Mapping a Circular RNA–microRNA–mRNA-Signaling Regulatory Axis that Modulates Stemness Properties of Cancer Stem Cell Populations in Colorectal Cancer Spheroid Cells
Abstract
:1. Introduction
2. Results
2.1. Generation of CRC Spheroid Cells That Showed Enhanced Stemness-Related Properties
2.2. Differentiation Abilities and Chemoresistance of the CRC Spheroid CSC Cells
2.3. Genome-Wide Expression Profiling of circRNAs in the CSC-Like Cells in the CRC Spheroids
2.4. The Top Eight Differentially Expressed circRNAs Form A Core circRNA–miRNA–mRNA Network to Regulate CSC Stemness Properties
3. Discussion
4. Materials and Methods
4.1. Cell Lines and Cell Culture
4.2. Spheroid Cell Culture
4.3. Immunofluorescence Analysis
4.4. Colony Forming
4.5. Migration and Invasion Assay
4.6. Spontaneous and Lineage-Directed Differentiation Assay
4.7. Chemosensitivity Assay
4.8. Stem-Loop RT-qPCR
4.9. Western Blot Analysis
4.10. RNA Extraction and circRNA Profiling
4.11. Bioinformatic Analysis
4.12. Selection of Top Four Up- and Down-Regulated circRNAs
4.13. Quantitative Real-Time RT-PCR
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
3D | three dimensional |
5-FU | 5-fluorouracil |
ALDH1 | aldehyde dehydrogenase 1 |
ATCC | American Type Culture Collection |
bFGF | basic fibroblast growth factor |
BM-MSC | bone marrow-derived mesenchymal stem cells |
BMP4 | bone morphogenetic protein 4 |
BSA | bovine serum albumin |
circRNA | circular RNA |
CRC | colorectal cancer |
CrCSC | colorectal cancer stem cells |
CSC | cancer stem cells |
DMEM | Dulbecco’s modified Eagle’s Medium |
EGF | epidermal growth factor |
FBS | fetal bovine serum |
GAPDH | glyceraldehyde 3-phosphate dehydrogenase |
GO | gene ontology |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
L-OHP | oxaliplatin |
lincRNA | long intervening non-coding RNA |
miRNA | microRNA |
polyHEMA | polyhydroxyethylmethacrylate. |
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CircRNA ID | HCT-15 | Fold Change (log2) | p-Value | WiDr | Fold Change (log2) | p-Value | ||
---|---|---|---|---|---|---|---|---|
Parental | Spheroid | Parental | Spheroid | |||||
Upregulated circRNA | ||||||||
hsa_circ_0002970 | 0 | 70.041 | 6.96 | 7.34 × 10−6 | 31.892 | 84.479 | 1.39 | 0.0440 |
hsa_circ_0008599 | 43.960 | 139.655 | 1.63 | 0.0017 | 15.380 | 67.332 | 2.10 | 0.0030 |
hsa_circ_0066631 | 37.906 | 117.341 | 1.62 | 0.000066 | 21.074 | 83.910 | 2.00 | 0.0011 |
hsa_circ_0082096 | 5.878 | 38.815 | 2.60 | 0.0030 | 9.682 | 47.797 | 2.26 | 0.0130 |
Downregulated circRNA | ||||||||
hsa_circ_0000400 | 445.113 | 121.834 | −1.83 | 0.0085 | 488.659 | 149.084 | −1.70 | 2.21 × 10−8 |
hsa_circ_0005174 | 19.487 | 8.0366 | −1.20 | 0.6087 | 11.391 | 1.455 | −2.54 | 0.6759 |
hsa_circ_0005507 | 107.582 | 29.003 | −1.87 | 7.57 × 10−6 | 17.089 | 3.568 | −2.08 | 0.5388 |
hsa_circ_0040238 | 138.420 | 65.182 | −1.08 | 0.0051 | 37.588 | 11.649 | −1.59 | 0.7378 |
CircRNA | miRNA | mRNA | |||||
---|---|---|---|---|---|---|---|
ACVR1C | FZD3 | IL6ST | SKIL | SMAD2 | WNT5A | ||
hsa_circ_0066631 | miR-140-3p | − | − | − | + | − | + |
miR-224 | − | + | − | − | + | + | |
miR-548c-3p | + | + | − | + | + | + | |
has-miR-579 | − | + | + | + | − | − | |
hsa_circ_0082096 | miR-140-3p | − | − | − | + | − | + |
miR-382 | + | + | + | − | − | + | |
miR-579 | − | + | + | + | − | − |
CircRNA miRNA | CircRNA—miRNA Pairing | Minimal Free Energy (kcal/mol) | Binding Pairing |
---|---|---|---|
hsa_circ_0082096 miR-579 | 5′-AGGGAACAAAUCAUUCAAAUGAA-3 ′3′-UUAGCGCCAAAUAUGGUUUACUU-5′ | −18.9 | |
hsa_circ_0082096 miR-382 | 5′-AACUAAACAACUUA-3′ 3′-GCUUAGGUGGUGCUUGUUGAAG-5′ | −22.6 | |
hsa_circ_0082096 miR-140-3p | 5′-UGAUACGACAUAUAACUGUGGUU-3′ 3′-GGCACCAAGAUGGGACACCAU-5′ | −21.6 | |
hsa_circ_0066631 miR-140-3p | 5′-UGAAAUAGGCAAAUACUGUGGUC-3′ 3′-GGCACCAAGAUGGGACACCAU-5′ | −18.8 | |
hsa_circ_0066631 miR-224 | 5′-UUCGCAUCAAAUUUGGUGACUUU-3′ 3′-UUGCCUUGGUGAUCACUGAAC-5′ | −19.0 | |
hsa_circ_0066631 miR-548c-3p | 5′-GGCAAAUACUGUGGUCUGGGGUUG-3′ 3′-CGUUUUCAUUAACUCUAAA-5′ | −18.0 | |
hsa_circ_0066631 miR-579 | 5′-UGUGGUCUGGGGUUGCAAAUGAA-3′ 3′-UUAGCGCCAAAUAUGGUUUACUU-5′ | −24.1 |
mRNA (Alias) | Signaling Pathway | CSC-Related Properties | References |
---|---|---|---|
ACVR1C (ALK7) | TGF-β/Activin/Nodal/Smad2 | Proliferation and invasion | [77,84] |
FZD3v (Fz-3) | Wnt/β-catenin | Proliferation | [79] |
IL6ST (Gp-130) | Stat3 signaling | Tumorigenesis initiation | [85] |
Stat3/Wnt/β-catenin | Tumorigenesis initiation | [86] | |
Jak/Stat3 and PI3K/AKT/mTOR | Chemoresistance | [87] | |
Stat3 | Naïve pluripotency | [69] | |
Jak/Stat3 | Self-renewal | [75] | |
SKIL (SnoN) | TGF-β | Chemoresistance | [88] |
TGF-β | Tumorigenesis | [89] | |
PI3K/AKT | Cancer cell proliferation | [90] | |
Activin/Nodal | Pluripotency maintenance | [70] | |
TGF-β | Epithelial-mesenchymal transition | [91] | |
SMAD2 | TGF-β | Pluripotency maintenance | [71,72] |
TGF-β/Akt | Stemness maintenance | [92,93] | |
TGF-β/Smad/Snail | Epithelial-mesenchymal transition | [94] | |
Nodal/Activin | Pluripotency maintenance | [73] | |
Wnt5A | TGF-β | Migration and invasion | [95] |
TGF-β | Migration and self-renewal | [96] | |
TGF-β/Smad | Tumorigenesis and migration | [97] |
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Rengganaten, V.; Huang, C.-J.; Tsai, P.-H.; Wang, M.-L.; Yang, Y.-P.; Lan, Y.-T.; Fang, W.-L.; Soo, S.; Ong, H.T.; Cheong, S.K.; et al. Mapping a Circular RNA–microRNA–mRNA-Signaling Regulatory Axis that Modulates Stemness Properties of Cancer Stem Cell Populations in Colorectal Cancer Spheroid Cells. Int. J. Mol. Sci. 2020, 21, 7864. https://doi.org/10.3390/ijms21217864
Rengganaten V, Huang C-J, Tsai P-H, Wang M-L, Yang Y-P, Lan Y-T, Fang W-L, Soo S, Ong HT, Cheong SK, et al. Mapping a Circular RNA–microRNA–mRNA-Signaling Regulatory Axis that Modulates Stemness Properties of Cancer Stem Cell Populations in Colorectal Cancer Spheroid Cells. International Journal of Molecular Sciences. 2020; 21(21):7864. https://doi.org/10.3390/ijms21217864
Chicago/Turabian StyleRengganaten, Vimalan, Chiu-Jung Huang, Ping-Hsing Tsai, Mong-Lien Wang, Yi-Ping Yang, Yuan-Tzu Lan, Wen-Liang Fang, Shelly Soo, Hooi Tin Ong, Soon Keng Cheong, and et al. 2020. "Mapping a Circular RNA–microRNA–mRNA-Signaling Regulatory Axis that Modulates Stemness Properties of Cancer Stem Cell Populations in Colorectal Cancer Spheroid Cells" International Journal of Molecular Sciences 21, no. 21: 7864. https://doi.org/10.3390/ijms21217864