Let-7b-5p sensitizes breast cancer cells to doxorubicin through Aurora Kinase B

Abstract

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the expression level of the target genes in the cell. Breast cancer is responsible for the majority of cancer-related deaths among women globally. It has been proven that deregulated miRNAs may play an essential role in the progression of breast cancer. It has been shown in many cancers, including breast cancer, that aberrant expression of miRNAs may be associated with drug resistance. This study investigated the effect of let-7b-5p, detected by bioinformatics methods, on Dox resistance through the Aurora Kinase B (AURKB) gene. In silico analysis using publicly available miRNA expression, GEO datasets revealed that let-7b-5p significantly downregulated in BC. Further in silico studies revealed that of the genes among the potential targets of let-7b-5p, AURKB was the most negatively correlated and may be closely associated with Dox resistance. Expression analysis via quantitative PCR confirmed that let-7b-5p was downregulated and AURKB was upregulated in breast cancer tissue samples. Later, functional studies conducted with MCF-10A, MCF-7, and MDA-MB-231 cell lines demonstrated that let-7b-5p inhibits cancer cells through AURKB and sensitizes them to Dox resistance. In conclusion, it has been shown that the let-7b-5p/AURKB axis may be significant in breast cancer progression and the disruption in this axis may contribute to the trigger of Dox resistance.

Fig 1

(A) DEmiRNAs shared between the three datasets (B) let-7b-5p had the potential to target a total of 1215 genes, including 14 overlapping genes, according to the miRNet tool. C) Venn diagram of overlapping genes between TCGA-BC data, GSE100925, GSE229571 geo datasets and miRNet-let-7b-5p potential targets. (D) To identify the potential transcription factors associated with overlapping 14 genes, we employed official human gene names, RegNetwork, and P<0.05 criteria in the miRNet tool. The results revealed that there were 80 transcription factors (with 467 edge connections). Nodes in pink: Overlapping genes, nodes on green: Transcription factors.

Fig 2. miRNAs that may have prognostic significance based on both TCGA-BC and METABRIC datasets.

The diminished expression of let-7b-5p (A-B) and let-7c-5p (C-D) miRNAs has a negative effect on BC-OS. The increased expression of miR-1290-5p (E-F) also had a harmful impact on BC-OS. Figures (A), (C), and (E) were produced using TCGA-BC data. Figures (B), (D), and (F) were created based on METABRIC data.

Fig 3

(A) STRING Analysis results: Nodes: 14, edges: 89, average node degree: 12.7, avg. local clustering coefficient: 0.98, the expected edges: 8, PPI p-value: < 1.0e-16 (B) Enrichment analysis was performed with 14 official gene names using Enrichr. According to the KEGG 2021 pathway analysis results, it was determined that 14 genes may play a role in important pathways, such as the oocyte maturation p53 signal pathway. (C) Schematic depiction of AURKB, showing the probable let-7b-5p target site. (D) According to TCGA-BC data, the Pearson correlation between let-7b-5p and AURKB expressions was significant (investigated using the CancermiRNome tool). (E) According to TCGA-BC data, AURKB expression was found to be significantly increased in all stages compared to normal tissue (analysis was performed using ExplORRnet).

Fig 4. In BC tissue samples and BC cell lines, the expression of let-7b-5p decreased while the expression of AURKB increased.

Quantification of the relative mRNA expression level of (A) let-7b-5p and (B) AURKB in 72 BC tumor specimens and their matched paracancerous tissue specimens using quantitative PCR. Examination of the (C) let-7b-5p miRNA (D) AURKB mRNA expression levels in different parental and doxorubicin-resistant BC cells. ROC analysis of (E) let-7b-5p and (F) AURKB expressions in BC tissue samples. RNU43 was used as an internal control for miRNA evaluation, and GAPDH was used as an internal control for mRNA evaluation.

Fig 5. Restoration of let-7b-5p miRNA expression or inhibition of AURKB expression in parental and doxorubicin-resistant BC cells.

Overexpression of let-7b-5p reduced AURKB expression in BC parental and doxorubicin-resistant cells. (A) The let-7b-5p mimic transfection efficiency was examined by qRT-PCR 24h after transfection. RNU43 was used as an internal control. (B) and (C) Determination of the AURKB mRNA expression levels in parental and doxorubicin-resistant BC cell lines. (D) The protein expression level of AURKB was detected by western blot in cells transfected with let-7b-5p mimics, or miR-NC. GAPDH was used as an internal control in mRNA and protein detection experiments. *P<0.05, **P<0.01, compared with the miR-NC or NC group. MiR-NC: miRNA negative control, NC: Negative control, AURKB inh.: AURKB inhibitor (Barasertib), MCF-7/Dox.: MCF-7 Doxorubicin-resistant cells, MDA-MB-231/Dox.: MDA-MB-231 Doxorubicin-resistant cells.

Fig 6. let-7b-5p restoration or AURKB inhibition sensitized BC cells to doxorubicin.

IC50 values of (A) Doxorubicin and (B) AURKB inhibitor (Barasertib) in parental and doxorubicin-resistant cells. Relative cell viability (mean ± SD) (C) MCF-7, (D) MCF-7/Dox. (Doxorubicin resistant), (D) MDA-MB-231, (E) MDA-MB-231/Dox. (Doxorubicin resistant) cells of let-7b-5p mimic transfected or AURKB inhibitor-treated with doxorubicin for 48 h.

Fig 7. Cell viability imaging in parental and doxorubicin-resistant cells.

Light microscopy images of (A) MCF-7, (B) MCF-7/Dox, (C) MDA-MB-231, and (D) MDA-MB-231/Dox cells transfected with let-7b-5p or miR-NC after 48 hours. Let-7b-5p overexpression significantly reduced parental and doxorubicin-resistant cell proliferation compared to control groups. The images were captured at 10x magnification. The bars represent mean ± SD, *p < 0.05. Dox.: Doxorubicin resistant.

Fig 8

The AURKB inhibitor (Barasertib) reduced migration in (A) doxorubicin-resistant MCF-7 cells and (B) MDA-MB-231 doxorubicin-resistant cells. Furthermore, migration was dramatically reduced in doxorubicin-resistant cells with the combination of transfection of a let-7b-5p mimic and AURKB inhibitor treatment.

Fig 9. Wound healing assay on the cell migration capability of parental and doxorubicin-resistant BC cells following transfection of let-7b-5p mimic.

Evaluation of the cell migration distances in (A) MCF-7 (B) MCF-7/Dox. cells. Let-7b-5p overexpression in both the parental and doxorubicin-resistant cells significantly reduced migration compared to the control groups.

Fig 10. The effect of let-7b-5p mimic transfection on apoptosis of BC cells.

Overexpression of let-7b-5p promoted apoptosis of (A) MCF-7 and (B) MCF-7/Dox cells **; P < 0.01.

Fig 11. Graphical abstract of the study.

The relationship between miRNAs and target genes in BC doxorubicin resistance was investigated following bioinformatics and wet lab study. In the bioinformatics part publicly available geo datasets were used for DEmiRNAs and DEMs selection. We have identified 19 shared DEmiRNAs in the 3 geo datasets. OS analysis was performed with the Kmplot tool, and it was found that let-7b-5p, miR-1290-5p, and let-7c-5p were significant in BC in both TCGA and METABIRIC data. According to Pubmed, let-7b-5p was chosen for wet lab research because it contained more results about BC associations than the other two miRNAs. DEMs were selected using TCGA-BC, GSE100925 and GSE229571 geo datasets. 14 shared genes (let-7b-5p potential targets) were found. According to the literature review and correlation analysis results, the AURKB gene was chosen as a candidate for wet lab research because it was seen to be more closely related to let-7b-5p among the overlapping genes. In the wet lab part of the study, let-7b-5p and AURKB expression levels were investigated in breast cancer tissue samples and cell lines (parental and/or doxorubicin-resistant). The effect of the let-7b-5p/AURKB axis on cellular processes was investigated by transfecting let-7b-5p mimic parental cells and doxorubicin-resistant cells and/or treating AURKB inhibitor (Barasertib). As a result, our findings showed that let-7b-5p sensitized breast cancer cells to doxorubicin resistance through the AURKB gene.