Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Western Blotting
2.3. RNA Isolation, Small RNA Library Preparation, and Deep Sequencing
2.4. Quantitative Real Time PCR
2.5. Prediction of Novel MiRNAs
2.6. miRNA Target Prediction and Pathway Analysis in IPA
2.7. Analysis of Publicly Available MiRNA Datasets
2.8. Cloning of Novel MiRNA
2.9. Cell Proliferation Assay
2.10. Intracellular Ki-67 Staining
2.11. Colony Formation Assay
2.12. Monolayer Wound Healing Assay
2.13. Availability of Data and Materials
3. Results
3.1. Small RNA Sequencing Identifies MiRNAs Deregulated in GOF Mutant p53 Cancer Cells
3.2. Validation of GOF Mutant p53-Regulated MiRNAs in Lung Adenocarcinoma (LUAD) Patients
3.3. Mutant p53-Regulated MiR-194 and MiR-378a Predict EMT Phenotype in Cancer Cells
3.4. Discovery of a Novel MiRNA
3.5. Novel miR-X Promotes Oncogenic Properties in Lung Adenocarcinoma Cells
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Samples | H1299/EV Replicate 1 | H1299/EV Replicate 2 | H1299/R273H Replicate 1 | H1299/R273H Replicate 2 | |
---|---|---|---|---|---|
Total Raw Reads | 1,442,378 | 751,538 | 1,292,998 | 874,753 | |
Reads after pre-processing | 753,866 | 340,244 | 566,961 | 500,523 | |
Mapping to References (miRBase, rRNA, tRNA & Adaptor) | Total Reads | 582,481 | 261,368 | 462,283 | 420,018 |
Percentage | 77.27 | 76.82 | 81.54 | 83.92 | |
Reads mapped to miRNA | Total Reads | 540,145 | 251,022 | 453,934 | 408,055 |
Percentage | 71.65 | 73.78 | 80.06 | 81.53 | |
Reads mapped to tRNA | Total Reads | 40,071 | 9763 | 7932 | 11,191 |
Percentage | 5.32 | 2.87 | 1.40 | 2.24 | |
Reads mapped to rRNA | Total Reads | 2235 | 575 | 406 | 767 |
Percentage | 0.30 | 0.17 | 0.07 | 0.15 | |
Reads mapped to adaptor | Total Reads | 30 | 8 | 11 | 5 |
Percentage | 0.0040 | 0.0024 | 0.0019 | 0.0010 | |
Total expressed miRNA (1 or more reads) | 692 | 512 | 507 | 518 | |
miRNA (3 or more reads) | 363 | 245 | 299 | 306 |
H1299/EV vs H1299/mutant p53R273H | ||
---|---|---|
Molecular and Cellular Functions | ||
Name | p-Value | # Molecules |
Cell Cycle | 7.36E-06-4.52E-02 | 8 |
Cellular Development | 7.50E-05-4.82E-02 | 13 |
Cellular Growth and Proliferation | 7.50E-05-4.82E-02 | 12 |
Cellular Movement | 2.72E-04-3.14E-02 | 10 |
DNA Replication, Recombination and Repair | 3.67E-04-4.82E-02 | 5 |
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Datta, A.; Das, P.; Dey, S.; Ghuwalewala, S.; Ghatak, D.; Alam, S.K.; Chatterjee, R.; Roychoudhury, S. Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53. Genes 2019, 10, 852. https://doi.org/10.3390/genes10110852
Datta A, Das P, Dey S, Ghuwalewala S, Ghatak D, Alam SK, Chatterjee R, Roychoudhury S. Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53. Genes. 2019; 10(11):852. https://doi.org/10.3390/genes10110852
Chicago/Turabian StyleDatta, Arindam, Pijush Das, Sanjib Dey, Sangeeta Ghuwalewala, Dishari Ghatak, Sk. Kayum Alam, Raghunath Chatterjee, and Susanta Roychoudhury. 2019. "Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53" Genes 10, no. 11: 852. https://doi.org/10.3390/genes10110852
APA StyleDatta, A., Das, P., Dey, S., Ghuwalewala, S., Ghatak, D., Alam, S. K., Chatterjee, R., & Roychoudhury, S. (2019). Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53. Genes, 10(11), 852. https://doi.org/10.3390/genes10110852