Persistence of Different Forms of Transient RNAi during Apoptosis in Mammalian Cells

Gene silencing by transient or stable RNA-interference (RNAi) is used for the study of apoptosis with an assumption that apoptotic events will not influence RNAi. However, we recently reported that stable RNAi, i.e., a permanent gene-knockdown mediated by shRNA-generating DNA vectors that are integrated in the genome, fails rapidly after induction of apoptosis due to caspase-3-mediated cleavage and inactivation of the endoribonuclease Dicer-1 that is required for conversion of shRNA to siRNA. Since apoptosis studies also increasingly employ transient RNAi models in which apoptosis is induced immediately after a gene is temporarily knocked down within a few days of transfection with RNAi-inducing agents, we examined the impact of apoptosis on various models of transient RNAi. We report here that unlike the stable RNAi, all forms of transient RNAi, whether Dicer-1-independent (by 21mer dsRNA) or Dicer-1-dependent (by 27mer dsRNA or shRNA-generating DNA vector), whether for an exogenous gene GFP or an endogenous gene poly(ADP-ribose) polymerase-1, do not fail for 2–3 days after onset of apoptosis. Our results reflect the differences in dynamics of achieving and maintaining RNAi during the early phase after transfection in the transient RNAi model and the late steady-state phase of gene-knockdown in stable RNAi model. Our results also sound a cautionary note that RNAi status should be frequently validated in the studies involving apoptosis and that while stable RNAi can be safely used for the study of early apoptotic events, transient RNAi is more suitable for the study of both early and late apoptotic events

The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing

<p>Abstract</p> <p>Background</p> <p>The use of RNAi to analyse gene function <it>in vitro </it>is now widely applied in biological research. However, several difficulties are associated with its use <it>in vivo</it>, mainly relating to inefficient delivery and non-specific effects of short RNA duplexes in animal models. The latter can lead to false positive results when real-time RT-qPCR alone is used to measure target mRNA knockdown.</p> <p>Findings</p> <p>We observed that detection of an apparent siRNA-mediated knockdown <it>in vivo </it>was dependent on the primers used for real-time RT-qPCR measurement of the target mRNA. Two siRNAs specific for <it>RRM1 </it>with equivalent activity <it>in vitro </it>were administered to A549 xenografts via intratumoural injection. In each case, apparent knockdown of <it>RRM1 </it>mRNA was observed only when the primer pair used in RT-qPCR flanked the siRNA cleavage site. This false-positive result was found to result from co-purified siRNA interfering with both reverse transcription and qPCR.</p> <p>Conclusions</p> <p>Our data suggest that using primers flanking the siRNA-mediated cleavage site in RT-qPCR-based measurements of mRNA knockdown <it>in vivo </it>can lead to false positive results. This is particularly relevant where high concentrations of siRNA are introduced, particularly via intratumoural injection, as the siRNA may be co-purified with the RNA and interfere with downstream enzymatic steps. Based on these results, using primers flanking the siRNA target site should be avoided when measuring knockdown of target mRNA by real-time RT-qPCR.</p

Effective Treatment of Respiratory Alphaherpesvirus Infection Using RNA Interference

BACKGROUND: Equine herpesvirus type 1 (EHV-1), a member of the Alphaherpesvirinae, is spread via nasal secretions and causes respiratory disease, neurological disorders and abortions. The virus is a significant equine pathogen, but current EHV-1 vaccines are only partially protective and effective metaphylactic and therapeutic agents are not available. Small interfering RNAs (siRNA's), delivered intranasally, could prove a valuable alternative for infection control. siRNA's against two essential EHV-1 genes, encoding the viral helicase (Ori) and glycoprotein B, were evaluated for their potential to decrease EHV-1 infection in a mouse model. METHODOLOGY/PRINCIPAL FNDINGS: siRNA therapy in vitro significantly reduced virus production and plaque size. Viral titers were reduced 80-fold with 37.5 pmol of a single siRNA or with as little as 6.25 pmol of each siRNA when used in combination. siRNA therapy in vivo significantly reduced viral replication and clinical signs. Intranasal treatment did not require a transport vehicle and proved effective when given up to 12 h before or after infection. CONCLUSIONS/SIGNIFICANCE: siRNA treatment has potential for both prevention and early treatment of EHV-1 infections

A locus at 19q13.31 significantly reduces the <em>ApoE</em> ε4 risk for Alzheimer\u27s Disease in African Ancestry

Copyright: \ua9 2022 Rajabli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. African descent populations have a lower Alzheimer disease risk from ApoE ε4 compared to other populations. Ancestry analysis showed that the difference in risk between African and European populations lies in the ancestral genomic background surrounding the ApoE locus (local ancestry). Identifying the mechanism(s) of this protection could lead to greater insight into the etiology of Alzheimer disease and more personalized therapeutic intervention. Our objective is to follow up the local ancestry finding and identify the genetic variants that drive this risk difference and result in a lower risk for developing Alzheimer disease in African ancestry populations. We performed association analyses using a logistic regression model with the ApoE ε4 allele as an interaction term and adjusted for genome-wide ancestry, age, and sex. Discovery analysis included imputed SNP data of 1,850 Alzheimer disease and 4,331 cognitively intact African American individuals. We performed replication analyses on 63 whole genome sequenced Alzheimer disease and 648 cognitively intact Ibadan individuals. Additionally, we reproduced results using whole-genome sequencing of 273 Alzheimer disease and 275 cognitively intact admixed Puerto Rican individuals. A further comparison was done with SNP imputation from an additional 8,463 Alzheimer disease and 11,365 cognitively intact non-Hispanic White individuals. We identified a significant interaction between the ApoE ε4 allele and the SNP rs10423769_A allele, (β = -0.54,SE = 0.12,p-value = 7.50x10-6) in the discovery data set, and replicated this finding in Ibadan (β = -1.32,SE = 0.52,p-value = 1.15x10-2) and Puerto Rican (β = -1.27,SE = 0.64,p-value = 4.91x10-2) individuals. The non-Hispanic Whites analyses showed an interaction trending in the “protective” direction but failing to pass a 0.05 significance threshold (β = -1.51,SE = 0.84,p-value = 7.26x10-2). The presence of the rs10423769_A allele reduces the odds ratio for Alzheimer disease risk from 7.2 for ApoE ε4/ε4 carriers lacking the A allele to 2.1 for ApoE ε4/ε4 carriers with at least one A allele. This locus is located approximately 2 mB upstream of the ApoE locus, in a large cluster of pregnancy specific beta-1 glycoproteins on chromosome 19 and lies within a long noncoding RNA, ENSG00000282943. This study identified a new African-ancestry specific locus that reduces the risk effect of ApoE ε4 for developing Alzheimer disease. The mechanism of the interaction with ApoEε4 is not known but suggests a novel mechanism for reducing the risk for ε4 carriers opening the possibility for potential ancestry-specific therapeutic intervention

miR-200 Enhances Mouse Breast Cancer Cell Colonization to Form Distant Metastases

BACKGROUND: The development of metastases involves the dissociation of cells from the primary tumor to penetrate the basement membrane, invade and then exit the vasculature to seed, and colonize distant tissues. The last step, establishment of macroscopic tumors at distant sites, is the least well understood. Four isogenic mouse breast cancer cell lines (67NR, 168FARN, 4TO7, and 4T1) that differ in their ability to metastasize when implanted into the mammary fat pad are used to model the steps of metastasis. Only 4T1 forms macroscopic lung and liver metastases. Because some miRNAs are dysregulated in cancer and affect cellular transformation, tumor formation, and metastasis, we examined whether changes in miRNA expression might explain the differences in metastasis of these cells. METHODOLOGY/PRINCIPAL FINDINGS: miRNA expression was analyzed by miRNA microarray and quantitative RT-PCR in isogenic mouse breast cancer cells with distinct metastatic capabilities. 4T1 cells that form macroscopic metastases had elevated expression of miR-200 family miRNAs compared to related cells that invade distant tissues, but are unable to colonize. Moreover, over-expressing miR-200 in 4TO7 cells enabled them to metastasize to lung and liver. These findings are surprising since the miR-200 family was previously shown to promote epithelial characteristics by inhibiting the transcriptional repressor Zeb2 and thereby enhancing E-cadherin expression. We confirmed these findings in these cells. The most metastatic 4T1 cells acquired epithelial properties (high expression of E-cadherin and cytokeratin-18) compared to the less metastatic cells. CONCLUSIONS/SIGNIFICANCE: Expression of miR-200, which promotes a mesenchymal to epithelial cell transition (MET) by inhibiting Zeb2 expression, unexpectedly enhances macroscopic metastases in mouse breast cancer cell lines. These results suggest that for some tumors, tumor colonization at metastatic sites might be enhanced by MET. Therefore the epithelial nature of a tumor does not predict metastatic outcome

Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening “superbugs” for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics

Role of miR-10b in breast cancer metastasis

Ninety percent of cancer-related mortality is caused by metastasis. Current cancer treatments can control many primary tumors but rarely stop the metastatic spread. Accumulating evidence demonstrates that miRNAs are involved in cancer initiation and progression. Furthermore, several miRNAs have been found to regulate metastasis. In particular, recent studies provide the first functional evidence that overexpression of a specific miRNA, miR-10b, can contribute to the development of metastasis, which can be exploited therapeutically in treating breast cancer metastasis in mice. Further in-depth analysis should provide more precise evaluation of the roles, mechanisms, and therapeutic utility of this miRNA in breast cancer

Silencing α-Synuclein Gene Expression Enhances Tyrosine Hydroxylase Activity in MN9D Cells

α-Synuclein has been implicated in the pathogenesis of Parkinson’s disease (PD). Previous studies have shown that α-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed α-synuclein stably silenced MN9D/α-SYN− cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/α-SYN− and MN9D/CON cells, suggesting that silencing α-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (l-DOPA) and DA levels were observed in MN9D/α-SYN− cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of α-synuclein, suggesting that α-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional α-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death

Enhancement of Allele Discrimination by Introduction of Nucleotide Mismatches into siRNA in Allele-Specific Gene Silencing by RNAi

Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs) against mutant alleles of the human Prion Protein (PRNP) gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the ‘seed region’ of microRNAs. Due to the essential role of the ‘seed region’ of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs), of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3′-ends of sense-strand siRNA elements, which possibly increase the assembly of antisense-strand (guide) siRNAs into RNA-induced silencing complexes (RISCs), may enhance ASP-RNAi in the case of inert siRNA duplexes. Therefore, the data presented here suggest that structural modification of functional portions of an siRNA duplex by base substitution could greatly influence allele discrimination and gene silencing, thereby contributing to enhancement of ASP-RNAi