Human Endogenous Retrovirus (HERV)-K env Gene Knockout Affects Tumorigenic Characteristics of nupr1 Gene in DLD-1 Colorectal Cancer Cells
Abstract
:1. Introduction
2. Results
2.1. Knockout of HERV-K env Gene in Human Colorectal Cancer Cell Lines
2.2. HERV-K env KO Reduced Tumorigenic Characteristics Including Proliferation, Invasion, Migration, and Tumor Colonization in DLD-1 Colorectal Cancer Cells
2.3. HERV-K env KO Reduced Tumor Growth in the In Vivo Xenograft Models
2.4. Next-Generation Sequencing (NGS) for Analysis of Gene Expression Profile
2.5. The Effect of NUPR1 on DLD-1 Colorectal Cancer Cells
2.6. The Effect of HERV-K env KO on Reactive Oxygen Species (ROS) Generation and Protein Expression Levels Involved in Apoptosis, Autophagy, and ER Stress
2.7. Confirmation of the Effect of HERV-K env KO on HCT116 Colorectal Cancer
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Transfection
4.2. Generation of Knockout Cell Line with CRISPR-Cas9
4.3. Plasmid Construct for Over-Expression
4.4. RT-PCR and Genomic PCR
4.5. Western Blot Analysis
4.6. Invasion and Migration Assays
4.7. Soft-Agar Colonogenic Assay
4.8. Cell Viability Assay
4.9. Immunofluorescence (IF) and Immunohistochemistry (IHC)
4.10. Tumor Xenograft Assays
4.11. RNA-Seq Data Analysis
4.12. Flow Cytometry Analysis for Measurement Generation of ROS
4.13. si RNA of NUPR1
4.14. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Conflicts of Interest
Abbreviations
HERV | human endogenous retrovirus |
HERV-K | HML-2 |
Env | envelope |
ROS | reactive oxygen species |
NUPR1 | nuclear protein-1 |
KO | knockout |
KD | knockdown |
NGS | next generation sequencing |
FC | fold change |
CRC | colorectal cancer |
siRNA | Small interfering RNA |
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HERV | Chromosomal Position (hg19) | Reverse Sequence 5′–3′ | Product Size (bp) |
---|---|---|---|
K101 | chr22:18,926,187–18,935,361 | TTCTTTCAAAATAATCCATGACTGG | 1480 |
K102 | chr1:155,596,457–155,605,636 | CATCTGAAAGGAGAACATAGGAGTG | 1444 |
K107 | chr5:156,084,717–156,093,896 | ATGCCTATGATCCCAGCACTTT | 1578 |
K109 | chr6:78,426,662–78,436,083 | GATGTCAAGCAAGGTAGAAAATGAT | 1455 |
K115 | chr8:7,355,397–7,364,859 | TCATTTAAAATTGTCTTCTCCATCC | 1594 |
K117 | chr3:185,280,336–185,289,515 | TTCCATGCCTTAGTTTAACAGGTAG | 1196 |
K119 | chr12:58,721,197–58,722,612 | TGACCCCTGTCACTCTAGTAA AACT | 1416 |
Case | Sample Name | Raw Data | Ensemble 72 (23,362 Coding Genes) Homo sapiens | ||
---|---|---|---|---|---|
Expressed Genes (FPKM > 0) | Unexpressed Genes | ||||
1 | Mock | CON | 69,280,600 | 17,135 | 6227 |
2 | HERV-K Env+ | POE (Positive − overexpressed) | 51,232,816 | 16,523 | 6839 |
3 | HERV-K Env- | NKO (Negative − knockout) | 65,934,080 | 17,216 | 6146 |
Target Gene | Fold Change (/CON) | Classification | Raw Data (FPKM) | |
---|---|---|---|---|
nupr1 | K env KO | 0.174 | Down –regulation | 285 |
K env Overexpression | 1.793 | Up-regulation | 2269 |
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Ko, E.-J.; Ock, M.-S.; Choi, Y.-H.; Iovanna, J.L.; Mun, S.; Han, K.; Kim, H.-S.; Cha, H.-J. Human Endogenous Retrovirus (HERV)-K env Gene Knockout Affects Tumorigenic Characteristics of nupr1 Gene in DLD-1 Colorectal Cancer Cells. Int. J. Mol. Sci. 2021, 22, 3941. https://doi.org/10.3390/ijms22083941
Ko E-J, Ock M-S, Choi Y-H, Iovanna JL, Mun S, Han K, Kim H-S, Cha H-J. Human Endogenous Retrovirus (HERV)-K env Gene Knockout Affects Tumorigenic Characteristics of nupr1 Gene in DLD-1 Colorectal Cancer Cells. International Journal of Molecular Sciences. 2021; 22(8):3941. https://doi.org/10.3390/ijms22083941
Chicago/Turabian StyleKo, Eun-Ji, Mee-Sun Ock, Yung-Hyun Choi, Juan L. Iovanna, Seyoung Mun, Kyudong Han, Heui-Soo Kim, and Hee-Jae Cha. 2021. "Human Endogenous Retrovirus (HERV)-K env Gene Knockout Affects Tumorigenic Characteristics of nupr1 Gene in DLD-1 Colorectal Cancer Cells" International Journal of Molecular Sciences 22, no. 8: 3941. https://doi.org/10.3390/ijms22083941