU, 5-Ethynyl-2’-deoxyuridine. FACS, fluorescence activated cell sorting. The data were representative of 3 independent experiments with the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 2

LINC01234 promoted TNBC cell migration. The migration capacity of MDA-MB-231 cell transfected with indicated vectors was detected by wound-healing assay (A) and transwell assay (B) Scale bar: 50 μm (200 ×). NC, negative control. The data were representative of 3 independent experiments with the mean ± standard deviation. *P<0.05, ***P<0.001.

Figure 3

LINC01234 functioned as a sponge for miR-429 in MDA-MB-231 cells. A. FISH analysis of the subcellular localization of LINC01234 in MDA-MB-231 cells. Scale bar: 50 μm (400 ×). B. The binding between LINC01234 and Ago2 detected by RIP. C. Co-location detection of LINC01234 and miR-429 using Co-FISH. Scale bar: 50 μm (400 ×). D. The relative luciferase activities were detected in 293T cells co-transfected with miR-429 mimic or NC mimic and LINC01234 WT or LINC01234 MUT. E. The expression of miR-429 was significantly downregulated in MDA-MB-231 cells compared with MCF-10A. NC, negative control. WT, wild type. MUT, mutant type. **P<0.01, ***P<0.001.

Figure 4

Effect of miR-429 on TNBC cells proliferation, apoptosis, and migration. (A) qRT-PCR analysis of miR-429 expression in MDA-MB-231 cells transfected with miR-429 mimic or miR-429 inhibitor. (B) The effect of miR-429 on cell proliferation was evaluated by CCK-8 assays. (C and D) EdU assays assessed proliferation of MDA-MB-231 cells transfected with miR-429 inhibitor or miR-429 mimic. Scale bar: 200 μm (100 ×). (E) The influence of miR-429 mimic on cell apoptosis was evaluated by flow cytometry. Cell migration was determined after infection with miR-429 inhibitor or miR-429 mimic using wound-healing assay (F) and transwell assay (G and H) Scale bar: 50 μm (200 ×). (I) CCK-8 assays were performed to analysis cell proliferation when MDA-MB-231 cells co-transfected with pcDNA-LINC01234, miR-429 or empty control. (J) Transwell assays of MDA-MB-231 cells co-transfected with pcDNA-LINC01234, miR-429 mimic, or empty control. Scale bar: 100 μm (100 ×). NC, negative control. EdU, 5-Ethynyl-2’-deoxyuridine. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 5

Effects of SYNJ1 on TNBC cells proliferation, apoptosis, and migration. (A) The relative luciferase activities were detected in 293T cells co-transfected with miR-429 mimic or NC mimic and SYNJ1 WT or SYNJ1 MUT. The expression level of SYNJ1 in MDA-MB-231 following knockdown (B) or overexpression (C) of LINC01234. (D) qRT-PCR analysis of SYNJ1 expression after co-transfection with si-LINC01234 and miR-429 inhibitor. (E) qRT-PCR analysis of SYNJ1 expression in MDA-MB-231 cells transfected with SYNJ1 siRNA. (F) The effect of SYNJ1 on cell proliferation was evaluated by CCK-8 assays. (G) The influence of SYNJ1 knockdown on cell apoptosis was evaluated by flow cytometry. Cell migration was determined after infection with SYNJ1 siRNA using wound-healing assay (H) and transwell assay (I) Scale bar: 100 μm (200 ×). NC, negative control. EdU, 5-Ethynyl-2’-deoxyuridine. WT, wild type. MUT, mutant type. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.