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. 2016 Feb 12;12(4):427-45.
doi: 10.7150/ijbs.12777. eCollection 2016.

V体育ios版 - MiRNA Transcriptome Profiling of Spheroid-Enriched Cells with Cancer Stem Cell Properties in Human Breast MCF-7 Cell Line

Lily Boo  1 , Wan Yong Ho  2 , Norlaily Mohd Ali  1 , Swee Keong Yeap  3 , Huynh Ky  4 , Kok Gan Chan  5 , Wai Fong Yin  5 , Dilan Amila Satharasinghe  6 , Woan Charn Liew  3 , Sheau Wei Tan  3 , Han Kiat Ong  1 , Soon Keng Cheong  7
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"VSports app下载" MiRNA Transcriptome Profiling of Spheroid-Enriched Cells with Cancer Stem Cell Properties in Human Breast MCF-7 Cell Line

Lily Boo et al. Int J Biol Sci. .

VSports注册入口 - Abstract

Breast cancer is the second leading cause of cancer-related mortality worldwide as most patients often suffer cancer relapse. The reason is often attributed to the presence of cancer stem cells (CSCs). Recent studies revealed that dysregulation of microRNA (miRNA) are closely linked to breast cancer recurrence and metastasis. However, no specific study has comprehensively characterised the CSC characteristic and miRNA transcriptome in spheroid-enriched breast cells. This study described the generation of spheroid MCF-7 cell in serum-free condition and the comprehensive characterisation for their CSC properties. Subsequently, miRNA expression differences between the spheroid-enriched CSC cells and their parental cells were evaluated using next generation sequencing (NGS). Our results showed that the MCF-7 spheroid cells were enriched with CSCs properties, indicated by the ability to self-renew, increased expression of CSCs markers, and increased resistance to chemotherapeutic drugs. Additionally, spheroid-enriched CSCs possessed greater cell proliferation, migration, invasion, and wound healing ability. A total of 134 significantly (p<0. 05) differentially expressed miRNAs were identified between spheroids and parental cells using miRNA-NGS. MiRNA-NGS analysis revealed 25 up-regulated and 109 down-regulated miRNAs which includes some miRNAs previously reported in the regulation of breast CSCs. A number of miRNAs (miR-4492, miR-4532, miR-381, miR-4508, miR-4448, miR-1296, and miR-365a) which have not been previously reported in breast cancer were found to show potential association with breast cancer chemoresistance and self-renewal capability VSports手机版. The gene ontology (GO) analysis showed that the predicted genes were enriched in the regulation of metabolic processes, gene expression, DNA binding, and hormone receptor binding. The corresponding pathway analyses inferred from the GO results were closely related to the function of signalling pathway, self-renewability, chemoresistance, tumorigenesis, cytoskeletal proteins, and metastasis in breast cancer. Based on these results, we proposed that certain miRNAs identified in this study could be used as new potential biomarkers for breast cancer stem cell diagnosis and targeted therapy. .

Keywords: MCF-7; breast cancer; cancer stem cells; miRNA transcriptome; next generation sequencing; spheroid culture. V体育安卓版.

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Conflict of interest statement

Competing Interests: The authors have declared no competing interest exists.

Figures

Figure 1
Figure 1
The morphology of MCF-7 monolayer and spheroid culture. (A, B) Appearance of MCF-7 monolayer under two-dimensional (2D) condition in tissue culture flask under bright field (magnification: 4X, scale bar: 100 µm) and scanning electron microscopy (magnification: 500X, scale bar: 10 µm) respectively. The cells appeared to be adherent and epithelial in shape. (C, D) Centrifugal force facilitated the cells to organize into a three-dimensional (3D) multicellular spheroidal structure in serum-free media. The structure became more rigid and compact 96 hours post-culture, where individual cells were indistinguishable (magnification: 4X, scale bar: 100 µm). (E) Appearance of the spheroidal structure under the scanning electron microscopy (magnification: 330X, scale bar: 100 µm). The cells appeared to be clumping to each other revealed that they were strongly adhered with one another to preserve the spheroid architecture. (F) Cells within the spheroid were connected to each other through cell-cell junction (arrows), responsible for maintaining the tight cell-cell contact. Present of micropores (arrowheads) on the interior of the spheroid, allowed the diffusion of nutrients and oxygen to the inner layer of the spheroid (magnification: 2000X, scale bar: 10 µm).
Figure 2
Figure 2
Secondary spheroids formation from human estrogen dependent breast cancer cell line MCF-7 at single cell and 200 cells/well dilution assays. (A) Microscopic images of the formation of the secondary spheroids demonstrated the propagation of the spheroid from a single cell from day 1 to day 14 (magnification: 20X, scale bar: 100 µm). (B) Progressive increase in size at day 3, day 7, day 10, and day 14 when the cells were seeded at higher cell seeding density at 200 cells per well (magnification: 20X, scale bar: 100 µm). Increment in size was recorded from three sets of experiments.
Figure 3
Figure 3
Spheroid-forming efficiency (SFE) of spheroids serially passaged from first to third generation. Bar graph represents the SFE mean calculated by counting the number of spheres formed in a given well and dividing by the total number of seeded cells in the well, represented as a percentage. Error bars represents standard deviation (SD) (n=3).
Figure 4
Figure 4
Immunophenotyping and ALDH1 staining analysis of spheroids compared to their parental cells. (A) The percentage of CD44+/CD24- cells of MCF-7 parental and spheroid cells was 0.63 ± 0.46% and 18.45 ± 0.51% (P = 0.0001), respectively. (B) The percentage of ALDH-positive cells was 1.81 ± 0.70% and 15.38 ± 0.50% (P = 0.0001) in MCF-7 parental and spheroid cells respectively. The proportion of subpopulation of cells expressing CD44+/CD24- and ALDH-positive cells in MCF-7 spheroid cells were significantly higher relative to the parental cells. (C) A set of representative flow cytometry dot plots of both experiments. Columns mean; bars, SD. R2 are the region of ALDH-positive cells. The data are representative of three independent experiments.
Figure 5
Figure 5
Immunofluorescent staining of CSC-related surface and internal markers of CD44, CD24, CD49f, Nanog, Sox2, and ALDH1 on spheroids and the monolayer cells. DAPI was used for nuclear counterstain. Magnifications at 4X and 10X.
Figure 6
Figure 6
Spheroid integrity following treatment with chemotherapeutic drugs at different inhibitory concentrations. Spheroids possessed higher drug resistance to conventional chemotherapeutics in vitro when compared to parental cells. The parental cells and spheroids were treated with tamoxifen, doxorubicin, and cisplatin for 96 hours and the morphology of the spheroids were shown. Each experiment was performed in triplicates, and only the represented images were shown. Magnification at 4X.
Figure 7
Figure 7
Cell proliferation, cell migration, wound healing, and cell invasion of spheroids cells compared to the parental cells. (A) Spheroids showed a higher cell proliferation rate as compared to their parental cells. Cell proliferation assay was carried out from day 3 to day 24 using alamarBlue assay. (B) Spheroids had a higher migration rate than their parental cells. Cells were seeded at 10,000 cells per insert of a 24-well plate and allowed to migrate toward serum-present medium for 24 hours. Migratory cells on the bottom of the PET membrane were then stained (right) and later dissolved and the percentage of migrated cells were quantified in a microplate reader (left). (C) Spheroids recovered the “gap” created during the scratch assay more efficiently than parental cells. The percentage of wound closure of parental and spheroids (left). Microscopic images of the wound closure at 0h, 6h, 12h, and 24h post-wound initiation (right). (D) Spheroids invaded more into the matrigel than parental cells as shown by microscopy. Quantification of cell invasion showed the mean ± standard deviations of three independent experiments. Invading cells were fixed in formaldehyde and stained with crystal violet. All data were expressed as mean ± standard deviations and *p< 0.05 compared with parental cells. Magnification at 10X.
Figure 8
Figure 8
Gene Ontology (GO) terms enriched in the up- and down- regulated predicted genes from the differentially expressed miRNAs between spheroids and parental. Only the top ten enriched GO terms are represented in the respective pie charts.
Figure 9
Figure 9
The enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the up- and down- regulated predicted genes of the differentially expressed miRNAs using DAVID. All the pathways are represented in the figure and include pathways in cancer, axon guidance, MAPK signalling pathway, endocytosis, Wnt signalling pathway, and regulation of actin cytoskeleton.
Figure 10
Figure 10
Validation of known miRNA with qRT-PCR. Comparison of the qRT-PCR and miRNA sequencing log-2 fold change for seven known miRNAs between parental and spheroids. A similar expression trends from both qRT-PCR and miRNA-NGS were observed.
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