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 a....

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 perfor.......

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

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/