Antisense Oligonucleotides as a Tool for Prolonged Knockdown of Nuclear lncRNAs in Human Cell Lines
Summary
To analyze the function of lncRNAs in time-dependent processes such as chromosomal instability, a prolonged knockdown effect must be achieved. To that purpose, presented here is a protocol that uses modified antisense oligonucleotides to achieve effective knockdown in cell lines for 21 days.
Abstract
Long noncoding RNAs (lncRNAs) play key regulatory roles in gene expression at the transcriptional level. Experimental evidence has established that a substantial fraction of lncRNA preferentially accumulates in the nucleus. For analysis of the function of nuclear lncRNAs, it is important to achieve efficient knockdown of these transcripts inside the nucleus. In contrast to the use of RNA interference, a technology that depends on the cytoplasmic silencing machinery, an antisense oligonucleotide (ASO) technology can achieve RNA knockdown by recruiting RNase H to the RNA-DNA duplexes for nuclear RNA cleavage. Unlike the use of CRISPR-Cas tools for genome engineering, where possible alterations in the chromatin state can occur, ASOs allow the efficient knockdown of nuclear transcripts without modifying the genome. Nevertheless, one of the major obstacles to ASO-mediated knockdown is its transitory effect. For the study of long-lasting effects of lncRNA silencing, maintaining efficient knockdown for a long time is needed. In this study, a protocol was developed to achieve a knockdown effect for over 21 days. The purpose was to evaluate the cis-regulatory effects of lncRNA knockdown on the adjacent coding gene RFC4, which is related to chromosomal instability, a condition that is observed only through time and cell aging. Two different human cell lines were used: PrEC, normal primary prostate epithelial cells, and HCT116, an epithelial cell line isolated from colorectal carcinoma, achieving successful knockdown in the assayed cell lines.
Introduction
The vast majority of the human genome is transcribed, giving rise to a wide variety of transcripts, including lncRNAs, which, in number, exceed the number of annotated coding genes in the human transcriptome1. LncRNAs are transcripts longer than 200 nucleotides that do not encode proteins2,3 and have recently been examined for their important regulatory functions in the cell4. Their functions have been shown to be dependent on their subcellular localization5, such as the nucleus where a significant fraction of lncRNAs accumulate and actively participate in transcriptional regulation6 and for nuclear architecture maintenance7, among other biological processes8,9,10.
For the functional characterization of nuclear lncRNAs, methods capable of inducing knockdown (KD) in the nucleus must be used, and ASOs are a powerful tool to silence nuclear transcripts. In general, ASOs are single-stranded DNA sequences ~20 base pairs in length that bind to complementary RNA by Watson-Crick base pairing11,12,13 and can modify the function of the RNA through mechanisms that depend on their chemical structure and modifications13,14. ASO chemistry modifications can be divided into 2 major groups: backbone modifications and 2' sugar ring modifications15, both of which are intended to increase stability by conferring high resistance to nucleases but also to enhance the intended biological effect13,16. Among backbone modifications, phosphoramidate morpholino (PMO), thiophosphoramidate, and morpholino bonds are widely used for purposes such as interference in splicing17,18 by serving as steric blocking agents19 but not to induce degradation of the transcript. Another backbone modification is the phosphorothioate (PS) bond, one of the most commonly used modifications in ASOs. In contrast to the previously mentioned modifications, PS bonds do not interfere with RNase H recruitment12,20, thus allowing RNA knockdown. However, there is also a wide variety of 2' sugar ring modifications21; nevertheless, for the purpose of RNA knockdown, among the modifications that induce efficient silencing effects are locked nucleic acids (LNAs)22, 23 and 2'-O-methyl modification24. Even though LNAs have proven to be highly effective for knockdown compared to other modifications25, they can induce unwanted effects such as hepatotoxicity26 and apoptosis induction in vivo and in vitro27.
For the purpose of RNA knockdown, ASOs with the proper modifications mentioned before can recruit RNase H1 and H220,28, and these enzymes are recruited to DNA-RNA hybrids and cleave the target RNA, releasing the ASO13. The RNA products of this cleavage are then processed by the RNA surveillance machinery, resulting in RNA degradation29 without modifying the genomic region of interest, in contrast to other techniques such as CRISPR-Cas systems, where modifications in the chromatin state can create unwanted biological effects30. Despite the advantages of ASO technology, the temporary silencing effects due to cell division or ASO degradation over time are an obstacle to overcome when studying time-dependent processes such as chromosomal instability (CIN)31.
In particular, CIN is defined as an increased rate of changes in chromosome number and structure compared to those of normal cells32 and arises from errors in chromosome segregation during mitosis, leading to genetic alterations that originate intratumor heterogeneity33 over time. Thus, CIN cannot be evaluated only by finding an aneuploid karyotype. For the proper study and evaluation of CIN in cell culture, it is important to monitor the cells over time. For study of the effects of a lncRNA KD on CIN, a methodology that allows a prolonged KD effect is needed. For this purpose, ASOs were used in this protocol, where lncRNA-RFC4 was successfully silenced in the human cell lines HCT115 and PrEC for 18 and 21 days, respectively. This transcript is an uncharacterized lncRNA of 1.2 kb in length, and its genomic location is on chromosome 3 (q27.3). It is adjacent to the protein coding gene RFC4, a gene associated with CIN in different types of human cancer34,35,36.
Protocol
Representative Results
Discussion
As previously mentioned, lncRNAs have key regulatory functions in the cell; thus, dysregulation of these transcripts may be involved in diseases. Cancer is one such disease characterized by lncRNA dysregulation43,44. In this disease, lncRNAs are known to play important regulatory roles as oncogenes45 or tumor suppressors46. Some of them are involved in the development of hallmarks of cancer, and they can regulate, f…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Montiel-Manriquez, Rogelio is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and has received CONACyT fellowship with CONACyT CVU number: 581151.
Materials
| 15ml Centrifuge Tubes – 15ml Conical Tubes | Thermo Fisher Scientific | 339650 | |
| Corning 100 mm TC-treated Culture Dish | Corning | 430167 | Surface area:55 cm2 |
| Corning 35 mm TC-treated Culture Dish | Corning | 430165 | Surface area: 9 cm2 |
| DPBS, no calcium, no magnesium | Thermo Fisher Scientific | 14190144 | |
| Fetal Bovine Serum (FBS) | ATCC | 30-2020 | |
| HCT 116 cell line | ATCC | CCL-247 | |
| HEPES, 1M Buffer Solution | Thermo Fisher Scientific | 15630122 | |
| Integrated DNA Technologies | NA | NA | https://www.idtdna.com/ |
| Lipofectamine RNAiMAX Reagent | Thermo Fisher Scientific | 13778150 | |
| McCoy's 5A medium | ATCC | 30-2007 | |
| Normal Human Primary Prostate Epithelial Cells (HPrEC) | ATCC | PCS-440-010 | |
| Nucleotide Blast NCBI | NA | NA | https://blast.ncbi.nlm.nih.gov/Blast.cgi |
| Opti-MEM Reduced Serum Media | Thermo Fisher Scientific | 31985070 | |
| PBS (10X), pH 7.4 | Thermo Fisher Scientific | ||
| Prostate Epithelial Cell Basal Medium | ATCC | PCS-440-030 | |
| Prostate Epithelial Cell Growth Kit | ATCC | PCS-440-040 | |
| Reverse complement online tool | NA | NA | https://www.bioinformatics.org/sms/rev_comp.html |
| RNAfold WebServer | NA | NA | http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi |
| RNase-free Microfuge Tubes, 1.5 mL | Thermo Fisher Scientific | AM12400 | |
| TrypLE Express Enzyme (1X), no phenol red | Thermo Fisher Scientific | 12604013 | Trypsin-EDTA solution |
| Trypsin Neutralizing Solution | ATCC | PCS-999-004 | |
| Trypsin-EDTA for Primary Cells | ATCC | PCS-999-003 | |
| UCSC Genome Browser, Human (GRCh38/hg38) | NA | NA | https://genome.ucsc.edu/ |
References
- Fang, S., et al. NONCODEV5: a comprehensive annotation database for long non-coding RNAs. Nucleic Acids Research. 46 (Database issue), D308-D314 (2018).
- Kopp, F., Mendell, J. T. Functional Classification and Experimental Dissection of Long Noncoding RNAs. Cell. 172 (3), 393-407 (2018).
- Palazzo, A. F., Koonin, E. V. Functional Long Non-coding RNAs Evolve from Junk Transcripts. Cell. 183 (5), 1151-1161 (2020).
- Ransohoff, J. D., Wei, Y., Khavari, P. A. The functions and unique features of long intergenic non-coding RNA. Nature Reviews Molecular Cell Biology. 19 (3), 143-157 (2017).
- Chen, L. L. Linking Long Noncoding RNA Localization and Function. Trends in Biochemical Sciences. 41 (9), 761-772 (2016).
- Basu, S., Larsson, E. A Catalogue of Putative cis-Regulatory Interactions Between Long Non-coding RNAs and Proximal Coding Genes Based on Correlative Analysis Across Diverse Human Tumors. G3: Genes|Genomes|Genetics. 8 (6), 2019-2025 (2018).
- Petermann, F., et al. The Magnitude of IFN-γ Responses Is Fine-Tuned by DNA Architecture and the Non-coding Transcript of Ifng-as1. Molecular Cell. 75 (6), 1229.e5-1242.e5 (2019).
- Tripathi, V., et al. The Nuclear-Retained Noncoding RNA MALAT1 Regulates Alternative Splicing by Modulating SR Splicing Factor Phosphorylation. Molecular Cell. 39 (6), 925-938 (2010).
- Chen, L. L., Carmichael, G. G. Decoding the function of nuclear long non-coding RNAs. Current Opinion in Cell Biology. 22 (3), 357-364 (2010).
- Vance, K. W., Ponting, C. P. Transcriptional regulatory functions of nuclear long noncoding RNAs. Trends in Genetics. 30 (8), 348-355 (2014).
- Ren, H., Zhang, Z., Zhang, W., Feng, X., Xu, L. Prodrug-type antisense oligonucleotides with enhanced nuclease stability and anti-tumour effects. European Journal of Pharmaceutical Sciences. 162, 105832 (2021).
- Kole, R. RNA therapeutics: beyond RNA interference and antisense oligonucleotides. DRUG DISCOVERY. 16, (2012).
- DeVos, S. L., Miller, T. M. Antisense Oligonucleotides: Treating Neurodegeneration at the Level of RNA. Neurotherapeutics. 10 (3), 486-497 (2013).
- Le, B. T., et al. Antisense Oligonucleotides Targeting Angiogenic Factors as Potential Cancer Therapeutics. Molecular Therapy Nucleic Acids. 14, 142-157 (2019).
- Shadid, M., Badawi, M., Abulrob, A. Antisense oligonucleotides: absorption, distribution, metabolism, and excretion. Expert Opinion on Drug Metabolism & Toxicology. 17 (11), 1281-1292 (2021).
- Roberts, T. C., Langer, R., Wood, M. J. A. Advances in oligonucleotide drug delivery. Nature Reviews Drug Discovery. 19 (10), 673-694 (2020).
- Havens, M. A., Hastings, M. L. Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Research. 44 (14), 6549-6563 (2016).
- Michaels, W. E., Pena-Rasgado, C., Kotaria, R., Bridges, R. J., Hastings, M. L. Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis. Proceedings of the National Academy of Sciences. 119 (3), e2114886119 (2022).
- Lim, K. H., et al. Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression. Nature Communications. 11 (1), 3501 (2020).
- Crooke, S. T. Molecular Mechanisms of Antisense Oligonucleotides. Nucleic Acid Therapeutics. 27 (2), 70-77 (2017).
- Monia, B. P., Johnston, J. F., Sasmor, H., Cummins, L. L. Nuclease Resistance and Antisense Activity of Modified Oligonucleotides Targeted to Ha. Journal of Biological Chemistry. 271 (24), 14533-14540 (1996).
- Grünweller, A., et al. Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2′-O-methyl RNA, phosphorothioates and small interfering RNA. Nucleic Acids Research. 31 (12), 3185-3193 (2003).
- Amodio, N., et al. Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity. Leukemia. 32 (9), 1948-1957 (2018).
- Yoo, B. H., Bochkareva, E., Bochkarev, A., Mou, T. C., Gray, D. M. 2′-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro. Nucleic Acids Research. 32 (6), 2008-2016 (2004).
- Chu, H. P., et al. TERRA RNA Antagonizes ATRX and Protects Telomeres. Cell. 170 (1), 86.e16-101.e16 (2017).
- Kasuya, T., et al. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides. Scientific Reports. 6 (1), 30377 (2016).
- Swayze, E. E., et al. Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals. Nucleic Acids Research. 35 (2), 687-700 (2007).
- Hyjek, M., Figiel, M., Nowotny, M. RNases H: Structure and mechanism. DNA Repair. 84, 102672 (2019).
- Lima, W. F., De Hoyos, C. L., Liang, X., Crooke, S. T. RNA cleavage products generated by antisense oligonucleotides and siRNAs are processed by the RNA surveillance machinery. Nucleic Acids Research. 44 (7), 3351-3363 (2016).
- Fu, Y., et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology. 31 (9), 822-826 (2013).
- Drews, R. M., et al. A pan-cancer compendium of chromosomal instability. Nature. 606 (7916), 976-983 (2022).
- Benhra, N., Barrio, L., Muzzopappa, M., Milán, M. Chromosomal Instability Induces Cellular Invasion in Epithelial Tissues. Developmental Cell. 47 (2), 161.e4-174.e4 (2018).
- Gronroos, E., López-García, C. Tolerance of Chromosomal Instability in Cancer: Mechanisms and Therapeutic Opportunities. Cancer Research. 78 (23), 6529-6535 (2018).
- Liu, L., et al. An RFC4/Notch1 signaling feedback loop promotes NSCLC metastasis and stemness. Nature Communications. 12 (1), 2693 (2021).
- Shiomi, Y., Nishitani, H. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication. Genes. 8 (2), (2017).
- Carter, S. L., Eklund, A. C., Kohane, I. S., Harris, L. N., Szallasi, Z. A signature of chromosomal instability inferred from gene expression profiles predicts clinical outcome in multiple human cancers. Nature Genetics. 38 (9), 1043-1048 (2006).
- Gruber, A. R., Lorenz, R., Bernhart, S. H., Neuböck, R., Hofacker, I. L. The Vienna RNA Websuite. Nucleic Acids Research. 36 (suppl_2), W70-W74 (2008).
- Wan, W. B., et al. Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages. Nucleic Acids Research. 42 (22), 13456-13468 (2014).
- Kanavarioti, A. HPLC methods for purity evaluation of man-made single-stranded RNAs. Scientific Reports. 9 (1), 1019 (2019).
- Zong, X., et al. Knockdown of Nuclear-Retained Long Noncoding RNAs Using Modified DNA Antisense Oligonucleotides. Nuclear Bodies and Noncoding RNAs: Methods and Protocols. , 321-331 (2015).
- Zhao, M., et al. Lipofectamine RNAiMAX: An Efficient siRNA Transfection Reagent in Human Embryonic Stem Cells. Molecular Biotechnology. 40 (1), 19-26 (2008).
- . . Nuclear bodies and noncoding RNAs: methods and protocols. , (2015).
- CAWG Drivers and Functional Interpretation Group. Cancer LncRNA Census reveals evidence for deep functional conservation of long noncoding RNAs in tumorigenesis. Communications Biology. 3 (1), 56 (2020).
- Schmitt, A. M., Chang, H. Y. Long Noncoding RNAs in Cancer Pathways. Cancer Cell. 29 (4), 452-463 (2016).
- Hosono, Y., et al. Oncogenic Role of THOR, a Conserved Cancer/Testis Long Non-coding RNA. Cell. 171 (7), 1559.e20-1572.e20 (2017).
- Cho, S. W., et al. Promoter of lncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element. Cell. 173 (6), 1398.e22-1412.e22 (2018).
- Ding, W., Ren, J., Ren, H., Wang, D. Long Noncoding RNA HOTAIR Modulates MiR-206-mediated Bcl-w Signaling to Facilitate Cell Proliferation in Breast Cancer. Scientific Reports. 7 (1), (2017).
- Huo, H., et al. Long non-coding RNA NORAD upregulate SIP1 expression to promote cell proliferation and invasion in cervical cancer. Biomedicine & Pharmacotherapy. 106, 1454-1460 (2018).
- Prensner, J. R., et al. The Long Non-Coding RNA PCAT-1 Promotes Prostate Cancer Cell Proliferation through cMyc. Neoplasia. 16 (11), 900-908 (2014).
- Li, H., et al. Long noncoding RNA NORAD, a novel competing endogenous RNA, enhances the hypoxia-induced epithelial-mesenchymal transition to promote metastasis in pancreatic cancer. Molecular Cancer. 16, (2017).
- Li, Q., et al. High expression of long noncoding RNA NORAD indicates a poor prognosis and promotes clinical progression and metastasis in bladder cancer. Urologic Oncology: Seminars and Original Investigations. 36 (6), 310.e15-310.e22 (2018).
- Katayama, H., et al. Long non-coding RNA HOTAIR promotes cell migration by upregulating insulin growth factor-binding protein 2 in renal cell carcinoma. Scientific Reports. 7 (1), (2017).
- Ling, H., et al. CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer. Genome Research. 23 (9), 1446-1461 (2013).
- Lee, S., et al. Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins. Cell. 164 (1-2), 69-80 (2016).
- Hanahan, D., Weinberg, R. A. Hallmarks of Cancer: The Next Generation. Cell. 144 (5), 646-674 (2011).
- Iwamoto, N., et al. Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides. Nature Biotechnology. 35 (9), 845-851 (2017).
- Naeem, M., Majeed, S., Hoque, M. Z., Ahmad, I. Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing. Cells. 9 (7), 1608 (2020).
- Vicente, M. M., Chaves-Ferreira, M., Jorge, J. M. P., Proença, J. T., Barreto, V. M. The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing. Frontiers in Cell and Developmental Biology. 9, (2021).
- Zhang, X. H., Tee, L. Y., Wang, X. G., Huang, Q. S., Yang, S. H. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering. Molecular Therapy – Nucleic Acids. 4, e264 (2015).
- Shen, X., Corey, D. R. Chemistry, mechanism, and clinical status of antisense oligonucleotides and duplex RNAs. Nucleic Acids Research. 46 (4), 1584-1600 (2018).
- Stein, C. A., Subasinghe, C., Shinozuka, K., Cohen, J. S. Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Research. 16 (8), 3209-3221 (1988).
- Crooke, S. T., Vickers, T. A., Liang, X. Phosphorothioate modified oligonucleotide-protein interactions. Nucleic Acids Research. 48 (10), 5235-5253 (2020).
- Manoharan, M. 2′-Carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation. Biochimica et Biophysica Acta (BBA) – Gene Structure and Expression. 1489 (1), 117-130 (1999).
- Maurisse, R., et al. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnology. 10 (1), 9 (2010).
- Bassett, A. R., et al. Considerations when investigating lncRNA function in vivo. eLife. 3, e03058 (2014).
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