RNA interference in mammalian cell systems

In the last decade, few areas of biology have been transformed as thoroughly as RNA molecular biology. Without any doubt, one of the most significant advances has been the discovery of small (20-30 nucleotide) noncoding RNAs that regulate genes and genomes. The effects of small RNAs on gene expression and control are generally inhibitory, and the corresponding regulatory mechanisms are therefore collectively subsumed under the heading of RNA silencing and/or RNA interference. Two primary categories of these small RNAs - short interfering RNAs (siRNAs) and microRNAs (miRNAs) - act in both somatic and germline lineages of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Recent advances have revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access. Our understanding of siRNA and miRNA-based regulation has direct implications for fundamental biology as well as disease aetiology and treatment as it is discussed in this review on 'new techniques in molecular biology'.

Improving gene silencing of siRNAs via tricyclo-DNA modification

;Small interfering RNAs (siRNAs) can be exploited for the selective silencing of disease-related genes via the RNA interference (RNAi) machinery and therefore raise hope for future therapeutic applications. Especially chemically modified siRNAs are of interest as they are expected to convert lead siRNA sequences into effective drugs. To study the potential of tricyclo-DNA (tc-DNA) in this context we systematically incorporated tc-DNA units at various positions in a siRNA duplex targeted to the EGFP gene that was expressed in HeLa cells. Silencing activity was measured by FACS, mRNA levels were determined by RT-PCR and the biostability of the modifed siRNAs was determined in human serum. We found that modifications in the 3'-overhangs in both the sense and antisense strands were compatible with the RNAi machinery leading to similar activities compared to wild type (wt) siRNA. Additional modifications at the 3'-end, the 5'- end and in the center of the sense (passenger) strand were also well tolerated and did not compromise activity. Extensive modifications of the 3'- and the 5'-end in the antisense (guide) strand, however, abolished RNAi activity. Interestingly, modifications in the center of the duplex on both strands, corresponding to the position of the cleavage site by AGO2, increased efficacy relative to wt by a factor of 4 at the lowest concentrations (2 nM) investigated. In all cases, reduction of EGFP fluorescence was accompanied with a reduction of the EGFP mRNA level. Serum stability analysis further showed that 3'-overhang modifications only moderately increased stability while more extensive substitution by tc-DNA residues significantly enhanced biostability.

RNA interference screening identifies a novel role for autocrine fibroblast growth factor signaling in neuroblastoma chemoresistance

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLX(L). Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.Oncogene advance online publication, 1 October 2012; doi:10.1038/onc.2012.416.

Transcriptional silencing of nonsense codon-containing immunoglobulin micro genes requires translation of its mRNA

Eukaryotes have evolved quality control mechanisms that prevent the expression of genes in which the protein coding potential is crippled by the presence of a premature translation-termination codon (PTC). In addition to nonsense-mediated mRNA decay (NMD), a well documented posttranscriptional consequence of the presence of a PTC in an mRNA, we recently reported the transcriptional silencing of PTC-containing immunoglobulin (Ig) mu and gamma minigenes when they are stably integrated into the genome of HeLa cells. Here we demonstrate that this transcriptional silencing of PTC-containing Ig-mu constructs requires active translation of the cognate mRNA, as it is not observed under conditions where translation of the PTC-containing mRNA is inhibited through an iron-responsive element in the 5'-untranslated region. Furthermore, RNA interference-mediated depletion of the essential NMD factor Upf1 not only abolishes NMD but also reduces the extent of nonsense-mediated transcriptional gene silencing (NMTGS). Collectively, our data indicate that NMTGS and NMD are linked, relying on the same mechanism for PTC recognition, and that the NMTGS pathway branches from the NMD pathway at a step after Upf1 function.

Silencing of microRNAs in vivo with 'antagomirs'

MicroRNAs (miRNAs) are an abundant class of non-coding RNAs that are believed to be important in many biological processes through regulation of gene expression. The precise molecular function of miRNAs in mammals is largely unknown and a better understanding will require loss-of-function studies in vivo. Here we show that a novel class of chemically engineered oligonucleotides, termed 'antagomirs', are efficient and specific silencers of endogenous miRNAs in mice. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals. The silencing of endogenous miRNAs by this novel method is specific, efficient and long-lasting. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 3' untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. Our findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease.

Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism.

An intergenic non-coding RNA targets and regulates the ribosome in H. volcanii

As translation is the final step in gene expression it is particularly important to understand the processes involved in translation regulation. It was shown in the last years that a class of RNA, the non-protein-coding RNAs (ncRNAs), is involved in regulation of gene expression via various mechanisms (e.g. gene silencing by microRNAs). Almost all of these ncRNA discovered so far target the mRNA in order to modulate protein biosynthesis, this is rather unexpected considering the crucial role of the ribosome during gene expression. However, recent data from our laboratory showed that there is a new class of ncRNAs, which target the ribosome itself [Gebetsberger et al., 2012/ Pircher et al, 2014]. These so called ribosome-associated ncRNAs (rancRNAs) have an impact on translation regulation, mainly by interfering / modulating the rate of protein biosynthesis. The main goal of this project is to identify and describe novel potential regulatory rancRNAs in H. volcanii with the focus on intergenic candidates. Northern blot analyses already revealed interactions with the ribosome and showed differential expression of rancRNAs during different growth phases or under specific stress conditions. To investigate the biological relevance of these rancRNAs, knock-outs were generated in H. volcanii which were used for phenotypic characterization studies. The rancRNA s194 showed association with the 50S ribosomal subunit in vitro and in vivo and was capable of inhibiting peptide bond formation and seems to inhibit translation in vitro. These preliminary data for the rancRNA s194 make it an interesting candidate for further functional studies to identify the molecular mechanisms by which rancRNAs can modulate protein biosynthesis. Characterization of further rancRNA candidates are also underway.

An intergenic non-coding RNA targets and regulates the ribosome in H. volcanii.

As translation is the final step in gene expression it is particularly important to understand the processes involved in translation regulation. It was shown in the last years that a class of RNA, the nonprotein-coding RNAs (ncRNAs), is involved in regulation of gene expression via various mechanisms (e.g. gene silencing by microRNAs). Almost all of these ncRNA discovered so far target the mRNA in order to modulate protein biosynthesis, this is rather unexpected considering the crucial role of the ribosome during gene expression. However, recent data from our laboratory showed that there is a new class of ncRNAs, which target the ribosome itself [Gebetsberger et al., 2012/ Pircher et al, 2014]. These so called ribosome-associated ncRNAs (rancRNAs) have an impact on translation regulation, mainly by interfering / modulating the rate of protein biosynthesis. The main goal of this project is to identify and describe novel potential regulatory rancRNAs in H. volcanii with the focus on intergenic candidates. Northern blot analyses already revealed interactions with the ribosome and showed differential expression of rancRNAs during different growth phases or under specific stress conditions. To investigate the biological relevance of these rancRNAs, knock-outs were generated in H. volcanii which were used for phenotypic characterization studies. The rancRNA s194 showed association with the 50S ribosomal subunit in vitro and in vivo and was capable of inhibiting peptide bond formation. These preliminary data for the rancRNA s194 make it an interesting candidate for further functional studies to identify the molecular mechanisms by which rancRNAs can modulate protein biosynthesis. Characterization of further rancRNA candidates are also underway.

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue

The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months.

CXC receptor-4 mRNA silencing abrogates CXCL12-induced migration of colorectal cancer cells

Background Interactions between CXCR4 and its ligand CXCL12 have been shown to be involved in cancer progression in colorectal cancer (CRC). We performed a comparative CXCL12/CXCR4 expression analysis and assessed the effect of external CXCL12 stimulation on migration of CRC cells without and with CXCR4 inhibition. Methods Expression of CXCL12/CXCR4 was assessed by quantitative real-time PCR, ELISA and immunohistochemistry in resection specimens of 50 CRC patients as well as in the corresponding normal tissues and in three human CRC cell lines with different metastatic potential (Caco-2, SW480 and HT-29). Migration assays were performed after stimulation with CXCL12 and CXCR4 was inhibited by siRNA and neutralizing antibodies. Results In CRC tissues CXCL12 was significantly down-regulated and CXCR4 was significantly up-regulated compared to the corresponding normal tissues. In cell lines CXCR4 was predominantly expressed in SW480 and less pronounced in HT-29 cells. CXCL12 was only detectable in Caco-2 cells. CXCL12 stimulation had no impact on Caco-2 cells but significantly increased migration of CXCR4 bearing SW480 and HT-29 cells. This effect was significantly abrogated by neutralizing anti-CXCR4 antibody as well as by CXCR4 siRNAs (P < 0.05). Conclusions CXCR4 expression was up-regulated in CRC and CXCL12 stimulation increased migration in CXCR4 bearing cell lines. Migration was inhibited by both neutralizing CXCR4 antibodies and CXCR4 siRNAs. Thus, the expression and functionality of CXCR4 might be associated with the metastatic potential of CRC cells and CXCL12/CXCR4 interactions might therefore constitute a promising target for specific treatment interventions.

Cellular immune responses to HCV core increase and HCV RNA levels decrease during successful antiretroviral therapy

Background Hepatitis C virus (HCV) infection is a major cause of morbidity in HIV infected individuals. Coinfection with HIV is associated with diminished HCV-specific immune responses and higher HCV RNA levels. Aims To investigate whether long-term combination antiretroviral therapy (cART) restores HCV-specific T cell responses and improves the control of HCV replication. Methods T cell responses were evaluated longitudinally in 80 HIV/HCV coinfected individuals by ex vivo interferon-γ-ELISpot responses to HCV core peptides, that predominantly stimulate CD4+ T cells. HCV RNA levels were assessed by real-time PCR in 114 individuals. Results The proportion of individuals with detectable T cell responses to HCV core peptides was 19% before starting cART, 24% in the first year on cART and increased significantly to 45% and 49% after 33 and 70 months on cART (p=0.001). HCV-specific immune responses increased in individuals with chronic (+31%) and spontaneously cleared HCV infection (+30%). Median HCV RNA levels before starting cART were 6.5 log10 IU/ml. During long-term cART, median HCV-RNA levels slightly decreased compared to pre-cART levels (−0.3 log10 IU/ml, p=0.02). Conclusions Successful cART is associated with increasing cellular immune responses to HCV core peptides and with a slight long-term decrease in HCV RNA levels. These findings are in line with the favourable clinical effects of cART on the natural history of hepatitis C and with the current recommendation to start cART earlier in HCV/HIV coinfected individuals.