Downregulation of microRNA-30c-5p was responsible for cell migration and tumor metastasis via COTL1-mediated microfilament arrangement in breast cancer

Abstract

Background: Breast cancer metastasis is the main problem that affects the therapy and prognosis of breast cancer patients. Studies have indicated the role of microRNAs in breast cancer regulation, but the mechanisms are largely unknown.

Methods: In this study, we determined the expression of microRNA-30c-5p (miR-30c-5p) and coactosin-like protein 1 (COTL1) gene in breast cancer tissues, and revealed their effects on breast cancer metastasis regulation. Breast cancer and paracancerous tissues were collected. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression of miR-30c-5p and COTL1, and breast cancer cell line (MCF-7) was employed to verify the relationship between miR-30c-5p and COTL1. Western blot analysis and immunofluorescence were used for proteins analysis and microfilament observation, respectively. A dual-luciferase reporter gene was used for microRNA-gene interaction assay.

Results: The results showed that the expression of miR-30c-5p decreased, while the expression of COTL1 increased in breast cancer tissues. The results of luciferase reporting gene assay showed that, COTL1 was the target of miR-30c-5p. After miR-30c-5p was upregulated, the expression of COTL1 was reduced, microfilament arrangement was in disorder, and cell migration ability was inhibited. After miR-30c-5p was downregulated, the expression of COTL1 was increased, and the cell migration ability was enhanced. COTL1 protein expression levels were significantly higher in cancer tissues with lymph node metastasis.

Conclusions: These findings indicate that miR-30c-5p/COTL1 pathway regulates breast cancer metastasis and can be used as a potential therapy target.

Keywords: Breast cancer; cell migration ability; coactosin-like protein 1 (COTL1); microRNA-30c-5p (miR-30c-5p); microfilament.

Figure 1

Pathological examination of (A) paracancerous and (B) cancer tissues at magnification 40×. (C) The expression of miR-30C-5p in paracancerous and cancer tissues, with U6 being used as the loading control. (D) The expression of COTL1 mRNA in paracancerous and cancer tissues. (E) The expression of COTL1 proteins in paracancerous and cancer tissues, with GAPDH being employed as the loading control. All data are presented as mean ± SE. (F) The correlation between miR-30c-5p and COTL1 proteins in cancer tissues was analyzed by Pearson’s correlation analysis. *** means P<0.001.

Figure 2

The interaction between miR-30c-5p and COTL1, and their effects on cell invasion ability. (A) Bioinformatics prediction of the binding site of miR-30c-5p on the 3’ UTR region of the COTL1 gene. (B) Dual-luciferase reporter gene assay showed the average fluorescence intensity in the different groups. The data are presented as mean ± SE, and the results were from 3 independent assays. ** means P<0.01. (C) MiR-30c-5p expression levels were determined by RT-PCR after cells were transfected with miR-30c-5p mimics, with U6 being used for the loading control. (D) COTL1 mRNA and protein expression levels were determined by RT-PCR and Western blotting after cells were transfected with miR-30c-5p mimics; the data are presented as mean ± SE, and GAPDH was used as protein loading control. (E) Cell proliferation was evaluated by CCK8 assay kit after finished miR-30c-5p mimics transfection for another 24h, * and ** means P<0.05 and P<0.01, respectively. (F) Cell microfilaments were stained red by rhodamine phalloidin, while the nuclei were stained blue with DAPI. (G) Cell invasion ability was assessed by Transwell assay after cells were transfected with miR-30c-5p mimics. *** means P<0.001.

Figure 3

The effects of microRNA inhibitor on COTL1 expression and cell invasion ability. (A) MiR-30c-5p expression levels were determined by RT-PCR after cells were transfected with miR-30c-5p inhibitor, with U6 being used for the loading control. (B) mRNA and protein expression levels were determined by RT-PCR and Western blotting after cells were transfected with miR-30c-5p inhibitor. The data are presented as mean ± SE. GAPDH was employed as the loading control. (C) Cell invasion ability was assessed by Transwell assay after cells were transfected with miR-30c-5p inhibitor. *, P<0.05; *** means P<0.001.

Figure 4

Cell migration ability was assessed by wound healing assay after cells were transfected with miR-30c-5p mimics or inhibitor. The images were obtained under magnification 40×. Initial stage means 0 hour after cells finished transfection and scratch, finished stage means 12 hours after transfection and scratch.

Figure 5

The expression of miR-30c-5p and COTL1 in tissues with or with metastasis. (A) Scatterplot shows the levels of miR-30c-5p measured with RT-PCR in cancer tissues with or without lymph node metastasis. (B) COTL1 protein levels were detected by ELISA kit in tissues with or without lymph node metastasis. *** means P<0.001.

Figure 6

A sketch map was drawn to show how the miR-30c-5p/COTL1 pathway regulating breast cancer cell metastasis. Important molecules and structures are indicated. In normal breast cells, miR-30c-5p inhibited COTL1 expression and kept cell migration to a normal level. When miR-30c-5p was downregulated, COTL1 expression was up regulated, and cell migration was enhanced.