Circ_RNF13 Regulates the Stemness and Chemosensitivity of Colorectal Cancer by Transcriptional Regulation of DDX27 Mediated by TRIM24 Stabilization
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Collection of Clinical Specimens
2.2. Cell Culture, Treatment, and Transfection
2.3. qRT-PCR
2.4. Western Blot Analysis
2.5. Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF)
2.6. Subcellular Fractionation
2.7. Cell Counting Kit-8 (CCK-8) Assay
2.8. Sphere Formation Assay
2.9. Colony Formation Assay
2.10. Cell Apoptosis Assay
2.11. Flow Cytometry
2.12. RNA Pull-Down Assay
2.13. RNA Immunoprecipitation (RIP) Assay
2.14. Chromatin Immunoprecipitation (ChIP) Assay
2.15. Co-Immunoprecipitation (Co-IP)
2.16. Animal Study
2.17. Immunohistochemistry (IHC)
2.18. TUNEL Assay
2.19. Statistical Analysis
3. Results
3.1. Characterization of Circ_RNF13
3.2. Circ_RNF13 and DDX27 Are Elevated in CRC Tumor Samples and Cells
3.3. Knockdown of Circ_RNF13 Suppresses Stemness and Increases Chemosensitivity in CRC Cells
3.4. Knockdown of DDX27 Inhibits Stemness and Increases Chemosensitivity in CRC Cells
3.5. Circ_RNF13 Regulates DDX27 Expression via TRIM24-Mediated Transcriptional Regulation
3.6. Circ_RNF13 Stabilizes TRIM24 via Suppressing FBXW7-Mediated TRIM24 Degradation
3.7. Knockdown of Circ_RNF13 Impairs Stemness and Enhances Chemosensitivity of CRC In Vivo
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Primer | Sequence 5′-3′ |
---|---|
circ_RNF13 (divergent) sense | GTCCAGGATAGACATAGAGC |
circ_RNF13 (divergent) anti-sense | GTGTAGACTTGTGTGGCTGA |
circ_RNF13 (convergent) sense | GCTCTCCATAGGGATGCTCA |
circ_RNF13(convergent) anti-sense | GCAGGGAGGTCATCAAATGT |
RNF13 sense | CCTCCCTGCAAGATTTGGTT |
RNF13 anti-sense | TGTATCCTGCTCGTGCA |
DDX27 sense | AGCCCGTGGACTTGACATTG |
DDX27 anti-sense | GCATCTTCCGCTCATCTTCTC |
TRIM24 sense | AGCCACAAATGCCTAAGC |
TRIM24 anti-sense | AGGATGAGGAGGAAGAACTG |
U6 sense | CTCGCTTCGGCAGCACA |
U6 anti-sense | AACGCTTCACGAATTTGCGT |
GAPDH sense | CCAGGTGGTCTCCTCTGA |
GAPDH anti-sense | GCTGTAGCCAAATCGTTGT |
Antibody | Vendor | Catalog No. | Working Dilution |
---|---|---|---|
DDX27 | Invitrogen | PA5-61421 | WB (1:500); IHC (1:50) |
CD44 | Abcam | ab51037 | WB (1:2000); IF (1:100) |
CD133 | Abcam | ab19898 | WB (1:1000); IF (1:100) |
SOX2 | Abcam | ab97959 | WB (1:1000) |
OCT4 | Abcam | ab19857 | WB (1:1000) |
NANOG | Abcam | ab109250 | WB (1:2000) |
CD133-APC | Invitrogen | 17-1338-42 | Flow (0.125 μg)/test |
CD44-FITC | Invitrogen | 11-0441-82 | Flow (0.5 μg)/test |
TRIM24 | Abcam | ab70560 | WB (1:1000); IP/RIP (2 μg) |
FBXW7 | Abcam | ab109617 | WB (1:1000); IP (2 μg) |
β-tubulin | Abcam | ab6046 | WB (1:1000) |
GAPDH | Abcam | ab8245 | WB (1:2000) |
Variables | Cases | Expression | p Value | x2 | |
---|---|---|---|---|---|
Low (n = 25) | High (n = 25) | ||||
Gender | |||||
Male | 14 | 12 | 0.5713 | 0.3205 | |
Female | 11 | 13 | |||
Age | |||||
≤62 years | 10 | 11 | 0.7745 | 0.08210 | |
>62 years | 15 | 14 | |||
Tumor size | |||||
<5 cm | 8 | 16 | 0.0235 * | 5.128 | |
≥5 cm | 17 | 9 | |||
Distant metastasis | |||||
Negative | 19 | 9 | 0.0044 ** | 8.117 | |
Positive | 6 | 16 | |||
Differentiation | |||||
Poor | 16 | 12 | 0.2545 | 1.299 | |
Well to moderate | 9 | 13 | |||
Lymph node metastasis | |||||
Negative | 18 | 10 | 0.0227 * | 5.195 | |
Positive | 7 | 15 | |||
TNM stage | |||||
I+II | 20 | 11 | 0.0087 ** | 6.876 |
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Guo, Y.; Hu, G.; Tian, B.; Ma, M.; Long, F.; Chen, M. Circ_RNF13 Regulates the Stemness and Chemosensitivity of Colorectal Cancer by Transcriptional Regulation of DDX27 Mediated by TRIM24 Stabilization. Cancers 2022, 14, 6218. https://doi.org/10.3390/cancers14246218
Guo Y, Hu G, Tian B, Ma M, Long F, Chen M. Circ_RNF13 Regulates the Stemness and Chemosensitivity of Colorectal Cancer by Transcriptional Regulation of DDX27 Mediated by TRIM24 Stabilization. Cancers. 2022; 14(24):6218. https://doi.org/10.3390/cancers14246218
Chicago/Turabian StyleGuo, Yihang, Gui Hu, Buning Tian, Min Ma, Fei Long, and Miao Chen. 2022. "Circ_RNF13 Regulates the Stemness and Chemosensitivity of Colorectal Cancer by Transcriptional Regulation of DDX27 Mediated by TRIM24 Stabilization" Cancers 14, no. 24: 6218. https://doi.org/10.3390/cancers14246218