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.
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.
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.
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…
The authors have nothing to disclose.
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.
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/ |
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