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. 2019 Apr;23(4):2442-2456.
doi: 10.1111/jcmm.14090. Epub 2019 Feb 1.

An integrated analysis to predict micro-RNAs targeting both stemness and metastasis in breast cancer stem cells

Mahsa Rahimi  1   2 , Ali Sharifi-Zarchi  2   3 , Javad Firouzi  2 , Mahnaz Azimi  2 , Nosratollah Zarghami  1 , Effat Alizadeh  1   4 , Marzieh Ebrahimi  2
Affiliations

An integrated analysis to predict micro-RNAs targeting both stemness and metastasis in breast cancer stem cells

Mahsa Rahimi et al. J Cell Mol Med. 2019 Apr.

Abstract

Several evidences support the idea that a small population of tumour cells representing self-renewal potential are involved in initiation, maintenance, metastasis, and outcomes of cancer therapy. Elucidation of microRNAs/genes regulatory networks activated in cancer stem cells (CSCs) is necessary for the identification of new targets for cancer therapy. The aim of the present study was to predict the miRNAs pattern, which can target both metastasis and self-renewal pathways using integration of literature and data mining. For this purpose, mammospheres derived from MCF-7, MDA-MB231, and MDA-MB468 were used as breast CSCs model. They had higher migration, invasion, and colony formation potential, with increasing in stemness- and EMT-related genes expression. Our results determined that miR-204, -200c, -34a, and -10b contemporarily could target both self-renewal and EMT pathways. This core regulatory of miRNAs could increase the survival rate of breast invasive carcinoma via up-regulation of OCT4, SOX2, KLF4, c-MYC, NOTCH1, SNAI1, ZEB1, and CDH2 and down-regulation of CDH1. The majority of those target genes were involved in the regulation of pluripotency, MAPK, WNT, Hedgehog, p53, and transforming growth factor β pathways. Hence, this study provides novel insights for targeting core regulatory of miRNAs in breast CSCs to target both self-renewal and metastasis potential and eradication of breast cancer.

Keywords: EMT; breast cancer stem cell; metastasis; miRNAs; self-renewal.

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Conflict of interest statement

The authors confirm that there are no conflicts of interest.

Figures

Figure 1
Figure 1
Cell morphology and efficiency of mammospheres derived from MCF‐7 cultured in different media and coating layers. To optimize the culture medium for mammosphere culture, DMEM alone or in mixture of methylcellulose and matrigel was used in different groups. Moreover, p‐HEMA and Agar were used as coating layer to reduce cell attachment. (A) Compact mammospheres 10 days post culture. Scale bar represents 100 μm for 40× magnifications. (B) The mammospheres forming efficiency was higher in DMEM medium and (C) when plates were coated by agar. **P < 0.01
Figure 2
Figure 2
The sphere and colony formation ability of mammospheres derived from different breast cancer cell lines. (A) Morphology of mammospheres derived from MCF‐7, MDA‐MB231, and MDA‐MB468 cultured with DMEM and in agar‐coated plates. MCF‐7 formed the round and compact spheres, but other cell lines formed grape‐like spheres and looser over passages. (B) Mammosphere‐forming efficiency (MFE) based on the mean percentages of the number of spheres relative to the initial cell seeding number (means ± SD, N = 3). The sphere‐forming ability of mammospheres enhanced with increasing the passages. Bar indicated mean ± SD at least three different biological replicate. G indicated generation. (C) Colony number showed a significant increase under 3D culture conditions compare to adherent culture. The clonogenic ability of mammospheres was higher in MCF‐7‐spheroids (means ± SD, N = 3). (D) Morphology of colonies in mammospheres was mostly holoclones with define border and dense cellularity in all groups. *P < 0.05; **P < 0.01; ***P < 0.001
Figure 3
Figure 3
Cell migration and cell invasion of mammospheres compare to the parental cells. (Left) Cells were seeded at 100 000 cells per insert of a six‐well plate and allowed to migrate towards serum‐present medium for 10 h. Migratory cells on the bottom of the insert membrane were then fixed in formaldehyde and stained with crystal violet migrated and invaded cells that passed through 8 μm filters with/without matrigel. Magnification 10×. (Right) Quantification of cell migrated and invaded cells in spheroid and adherent cells determined higher ability of mammospheres in migration and invasion. Data indicated the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01 compared with parental cells.
Figure 4
Figure 4
Mean values of fold change for stemness‐, differentiation‐, and metastasis‐related genes in MCF‐7, MDA‐MB231, and MDA‐MB468. Expression of stemness genes (left part), differentiation genes (middle part), and metastasis genes (right part) in mammosphere relative to adherent cells (control) determined by qRTPCR. β‐ACTIN mRNA was used as the housekeeping gene. Levels of gene expression for adherent culture (the black line has started from one). Each cell line represents n ≥ 3. Statistically significant difference was determined by paired t test with GraphPad Prism 6 software. Results were mean ± SEM. **P < 0.01; ***P < 0.001
Figure 5
Figure 5
Gene Ontology (GO) and KEGG pathway analysis using Enrichr. The Stemness and EMT regulated genes from the differentially expressed miRNAs between mammospheres and adherent culture. Only the top ten enriched GO terms are represented in the respective pie charts. The enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the 35 selected target genes of 8 microRNAs
Figure 6
Figure 6
Expression (mean ± SD) of miR‐204, miR‐21, miR‐30c, miR‐34a, miR‐200c, miR‐10b, miR‐373, and miR‐520c between mammospheres derive of MCF‐7, MDA‐MB231, and MDA‐MB468 vs adherent culture (as control) determined by qRT‐PCR. The expression of each miRNA was normalized to the levels of u6. Each cell lines represent n ≥ 3. **P < 0.01; ***P < 0.001
Figure 7
Figure 7
Diagnostic plots created with PROGmiR for published signatures in breast invasive carcinoma (BRCA). (A) Kaplan‐Meier survival curve analysis for overall survival of breast invasive carcinoma patients using the five‐miRNA regulate stemness genes. (B) Prognostic evaluation of the three miRNAs that act as metastatic regulator genes were associated with overall survival in breast cancer patients. (C) The miRNAs that regulate both of stemness and metastasis genes. (D) The stemness‐ and metastasis‐related genes in the patients were stratified into a high‐risk group and a low‐risk group according to median of each miRNA
Figure 8
Figure 8
Schematic illustration of selected miRNAs on individual components of signalling pathways related to stemness and metastasis
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