The effects of miR-25 and the potential molecular mechanisms on migration and invasion of triple negative breast cancer

Abstract

Objective To explore the expression of tiny RNA-25 (microRNA-25, miR-25) in the plasma、tissues of triple-negative breast cancer (TNBC) patients and cell lines, to investigate the potential molecular mechanisms of miR-25 on migration and invasion of TNBC. Methods Real-time fluorescent quantitative PCR was used to detect the expression of miR-25 in the plasma of TNBC patients. Linked omics web platform was used to analyse miR-25 level in samples of TNBC and non-TNBC. Real-time fluorescent quantitative PCR was also used to detect the miR-25 level in TNBC cell lines. The wound healing and transwell assay was applied to assess the effects on migration and invasion of TNBC cell lines which transfected with miR-25 inhibitor or the negative control. The luciferase reporter assay was used to validate the relationship between miR-25 and the sphingosine-1-phosphate phosphatase 1 (SGPP1) in HEK293T cell. The wound healing and transwell assay was used to detect the migration and invasion ability of TNBC cell lines when cotransfected with pCMV6-SGPP1 and miR-25. Furthermore, Western blot was performed to detect the SGPP1 level in TNBC cell lines. Results The expression of miR-25 was significantly elevated in the plasma of 86 TNBC patients compared with the healthy controls (P value was 0.031). LinkedOmics web platform analysis showed that miR-25 expression was significantly higher in TNBC samples than in non-TNBC samples with Luminal A or Luminal B (P value was<0.001 and 0.006). The level of miR-25 was also elevated in TNBC cell lines HS578T, HCC1806, MDA-MB-231 and BT549(P value was 0.006, 0.01, 0.029 and 0.046). The MDA-MB-231 and HS578T cells which transfected with miR-25 inhibitor exhibited a significant slower wound healing rate than control (P value was 0.035 and 0.001). At the same time, when transfected with miR-25 inhibitor, MDA-MB-231 and HS578T both exhibited a decreased invasion ability compared with the control group(P value was 0.002 and 0.001). LinkedOmics web platform analysis showed that sphingosine-1-phosphate phosphatase 1 (SGPP1) gene level was negatively correlated with miR-25 in the tissues of TNBC patients (P value was 0.037). The luciferase reporter assay validated that SGPP1 was a directed target of miR-25. The western blot assay indicated that the SGPP1 level was increased in MDA-MB-231 and HS578T after transfection with miR-25 inhibitor. Over-expression of SGPP1 could abrogate the positive effects of miR-25 on migration and invasion when pCMV6-SGPP1 was cotransfected with miR-25 (P value was all 0.002). Conclusions MiR-25 was elevated in both plasma and tissues of TNBC patients and also increased in TNBC cell lines. Transfection of MDA-MB-231 and HS578T cells with miR-25 inhibitor resulted in reduced migration and invasion. Moreover, SGPP1 was identified as a novel target of miR-25. The ability of miR-25 to promote TNBC cell migration and invasion is attributable to its effect on SGPP1 suppression. Key words: Triple negative breast neoplasms; microRNAs; Membrane proteins; Phosphoric monoester hydrolases; Neoplasm metastasis