Mir-30b-5p Promotes Proliferation, Migration, and Invasion of Breast Cancer Cells via Targeting ASPP2

Abstract

Triple-negative breast cancer (TNBC) is the most aggressive subtypes of breast cancer, which has few effective targeted therapies. Various sources of evidence confirm that microRNAs (miRNAs) contribute to the progression and metastasis of human breast cancer. However, the molecular mechanisms underlying the changes in miRNAs expression and the regulation of miRNAs functions have not been well clarified. In this study, we found that the expression of miR-30b-5p was upregulated in breast cancer tissues and breast cancer cell lines, compared to paracancer tissues and normal breast cell lines. Moreover, induced overexpression of miR-30b-5p promoted the MDA-MB-231 and HCC 1937 cell growth, migration, and invasion and reduced the cellular apoptosis. Further studies confirmed that miR-30b-5p could directly target ASPP2 and then activate the AKT signaling pathway. Our results suggested that miR-30b-5p could act as a tumor promoter in TNBC. The newly identified miR-30b-5p/ASPP2/AKT axis represents a novel therapeutic strategy for treating TNBC.

Figure 1

MiR-30b-5p is significantly upregulated in breast cancer specimens and cell lines. (a) The relative mRNA expression of miR-30b-5p in breast cancer tissues. (b) The relative mRNA level of miR-30b-5p in breast cancer cell lines. Data are represented as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 2

MiR-30b-5p promotes proliferation of breast cancer cells. (a) MTT assay showed miR-30b-5p promoted cell growth of MDA-MB-231 and HCC 1937 cells. (b) MTT assay showed miR-30b-5p inhibitor inhibited cell growth of MDA-MB-231 and HCC 1937 cells. (c) MiR-30b-5p promoted colony formation of MDA-MB-231 and HCC 1937 cells. (d) MiR-30b-5p inhibitor reduced colony formation of MDA-MB-231 and HCC 1937 cells. Data was represented as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 3

MiR-30b-5p reduces cell apoptosis of breast cancer cells. (a) Overexpression of miR-30b-5p suppressed cell apoptosis of MDA-MB-231 and HCC 1937 cells. (b) Inhibition of miR-30b-5p increased cell apoptosis of MDA-MB-231 and HCC 1937 cells. Data are represented as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 4

MiR-30b-5p promotes motility of breast cancer cells. (a) Wound healing assay indicated that miR-30b-5p increased migration of MDA-MB-231 and HCC 1937 cells. (b) Wound healing assay indicated that miR-30b-5p inhibitor inhibited migration of MDA-MB-231 and HCC 1937 cells. (c) Transwell assay showed that miR-30b-5p promoted MDA-MB-231 and HCC 1937 cells invasion. (d) Transwell assay showed that miR-30b-5p inhibitor decreased MDA-MB-231 and HCC 1937 cells invasion. Data are represented as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 5

Effects of miR-30b-5p on the expression of apoptosis and EMT-related genes. (a) The expressions of apoptosis and EMT related proteins in MDA-MB-231 and HCC 1937 cells transfected with miR-30b-5p or NC mimics, respectively. (b) The expressions of apoptosis and EMT related proteins in MDA-MB-231 and HCC 1937 cells transfected with miR-30b-5p inhibitor or NC inhibitor, respectively. Data was represented as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 6

ASPP2 is a target gene of miR-30b-5p. (a) Binding sites of miR-30b-5p and ASPP2 3′-UTR. (b) MiR-30b-5p decreased luciferase activity in wild-type group. Data was represented as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 7

MiR-30b-5p regulates the expression of ASPP2/AKT axis. (a) MiR-30b-5p suppressed mRNA expression of ASPP2 in MDA-MB-231 and HCC 1937 cells. (b) MiR-30b-5p inhibitor increased mRNA expression of ASPP2. (c) MiR-30b-5p suppressed protein expression of ASPP2 but contributed to the activation of p-AKT in MDA-MB-231 and HCC 1937 cells. (d) MiR-30b-5p inhibitor increased protein expression of ASPP2 and prevented the activation of p-AKT. Data are represented as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.