Tumor-suppressive microRNA-452 inhibits migration and invasion of breast cancer cells by directly targeting RAB11A

Abstract

Breast cancer is the most common type of malignant tumor in females, and metastasis is the most common cause of breast cancer-associated mortality. Previous studies have identified that abnormal expression of microRNAs is commonly observed in human cancer and may be crucial for cancer metastasis. In the present study, microRNA-452 (miR-452) was investigated for its ability to act as a tumor suppressor in breast cancer. miR-452 expression was quantified in breast cancer tissue samples and cell lines with reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Transwell migration and invasion assays were used to investigate the effect of miR-452 on the migration and invasion capabilities of breast cancer cells. Potential target genes of miR-452 were identified with miRanda and TargetScan. A luciferase reporter assay was performed to validate RAB11A as a putative target of miR-452, and was corroborated by RT-qPCR and western blot analyses. Finally, small interfering RNA (siRNA) was used to knockdown RAB11A expression and confirm whether miR-452 inhibited breast cancer cell migration and invasion via the negative regulation of RAB11A. The results revealed that miR-452 was downregulated in breast cancer tissues and cell lines, and that its downregulation may be associated with breast cancer metastasis, as miR-452 expression inhibited the migration and invasion capacities of breast cancer cells. RT-qPCR and western blot analyses indicated that miR-452 negatively regulated the expression of RAB11A mRNA and protein. The luciferase reporter assay revealed that miR-452 specifically bound to the 3'-untranslated region of RAB11A. Furthermore, inhibition of RAB11A with siRNA inhibited breast cancer cell migration and invasion. In conclusion, the present study has demonstrated that miR-452 may act as a tumor suppressor gene via inhibition of cell migration and invasion by targeting RAB11A in breast cancer.

Keywords: RAB11A; breast cancer; metastasis; microRNA-452.

Figure 1.

miR-452 mimic sequence alongside RAB11A-3′-UTR-WT and RAB11A-3′-UTR-Mut mRNA sequences, including the miR-452-binding site of the WT sequence, and the equivalent area from the Mut sequence. miR-452, microRNA-452; 3′-UTR, 3′-untranslated region; WT, wild type; Mut, mutant.

Figure 2.

Expression levels of miR-452 in breast cancer tissues assessed by reverse transcription-quantitative polymerase chain reaction. (A) miR-452 was downregulated in breast cancer tissue, compared with paired adjacent normal tissue. *P<0.05 vs. normal tissues. (B) miR-452 levels in breast cancer with metastasis were significantly lower than in those without metastasis. Data are presented as the mean ± standard deviation. *P<0.05 vs. no metastasis. miR-452, microRNA-452.

Figure 3.

Expression levels of miR-452 in breast cancer cell lines detected by RT-qPCR. (A) The relative expression of miR-452 was downregulated in breast cancer cell lines compared with the normal breast epithelial cell line, MCF-10A. (B) The transfection efficiency of an miR-452 mimic was assessed by comparing the detected miR-452 levels of mimic-transfected cells, with miR-NC-transfected cells, using RT-qPCR. Data are presented as the mean ± standard deviation. *P<0.05 vs. MCF-10A or miR-NC. miR-452, microRNA-452; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR-NC, negative control microRNA.

Figure 4.

miR-452 inhibits the migration and invasion abilities of breast cancer cells. (A) Transwell migration and (B) Transwell invasion assays were performed to investigate the effects of miR-452 on MCF-7 and MDA-MB-231 breast cancer cells, revealing reduced migration and invasion capabilities in miR-452 mimic-transfected cells. Data are presented as the mean ± standard deviation. *P<0.05 vs. corresponding miR-NC group. miR-452, microRNA-452; miR-NC, negative control microRNA.

Figure 5.

miR-452 directly targets RAB11A in breast cancer cells. The relative RAB11A (A) mRNA and (B) protein expression levels were significantly reduced in the miR-452 mimic-transfected breast cancer cells, compared with miR-NC-transfected cells. (C) A luciferase reporter assay was performed on HEK293T cells co-transfected with an miR-452 mimic or miR-NC, and PmirGLO-RAB11A-3′-UTR-WT or PmirGLO-RAB11A-3′-UTR-Mut, revealing that miR-452 decreased activity only in cells transfected with the WT RAB11A 3′-UTR. Data are presented as the mean ± standard deviation. *P<0.05 vs. corresponding miR-NC group. miR-452, microRNA-452; miR-NC, microRNA negative control; 3′-UTR, 3′-untranslated region; WT, wild type; Mut, mutant.

Figure 6.

Inhibition of RAB11A inhibits migration and invasion of MCF-7 and MDA-MB-231 breast cancer cells. (A) Compared with siR-NC, RAB11A siRNA decreased RAB11A protein expression in breast cancer cells. (B) Transwell migration and (C) Transwell invasion assays were performed, revealing that RAB11A knockdown decreased breast cancer cell migration and invasion capacities. Data are presented as the mean ± standard deviation. *P<0.05 vs. corresponding siR-NC group. siRNA, small interfering RNA; siR-NC, negative control siRNA.