LINC01234 Accelerates the Progression of Breast Cancer via the miR-525-5p/Cold Shock Domain-Containing E1 Axis

Abstract

Backgrounds: Long noncoding RNAs (lncRNAs) are strongly associated with the development of breast cancer (BC). As yet, the function of LINC01234 in BC remains unknown.

Methods: Using biological information, the potential lncRNA, miRNA, and target gene were predicted. LINC01234 and miR-525-5p expression in BC tissues was detected using quantitative real-time reverse transcription polymerase chain reaction. Fluorescence in situ hybridization was used to determine the distribution of LINC01234. Cell proliferation was analyzed using CCK-8 assay, colony formation, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and apoptosis evaluated using flow cytometry. Western blotting was used to evaluate protein expression. Dual-luciferase® reporter, RNA pull-down, and RNA immunoprecipitation assays were performed to analyze the binding relationships among LINC01234, miR-525-5p, and cold shock domain-containing E1 (CSDE1).

Results: We screened out LINC01234, found to be significantly increased in BC tissues, associated with a poor prognosis, and positively correlated with tumor size of BC. Knockdown of LINC01234 suppressed BC cell growth and facilitated apoptosis. Dual-luciferase reporter®, RNA pull-down, and RNA immunoprecipitation assays confirmed that LINC01234 and CSDE1 directly interacted with miR-525-5p. Upregulation of miR-525-5p and suppression of CSDE1 inhibited BC cell growth and induced cell apoptosis.

Conclusion: Upregulation of LINC01234 contributes to the development of BC through the miR-525-5p/CSDE1 axis. LINC01234 may be one of the potential diagnostic and treatment targets for BC.

Figure 1

LINC01234 is increased and correlated with poor prognosis in breast cancer (BC). (a, b) A heat map and volcano plot chart showing the distribution of the DEG TCGA datasets. Green: downregulated genes; red: upregulated genes. (c) The correlation of gene expression between tumor and normal groups. (d) The patients with high LINC01234 expression have poor overall survival (OS). (e) The expression level of LINC01234 using quantitative real-time reverse transcription polymerase chain reaction in BC tissues (n = 101). ^∗∗p < 0.01.

Figure 2

Inhibition of LINC01234-suppresses BC cell proliferation. (a) LINC01234 expression in breast cancer (BC) cells is notably higher than that in MCF-10A. (b) Fluorescence in situ hybridization shows that LINC01234 is mainly expressed in the cytoplasm. (c) Quantitative real-time reverse transcription polymerase chain reaction demonstrates that LINC01234 is successfully inhibited or overexpressed in BC cells. (d, e) CCK-8 assay verifies that the knockdown of LINC01234 notably suppresses cell growth, while overexpression of LINC01234 promotes it. (f, g) Colony formation assay verifies that knockdown of LINC01234 notably suppresses colony-forming activity and overexpression of LINC01234 promotes it. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 3

Inhibition of LINC01234 promotes breast cancer cell apoptosis. (a) Flow cytometry results display that downregulation of LINC01234 markedly promotes cell apoptosis. Meanwhile, overexpression of LINC01234 shows the opposite effect. (b) Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining results show that downregulation of LINC01234 markedly promotes cell apoptosis. Meanwhile, overexpression of LINC01234 shows the opposite effects. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 4

LINC01234 directly interacts with miR-525-5p as a sponge in breast cancer cells. (a) qRT-PCR results show that miR-525-5p is downregulated in breast cancer (BC) tissues. (b) miR-525-5p expression levels are negatively correlated with those of LINC01234. (c) Overexpression of LINC01234 significantly attenuates miR-525-5p expression in BC cells, while downregulation LINC01234 promotes miR-525-5p expression. (d) The potential binding sites of LINC01234 within the miR-525-5p sequence. (e) The luciferase activity is significantly suppressed on cotransfecting LINC01234-WT and miR-525-5p mimics in cells. (f) RNA pull-down results show that miR-525-5p can band with LINC01234. (g) RNA immunoprecipitation results showed that LINC01234 and miR-525-5p are specifically enriched in the AGO2 antibody immunoprecipitated complexes. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 5

Upregulation of miR-525-5p suppresses breast cancer cell proliferation. (a) Quantitative real-time reverse transcription polymerase chain reaction shows that miR-525-5p is successfully inhibited or overexpressed in MCF-7 and MDA-MB-468 cells. (b–d) CCK-8 and colony formation assays verify that overexpression of miR-525-5p notably suppresses cell growth and colony-forming activity, while suppression of LINC01234 promotes it. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 6

Overexpression of miR-525-5p promotes breast cancer cell apoptosis. (a, b) Flow cytometry and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining results display that upregulation of miR-525-5p markedly promotes cell apoptosis. Conversely, downregulation of miR-525-5p shows the opposite effect. ^∗∗p < 0.01.

Figure 7

CSDE1 is a target gene of miR-525-5p. (a) On miRDB, miRTarBase, and TargetScan, 32 overlapping target mRNA are identified, and CSDE1 is chosen as the gene potentially downstream of miR-525-5p. (b) Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analyses validate that CSDE1 is increased and reduced after transfection with LINC01234 overexpression plasmid and small interfering RNA in breast cancer (BC) cells. (c) qRT-PCR analysis of the expression of CSDE1. (d) Immunohistochemical (IHC) analysis of the expression of CSDE1 in BC tissues and normal tissues. (e) Quantification of IHC results. (f) Correlation between CSDE1 expression and LINC01234 expression in BC tissues. (g) The potential binding sequences between miR-525-5p and 3′UTR of CSDE1. (h) miR-525-5p mimics cotransfected with CSDE1-WT notably reduce luciferase activity. (i) RNA pull-down results show that miR-525-5p can band with the 3′UTR of CSDE1. (j) RNA immunoprecipitation results reveal that CSDE1 and miR-525-5p are specifically enriched in the AGO2 antibody immunoprecipitated complexes. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 8

Inhibition of CSDE1 suppresses breast cancer cell proliferation. (a, b) Quantitative real-time reverse transcription polymerase chain reaction and Western blot results show that CSDE1 is successfully inhibited or overexpressed in MCF-7 and MDA-MB-468 cells. (c, d) CCK-8 and colony formation assays verify that knockdown of CSDE1 notably suppresses cell growth and colony-forming activity, while overexpression of CSDE1 promotes them. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 9

Suppression of CSDE1 induces breast cancer cell apoptosis. (a, b) Flow cytometry and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining show that downregulation of CSDE1 markedly promotes cell apoptosis, while overexpression of CSDE1 has the opposite effect. ^∗∗p < 0.01.

Figure 10

LICN01234 promotes breast cancer progression by relieving the repression of miR-525-5p on CSDE1. (a, b) In the rescue experiments, CSDE1 is significantly decreased by miR-525-5p mimics, which are rescued by LINC01234 overexpression. (c, d) Overexpression of miR-525-5p inhibits cell proliferation, while overexpression of LINC01234 can reverse miR-525-5p mimic-mediated suppression of cell proliferation. (e) Overexpression of miR-525-5p promotes cell apoptosis, while overexpression of LINC01234 can reverse miR-525-5p mimic-mediated promotion of cell apoptosis. ^∗∗p < 0.01 vs. NC group; ^##p < 0.01 vs. miR-525-5p mimics group.