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 proteins^2,3 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 processes^8,9,10.

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 pairing^11,12,13 and can modify the function of the RNA through mechanisms that depend on their chemical structure and modifications^13,14. 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 effect^13,16. Among backbone modifications, phosphoramidate morpholino (PMO), thiophosphoramidate, and morpholino bonds are widely used for purposes such as interference in splicing^17,18 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 recruitment^12,20, 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 H2^20,28, 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 cancer^34,35,36.

Protocol

NOTE: This protocol is intended to be performed only by laboratory personnel with experience in laboratory safety procedures. It is essential to properly read the safety data sheets from all the reagents and materials used in this protocol prior to starting to handle hazardous materials and equipment. It is essential to read, understand and fulfill all the safety requirements indicated in your institution's laboratory safety manual along the whole protocol. Disposal of all biological and chemical waste must be performed according to the institution´s waste management and disposal manual. If not handled with care and according to safe laboratory practices, materials and equipment used in this protocol can cause serious injury. Always follow institution´s safety laboratory manual and safety procedures.

1. Design of ASOs

1. Manually design ASOs as described below.
   1. Obtain the complete sequence of the target transcript and analyze it in the RNAfold WebServer, University of Vienna³⁷ (Table of Materials), using the default parameters on the website to obtain the minimum free energy (MFE) secondary structure.
   2. When the results are ready, go to MFE plain structure drawing and click View In Forna to open the MFE secondary structure on the website, and the software will show the predicted secondary structure of the transcript analyzed.
   3. From the predicted MFE secondary structure, select a region of 20 bp length in the RNA, avoiding G-strings (sequences of 3 guanines in a row) when designing the oligonucleotides. For selection of the best target region for the ASO, use the regions predicted to have a lower probability of internal base pairing (Figure 1).
   4. From the selected region of 20 bp, create the reverse complement sequence. Manually create the reverse complement or use the reverse complement online tool (Table of Materials). The reverse complement sequence will be the ASO that must be synthesized for the experiment.
   5. Analyze the ASO sequence using Nucleotide Blast NCBI (blastn) and UCSC Genome Browser on Human (GRCh38/hg38). Ensure ASOs do not show homology to other transcripts or other genomic regions in the human genome.
      NOTE: ASO synthesis and purification were performed by a commercial company (Table of Materials).
2. Ensure the oligonucleotides synthesized have the following characteristics.
   1. Ensure PS bonds along the whole sequence of the oligonucleotide³⁸ (Figure 2A). Ensure ASOs are chimeric, with 5 nucleotides flanking the 5' and 3' ends with sugar rings modified with 2'-O-methyl (Figure 2B).
   2. Ensure the 10 nucleotides in the middle have an unmodified sugar ring to support RNase H binding (Figure 2C).
   3. Ask the company to perform purification using standard desalting and reverse-phase (RP) HPLC³⁹.
   4. Order ASO delivery in lyophilized format.
3. Dissolve the lyophilized ASOs in Dulbecco's phosphate-buffered saline (DPBS) without calcium and magnesium to a final concentration of 200 µM. Store at -20 °C.
4. Prepare the working solution of ASO by diluting the concentrated stock to a concentration of 20 µM. Prepare the dilution using DPBS. Store at -20 °C.
   NOTE: For this study, two ASOs targeting lncRNA-RFC4 were designed. For optimization of the ASO concentration for transfection and efficiency, the protocol by Zong et al.⁴⁰ was followed. Two ASOs were designed and experimentally optimized according to Zong's protocol: ASO-lncRFC4 and ASO-lncRFC4-2. After optimization, one effective ASO targeting lncRNA-RFC4, ASO-lncRFC4, was selected for the prolonged knockdown protocol (Supplementary Figure 1). As a positive control, a previously confirmed efficient ASO targeting lncRNA MALAT1⁸ was used: ASO-MALAT1. For the negative control, an ASO targeting the Escherichia coli lacZ gene, ASO-lacZ (NCBI accession number: FN297865), was designed.

2. Preparation of cells

NOTE: Work inside the cell culture hood every time cell lines, solutions, material, or any product to be used during cell culture manipulation is handled. When manipulating liquids for cell culture, always use sterilized serological pipettes or micropipettes with sterilized tips. Always fulfill and follow all the safety requirements indicated in the institution's laboratory safety manual during the whole protocol.

1. Prepare the complete cell culture media for each cell line as described below.
   1. For HCT116, prepare McCoy's 5A medium with 10% of fetal bovine serum (FBS).
   2. For PrEC, prepare prostate epithelial cell basal medium with the prostate epithelial cell growth kit.
2. Use three 100 mm culture plates (55 cm² surface area) for KD experiments, one for each oligonucleotide to be used: ASO targeting the lncRNA, ASO to be used as a positive control (ASO-MALAT1) and ASO to be used as a negative control (ASO-lacZ).
3. Use two 35 mm culture plates (9 cm² in surface area) to be used as checkpoints for KD between cell passages, one of which will be transfected with complete transfection media for ASO-lncRFC4 and the other for ASO-lacZ.
   NOTE: Transfection was planned according to the lipid-based transfection reagent (see Table of Materials) manufacturer´s procedures and protocols previously reported^41,42.
4. Seed cells to a density of 1 x 10⁴ cells/cm² in cell culture dishes. Incubate at 37 °C in the incubator using 5% CO₂ in air until cells reach 50% confluency (when 50% of the surface is covered by the cell monolayer). With a confluence of 50%, cells are ready to be transfected for this protocol.
   ​NOTE: For transfection of ASO-lncRFC4 and ASO-lacZ, one 100 mm dish and one 35 mm dish were seeded. For transfection of ASO-MALAT1, only a 100 mm dish was seeded (Figure 3A).

3. Transfection

1. Warm up reduced serum medium to 37 °C prior to the start of the procedure.
2. For transfection with ASO-lacZ and ASO-lncRFC4, follow the steps described below.
   NOTE: Transfection with ASO-lacZ and ASO-lncRFC4 is planned for cells in a surface area of 55 cm²; when different areas are used, volumes can be adjusted accordingly. This procedure must be performed for each ASO used under the same conditions.
   1. Prepare tube 1 inside the cell culture hood by dissolving 43.75 µL of ASO (20 µM) in 1.4 mL of warm reduced serum medium in a 15 mL conical centrifuge tube.
   2. Prepare tube 2 inside the cell culture hood by mixing 18.66 µL of lipid-based transfection reagent with 1.4 mL of warm reduced serum medium in a 1.5 mL RNase-free microfuge tube.
   3. Incubate tubes 1 and 2 inside the cell culture hood at room temperature for 10 min.
   4. Add the solution contained in tube 2 (transfection reagent + reduced serum) directly to tube 1 (ASO + reduced serum) slowly, drop by drop, to avoid spreading the reagents over the surface of the tube.
   5. Incubate the mixture contained in tube 1 for 20 min inside the culture hood at room temperature.
   6. Add warm reduced serum medium to tube 1 to a final volume of 8.75 mL.
      NOTE: The final transfection media contained ASO at a concentration of 100 nM.
   7. Remove the culture media from the cell culture dish containing the cells to be transfected and add 7.5 mL of the transfection media to the 100 mm culture plate and 1 mL to the 35 mm culture plate directly in the cell monolayer, drop by drop. Make sure the media is evenly distributed along the whole surface by gentle shaking of the dish.
   8. Incubate in an incubator at 37 °C with 5% CO₂ in air for 6 h.
   9. After 6 h of incubation in the transfection media, add 7.5 mL of complete media to the cells in the 100 mm culture plate and 1 mL of complete media to the cells in the 35 mm culture plate. Incubate at 37 °C and 5% CO₂.
3. For transfection with ASO-MALAT1, perform the experiments as described below.
   NOTE: Transfection with ASO-MALAT1 is planned for cells in a surface area of 55 cm².
   1. Prepare tube 1 inside the cell culture hood by dissolving 37.5 µL of ASO (20 µM) in 1.2 mL of warm reduced serum medium in a 15 mL conical centrifuge tube.
   2. Prepare tube 2 by mixing 16 µL of lipid-based transfection reagent with 1.2 mL of warm reduced serum medium.
   3. Incubate tubes 1 and 2 at room temperature for 10 min inside the cell culture hood.
   4. Add the solution contained in tube 2 (lipid-based transfection reagent + reduced serum) directly to tube 1 (ASO + reduced serum) slowly, drop by drop, to avoid spreading the reagents over the surface of the tube.
   5. Incubate the mixture contained in tube 1 for 20 min inside the culture hood at room temperature.
   6. Add warm reduced serum medium to tube 1 to a final volume of 7.5 mL. The final transfection media contained ASO at a concentration of 100 nM.
   7. Remove the culture media from the cell culture dish containing the cells to be transfected and add 7.5 mL of the transfection media directly to the cell monolayer dropwise to ensure that the medium is evenly distributed along the whole surface by gentle shaking of the dish.
   8. Incubate at 37 °C in an incubator using 5% CO₂ in air for 6 h.
   9. After 6 h of incubation with the transfection media, add 7.5 mL of complete media to the cells and incubate in an incubator at 37 °C and 5% CO₂.
4. Assess cells microscopically every 12-24 h until cells are ready for the next transfection round.
   NOTE: Next, transfection must be performed when cells achieve confluence of 70%. If needed, exchange media replacing with complete culture medium.
5. When cells are 70% confluent in the cell culture dish, repeat the procedure (steps 3.1 to 3.4) and perform the next round of transfection.
   ​NOTE: After the second transfection, cells should be assessed microscopically every 12-24 h. Cell harvest and passaging must be performed when cells achieve confluence of 80%-85%. If needed, exchange media replacing with complete culture medium.

4. Cell harvest and passaging

NOTE: Cell harvest and passaging are performed after every 2 rounds of transfection and after the 2^nd, 4^th, and 6^th rounds of transfection. The mean time for harvest in HCT116 cells was 6 days after the 1^st, 3^rd, and 5^th transfections, and for PrEC cells, the mean time was 7 days after the 1^st, 3^rd, and 5^th transfections. The timepoint for transfection differs for both cell lines used in this protocol. For HCT116, the 2^nd, 3^rd, 4^th, 5^th, and 6^th transfections are performed 3, 6, 9, 12 and 15 days after the first transfection, respectively. For PrEC, the 2^nd, 3^rd, 4^th, 5^th, and 6^th transfections are performed 3, 7, 10, 14 and 18 days after the first transfection, respectively. Refer to the timeline in Figure 3 to check timepoints for transfection, passaging, and harvesting.

1. Perform cell harvesting and passaging for the KD experiments in 100 mm culture plates as described below.
   1. Proceed to harvest cells and passage when cells achieve confluence of 80%-85%.
   2. Warm up the complete medium and wash solutions to 37 °C. Warm up the dissociation reagent to room temperature. Warm up the neutralizing solution for the dissociation reagent to room temperature.
      NOTE: Washing solutions, dissociation reagents and neutralizing solutions differ for the two cell lines used in this protocol. HCT116 cells are washed with PBS (phosphate buffered saline) and dissociated with trypsin-EDTA solution. Neutralization of the dissociation reagent is performed with complete culture media for HCT116 cells. For PrEC, HEPES-buffered saline solution is used as the washing solution. Trypsin-EDTA is used as the dissociation reagent. For neutralization of the dissociation reagent, use trypsin neutralizing solution.
   3. Inside the culture hood, remove culture media from the cell culture dish and wash gently with 3 mL of washing solution, followed by discarding the washing solution. Repeat this step 2x.
   4. Add 2 mL of dissociation reagent according to the cell line to the cell monolayer and incubate at 37 °C for 3-5 min. Assess cells every 2 min until dissociation is complete.
   5. Add 2 mL of neutralizing solution according to the cell line and transfer the whole volume of the cell suspension from the cell culture dish to a 15 mL conical centrifuge tube.
   6. For RNA extraction, take 500 µL of the cell suspension and transfer it to a 1.5 mL centrifuge tube. Place the tube on ice and proceed to step 5.1 for RNA extraction and RT-qPCR.
   7. Centrifuge the rest of the cell suspension at 120 x g for 5 min at room temperature and discard the supernatant.
   8. Resuspend the cell pellet in 2 mL of complete culture media and proceed to cell counting according to standard procedures.
   9. For karyotyping, seed cells to a density of 1 x 10⁴ cells/cm² in a cell culture dish of 35 mm in diameter (surface area of 9 cm²), incubate in an incubator at 37 °C at 5% CO₂ in air for 24 h and proceed to karyotyping according to standard procedures.
   10. To continue the KD experiments in the next cellular passage, seed cells to a density of 1 x 10⁴ cells/cm² in a new cell culture dish 100 mm in diameter. This dish will be passage number 2 for the experiment. Incubate at 37 °C in incubator using 5% CO₂ in air until cells achieve confluence of 50%.
       NOTE: If desired, the remaining cells can be used for protein extraction and/or freezing according to standard procedures.
   11. Repeat steps 3.1 to 4.1.10 for transfection and harvesting from passage 2 and from passage 3.
2. Follow the next procedure for cell harvest for the checkpoints of the KD experiments in 35 mm culture dishes.
   NOTE: The harvest for the first checkpoint in the KD experiment is performed 2 days after the 2^nd and 4^th transfection procedures.
   1. Warm up the complete media and washing solutions to 37 °C. Warm up the dissociation reagent to room temperature. Warm up the neutralizing solution for the dissociation reagent to room temperature.
   2. Inside the culture hood, remove culture media from the cell culture dish and wash gently with 0.5 mL of washing solution and then discard the washing solution. Repeat this step 2x.
   3. After discarding the washing solutions, add 0.5 mL of dissociation reagent according to the cell line and incubate at 37 °C for 3-5 min. Assess cells every 2 min until the dissociation is complete.
   4. Add 0.5 mL of neutralizing solution according to the cell line and transfer the whole volume of the cell suspension from the cell culture dish to a 1.5 mL microcentrifuge tube and place it on ice.
   5. Proceed to step 5.1 for RNA extraction and qPCR.

5. RNA extraction and RT-PCR

1. Centrifuge the cell suspension at 200 x g for 2 min at 4 °C and discard the supernatant. Wash the cell pellet with 600 µL of cold PBS (4 °C).
2. Centrifuge the cell suspension at 200 x g for 2 min at 4 °C and discard the supernatant.
   Use the cell pellet for RNA extraction according to standard procedures.
3. Proceed to DNase treatment of the purified RNA according to the standard procedure followed in the laboratory. At the pause point, store purified RNA at -80 °C for long periods.
4. Use the RNA for standard cDNA synthesis with every RNA sample obtained. At the pause point, store cDNA at -20 °C for long periods.
5. Perform standard qPCR as per the steps described below.
   1. For qPCR, oligonucleotides amplify the following transcripts: lncRNA of interest, MALAT1 and a constitutively expressed gene as an internal control (Table 1). For this protocol, the expression RPS28 was used as an internal control.
   2. Perform qPCR reactions to amplify the internal control, the lncRNA of interest and MALAT1 using cDNA from the ASO-LacZ sample. This sample will be used to normalize the expression from the other samples.
   3. Perform qPCR reactions to amplify the internal control RPS28 and the lncRNA of interest using cDNA from the ASO-lncRNA sample.
   4. Perform qPCR reactions to amplify the internal controls RPS28 and MALAT1 using cDNA from the ASO-MALAT1 sample. Follow reaction set up and thermocycler conditions as described in Table 2.
   5. Analyze qPCR data by ΔΔCt normalization of the expression of the transcript of interest against the internal control, and then, normalize the expression of the transcript of interest (lncRNA or MALAT1) against the expression of the same transcript in the ASO-LacZ sample to obtain the relative expression level of the transcript.

Results

In the present protocol, the use of ASOs was adapted to the KD of a nuclear lncRNA for a prolonged time in the human cell lines PrEC and HCT116.

Certainly, the KD experiment was successful in the cell line PrEC for 21 days of the experiment, as observed in Figure 4. To confirm this statement, in addition to analyzing expression in the days of cell passaging (Figure 4 A-C), we analyzed the checkpoints established betwe...

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 dysregulation^43,44. 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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/