MiR-890 inhibits proliferation and invasion and induces apoptosis in triple-negative breast cancer cells by targeting CD147

Abstract

Background: Triple-negative breast cancer (TNBC) is a type of breast cancer with a high degree of malignancy. Because of the remarkable biological characteristics of high invasion, metastasis and recurrence, TNBC is often accompanied by a poor prognosis. As a molecular characteristic of TNBC, high expression of CD147 has been confirmed by a large number of studies. However, the mechanism of CD147 expression regulation in TNBC remains elusive. In this study, we investigated the roles of miR-890 in inhibiting CD147.

Methods: Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to detect CD147 mRNA and miR-890 level, and western blotting was used to detect CD147 protein. Bioinformatics screening and 3'-Untranslated Region (3'-UTR) luciferase assays were used to analyze the microRNAs (miRNA) binding site. Cell proliferation, apoptosis and invasion were assessed by using CCK-8, flow cytometry and transwell assays.

Results: The upregulation of miR-890 inhibited cell proliferation and invasion, induced apoptosis in MDA-MB-231 and HCC-70 TNBC cells by negatively regulating its target gene, CD147, and the upregulation of CD147 rescued the inhibitory effects of miR-890. miR-890 targeted CD147 by binding to its 3'-UTR. Further results showed that the upregulation of miR-890 also inhibited the expression of MMPs, the downstream genes of CD147, and promoted the cleavage of Caspase-3. The CD147 recovery experiment was further confirmed by the activity changes in the downstream MMPs of CD147. In addition, it was confirmed that the effect of CD147 in promoting TNBC cell proliferation and invasion, inhibiting apoptosis was related to the change in caspase-3 activity.

Conclusion: The downregulation of miR-890 is the potential cause of high CD147 expression in TNBC, which can promote the malignant transformation of TNBC.

Keywords: CD147; Invasion; Proliferation; TNBC; miR-890.

Fig. 1

Levels of miR-890, CD147 mRNA and protein in TNBC tissue and cells. a Determination of CD147 mRNA, CD147 protein and miR-890 expression in 20 pairs of TNBC tissues and adjacent tissues. U6 and β-actin served as internal reference for the determination of miR-890 and CD147 mRNA, and the relative expression values of miR-890 and CD147 mRNA content in TNBC tissue was used. For the determination of CD147 protein expression, 20 pairs of samples were pooled, and tested by western blotting, β-actin served as internal reference. b Correlation analysis of CD147 protein/mRNA and miR-890 in 20 TNBC tumors. c. Determination of CD147(left), CD147 protein (middle,42 kDa) and miR-890 (right) in MDA-MB-231 and HCC-70 and MCF-10A cells. ** P < 0.01, vs. MCF-10A. The tests were carried out on three biological triplicates, and data are expressed as the mean ± SD

Fig. 2

miR-890 binds to CD147 3’UTR. a Predicted binding site of miR-890 in 3′-UTR of CD147 gene; b Effects of miR-890 on the expression of a luciferase cassette with the CD147 3′-UTR. 293TN cells were transfected with pGL3-wt-CD147 or pGL3-mt- CD147 in the presence or absence of miR-890-mimic or inhibitor and subjected to luciferase activity assay 48 h later. The histogram shows the relative firefly luciferase activity for the different experimental groups. *, P < 0.05, and **, P < 0.01. Data are expressed as mean ± SD of at least three independent experiments

Fig. 3

Genetic intervention through a lentiviral approach and interaction detection. a GFP expression 72 h after MDA-MB-231 was infected with recombinant virus. The infection rate was estimated by observing the number of the cells expressing GFP in each view. Determination of miR-890 by qRT-PCR with U6 served as internal reference, and the CD147 protein level was detected by western blotting with β-actin (37 kDa) served as an internal reference (down). b The same experimental data appeared in HCC-70. ** P < 0.01. The tests were carried out on three biological triplicates, and data are expressed as the mean ± SD

Fig. 4

Effects of miR-890 upregulation on the proliferation, apoptosis and invasion in TNBC cells. a The proliferation of MDA-MB-231 and HCC-70 cells 24–72 h after being infected with the indicated virus determined by CCK-8 assay. b Apoptosis in MDA-MB-231 infected with indicated lentivirus. Left panel: representative plots of MDA-MB-231 undergoing indicated treatments, the x-coordinate is the FITC channel and the y-coordinate shows PI staining, the lower left panel represents living cells, the lower right panel shows early apoptotic cells, the upper left panel shows necrotic cells and the upper right panel shows late apoptotic cells. Right panel: Quantification of apoptosis for the indicated treatments (including early and late apoptotic). c Invasion data of the HCC-70 cells 72 h after being infected with the indicated virus determined by a transwell assay.** P < 0.01, *P < 0.05. The tests were carried out on three biological triplicates, and data are expressed as the mean ± SD

Fig. 5

Effects of miR-890 expression on MMP-9 and Caspase-3 cleaved. a MDA-MB-231 and HCC-70 infected with or without Lv-miR890 or Lv-CD147,72 h post infection were used for detecting MMP-9 (78 kDa) and Caspase-3 cleaved (17 kDa). β-actin as a loading control. Data were representative of at least three independent experiments. ** P < 0.01. b The same experimental data appeared in HCC-70