Knockdown of LINC00665 inhibits proliferation and invasion of breast cancer via competitive binding of miR-3619-5p and inhibition of catenin beta 1

Abstract

Background: Long intergenic non-protein coding RNA00665 (LINC00665) plays a crucial tumorigenic role in many cancers, such as gastric cancer and lung adenocarcinoma. However, its role and mechanism of action in the progression of breast cancer (BC) are unknown.

Methods: LINC00665 expression levels were determined using quantitative polymerase chain reaction analysis with BC tissues and cell lines. BC cell proliferation was tested by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, whereas BC cell migration and invasion capabilities were analyzed by performing transwell migration assays. Percentages of apoptotic cells were measured by flow cytometry. Interactions between LINC00665 and miR-3169-5p were examined by performing luciferase reporter assays, and the expression levels of proteins, such as β-catenin, were examined by western blot analysis.

Results: LINC00665 was expressed at high levels in BC tissues and cells. Upregulated LINC00665 expression correlated with tumor size and tumor, node, and metastasis stages, but not with the age of patients. LINC00665 knockdown inhibited BC cell proliferation, migration, and invasion, whereas it promoted apoptosis. Moreover, bioinformatics analysis and the luciferase reporter assay revealed that LINC00665 bound the microRNA (miR) miR-3619-5p. miR-3619-5p expression correlated negatively with LINC00665 expression in BC tissues. miR-3619-5p overexpression inhibited BC cell proliferation, migration, and invasion, but promoted apoptosis. Simultaneous knockdown of LINC00665 and miR-3619-5p led to increased cell proliferation, migration, and invasion, and inhibited apoptosis. Additionally, catenin beta 1, which encodes the β-catenin protein, was the target gene of miR-3619-5p. β-catenin expression clearly decreased after LINC00665 knockdown and miR-3619-5p overexpression, but increased after simultaneous knockdown of LINC00665 and miR-3619-5p.

Conclusion: LINC00665 knockdown inhibited BC cell proliferation and invasion by binding miR-3619-5p and inhibiting β-catenin expression.

Keywords: Breast cancer; CTNNB1; LINC00665; miR-3619-5p; β-Catenin.

Fig. 1

LINC00665 upregulation in breast cancer (BC) tissues and associations with different patient clinical characteristics. a LINC00665 expression levels in BC tissues (n = 1085) and normal breast tissues, as analyzed using gene expression profiling interactive analysis (GEPIA, n = 291). b LINC00665 expression in BC tissues and adjacent normal breast tissues, as measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). c The number of BC patients (x-axis) versus the distribution plot of the log2 values of the relative expression ratio of LINC00665 in BC tissues to that in adjacent normal breast tissues (y-axis). d Relationships between LINC00665 expression and different patient clinical characteristics. **P < 0.01, ***P < 0.001

Fig. 2

LINC00665 expression in breast cancer (BC) cells. a LINC00665 knockdown efficiency in BC cells and in normal cells, as measured by performing quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. b LINC00665 knockdown efficiency, as measured by qRT-PCR, 48 h after transfection. ***P < 0.001

Fig. 3

LINC00665 knockdown inhibited breast cancer (BC) cell proliferation, migration, and invasion, but promoted apoptosis. a BC cell proliferation, apoptosis, migration, and invasion were measured by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, flow cytometry, transwell migration assays, and invasion assays, respectively. The data shown are the mean ± standard deviation (SD). b Representative images showing cell apoptosis, migration, and invasion. ***P < 0.001

Fig. 4

miR-3619-5p is a direct binding target of LINC00665. a The relationships of miR-3619-5p, miR-761, and miR-296-3p expression with poor patient prognosis were analyzed using the Kaplan–Meier plotter (http://kmplot.com/analysis/index.php). b miR-3619-5p expression levels in breast cancer (BC) tissues. c Relationship between expression levels of LINC00665 and miR-3619-5p. d miR-3619-5p expression levels in BC cells. e miR-3619-5p expression levels in BC cells after LINC00665 was knocked down by transfecting cells with the si-LINC00665 plasmid. f Potential binding sites between LINC00665 and miR-3619-5p, as predicted using the LncBase Predicted v.2 website. g and h Luciferase reporter assays showing that LINC00665 has a direct binding site for miR-3619-5p. ***P < 0.001

Fig. 5

miR-3619-5p overexpression inhibited proliferation, migration, and invasion of breast cancer (BC) cells, but promoted apoptosis. a miR-3619-5p expression, as measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis at 48 h after transfecting the miR-3619-5p mimic. b–e Proliferation, apoptosis, migration, and invasion of BC cells measured by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, flow cytometry, transwell migration assays, and invasion assays, respectively. The data shown are the mean ± standard deviation (SD). f Representative images showing cell apoptosis, migration, and invasion. ***P < 0.001

Fig. 6

miR-3619-5p expression was significantly inhibited after simultaneous transfection of the si-LINC00665 plasmid and the miR-3619-5p inhibitor. miR-3619-5p expression, as measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) after simultaneous transfection of the si-LINC00665 plasmid and the miR-3619-5p (or NC) inhibitor at 48 h. ***P < 0.001

Fig. 7

Simultaneous knockdown of LINC00665 and miR-3619-5p promoted breast cancer (BC) cell proliferation, migration, and invasion, but inhibited apoptosis. a Proliferation, apoptosis, migration, and invasion, as measured by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, flow cytometry, transwell migration assays, and invasion assays, respectively. The data shown are the mean ± standard deviation (SD). b Representative images showing BC cell apoptosis, migration, and invasion. ***P < 0.001

Fig. 8

Effect of LINC00665/miR-3619-5p expression on β-catenin expression in breast cancer (BC) cells. a Potential binding sites between CTNNB1 and miR-3619-5p. b Luciferase reporter assay showing that the CTNNB1 3′-untranslated region has a direct binding site for miR-3619-5p. c Western blot showing LINC00665 knockdown and that miR-3619-5p overexpression inhibited expression of β catenin (encoded by CTNNB1). Simultaneous knockdown of LINC00665 and miR-3619-5p promoted β-catenin expression in MCF-7 and MDA-MB-231 cells