miRNA-337-3p suppresses neuroblastoma progression by repressing the transcription of matrix metalloproteinase 14
Xuan XiangHong MeiXiang ZhaoJiarui PuDan LiHongxia QuWanju JiaoJihe ZhaoKai HuangLiduan ZhengQiangsong Tong
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// Xuan Xiang 1, * , Hong Mei 1, * , Xiang Zhao 1, * , Jiarui Pu 1 , Dan Li 1 , Hongxia Qu 1 , Wanju Jiao 2 , Jihe Zhao 3 , Kai Huang 4 , Liduan Zheng 2, 4 , Qiangsong Tong 1, 4 1 Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, P. R. China 2 Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, P. R. China 3 Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, USA 4 Clinical Center of Human Genomic Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, P. R. China * These authors have contributed equally to this work Correspondence to: Qiangsong Tong, e-mail: qs_tong@hotmail.com Liduan Zheng, e-mail: ld_zheng@hotmail.com Keywords: neuroblastoma, microRNA-337-3p, matrix metalloproteinase 14, transcriptional repression Received: February 18, 2015 Accepted: June 03, 2015 Published: June 15, 2015 ABSTRACT Recent evidence shows the emerging roles of endogenous microRNAs (miRNAs) in repressing gene transcription. However, the miRNAs inhibiting the transcription of matrix metalloproteinase 14 ( MMP-14 ), a membrane-anchored MMP crucial for the tumorigenesis and aggressiveness, still remain largely unknown. In this study, through mining computational algorithm program and genome-wide Argonaute profiling dataset, we identified one binding site of miRNA-337-3p (miR-337-3p) within the MMP-14 promoter. We demonstrated that miR-337-3p was under-expressed and inversely correlated with MMP-14 expression in clinical specimens and cell lines of neuroblastoma (NB), the most common extracranial solid tumor in childhood. Patients with high miR-337-3p expression had greater survival probability. miR-337-3p suppressed the promoter activity, nascent transcription, and expression of MMP-14 , resulting in decreased levels of vascular endothelial growth factor, in cultured NB cell lines. Mechanistically, miR-337-3p recognized its binding site and recruited Argonaute 2 to facilitate the enrichment of repressive epigenetic markers and decrease the binding of RNA polymerase II and specificity protein 1 on the MMP-14 promoter. Gain- and loss-of-function studies demonstrated that miR-337-3p suppressed the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo . In addition, restoration of MMP-14 expression rescued the NB cells from changes in these biological features. Taken together, these data indicate that miR-337-3p directly binds the MMP-14 promoter to repress its transcription, thus suppressing the progression of NB.Keywords:
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Analysis at the individual level using genetically-engineered mice has allowed conclusions to be reached regarding the actual function of a target gene. In recent years, the “knockdown” method using RNA interference (RNAi) has been established as a powerful tool for analyzing gene function. In this review, we focus on RNAi knockdown technology for producing genetically-engineered mice and describe the value of this approach from the perspective of both basic research and therapeutic potential. First, we introduce the basic mechanism of RNAi and development of knockdown animals from worms to mice. Next, we describe strategies to produce knockdown mice using DNA-based expression vectors introduced into zygotes or embryonic stem cells. Finally, we refer to the trends of research for clinical application. By way of illustration, we show the production of knockdown mice for treatment of neurodegenerative disease and mention the prospect of therapeutic potential of RNAi technology.
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RNA Silencing
Rhodnius
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Genetic screening is the most powerful method through which to uncover gene function. It has been applied very successfully in lower organisms but seldom attempted in mammalian species because of their long generation time. In this study, we exploit RNA interference (RNAi) for its potential use in genetic screening in mice. We show that RNAi-induced gene knockdown can be generated through introducing small hairpin RNA-expressing constructs into the mouse as transgenes via conventional pronuclear injection. The knockdown effect can be transmitted for many generations in these transgenic animals. In a small-scale screening for developmental defects in the kidney, we uncovered a potential role of Id4 in the formation of the renal medulla. Our results demonstrate the feasibility of using RNAi for genetic screening in mice.
Genetic screen
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Background The approach of RNAi mediated gene knockdown, employing exogenous dsRNA, is being beneficially exploited in various fields of functional genomics. The immense utility of the approach came to fore from studies with model system C. elegans, but quickly became applicable with varied research models ranging from in vitro to various in vivo systems. Previously, there have been reports on the refractoriness of the neuronal cells to RNAi mediated gene silencing following which several modulators like eri-1 and lin-15 were described in C. elegans which, when present, would negatively impact the gene knockdown. Methodology/Principal Findings Taking a clue from these findings, we went on to screen hypothesis-driven- methodologies towards exploring the efficiency in the process of RNAi under various experimental conditions, wherein these genes would be knocked down preceding to, or concurrently with, the knocking down of a gene of interest. For determining the efficiency of gene knockdown, we chose to study visually stark phenotypes of uncoordinated movement, dumpy body morphology and blistered cuticle obtained by knocking down of genes unc-73, dpy-9 and bli-3 respectively, employing the RNAi-by-feeding protocol in model system C. elegans. Conclusions/Significance Our studies led to a very interesting outcome as the results reveal that amongst various methods tested, pre-incubation with eri-1 dsRNA synthesizing bacteria followed by co-incubation with eri-1 and gene-of-interest dsRNA synthesizing bacteria leads to the most efficient gene silencing as observed by the analysis of marker phenotypes. This provides an approach for effectively employing RNAi induced gene silencing while working with different genetic backgrounds including transgenic and mutant strains.
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RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo.
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Abstract The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNa v ) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNa v dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30–60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control.
RNA Silencing
Red flour beetle
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RNA Silencing
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Targeting genes via RNA interference (RNAi) has become a successful method to reduce pest populations. Ideally, the expression of a gene critical for a life function in the insect is targeted by specific dsRNA, via spray or oral delivery. Experts have developed working guidelines in the development and regulation of RNAi as a pesticide. We argue that an important tool in the validation of RNAi is genome-wide expression analysis in the targeted pest, and we name this approach RNAiSeq. We have used RNAiSeq in the coleopteran model Tribolium castaneum to validate knockdown of target genes, and to examine the effect of knockdown on other genes. With RNAiSeq, we identified compensation responses to the knockdown of a gene encoding a major digestive enzyme in larvae that correlate to the responses we have observed with ingested protease inhibitors. Compensation can mask RNAi phenotypic responses and are important to understand in the context of efficacy. RNAiSeq also has identified new gene interactions that were previously unassociated with the target gene, important in the context of the large number of genes without associated functions in insects and other organisms. We present other research where RNAiSeq has led to important findings. These data not only provide validation of target knockdown, but also further identify changes in the expression of other genes impacted by the knockdown. From the context of pest control, the information can be used to predict genetic changes that will impact the efficacy of RNAi products in target pests.
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Effective RNA interference (RNAi) methods have been developed in many pest species, enabling exploration of gene function. Until now RNAi had not been attempted in the cat flea, Ctenocephalides felis, although the development of RNAi approaches would open up potential avenues for control of this important pest. This study aimed to establish if an RNAi response occurs in adult C. felis upon exposure to double-stranded RNA (dsRNA), which administration methods for dsRNA delivery could bring about effective gene knockdown and to investigate dynamics of any RNAi response. Knockdown of 80% of GSTσ was achieved by intrahaemoceolic microinjection of dsGSTσ but this invasive technique was associated with relatively high mortality rates. Immersing C. felis in dsGSTσ or dsDicer-2 overnight resulted in 65% knockdown of GSTσ or 60% of Dicer-2, respectively, and the degree of knockdown was not improved by increasing the dsRNA concentration in the bathing solution. Unexpectedly, the greatest degree of knockdown was achieved with the continuous administration of dsRNA in whole blood via a membrane feeding system, resulting in 96% knockdown of GSTσ within 2 days and sustained up to, at least, 7 days. Thus, unlike in many other species, the gut nucleases do not impair the RNAi response to ingested dsRNA in C. felis. A modest, but significant, upregulation of Dicer-2 and Argonaute2 was detectable 3 h after exposure to exogenous dsRNA, implicating the short-interfering RNA pathway. To our knowledge this study represents the first demonstration of experimentally induced RNAi in the cat flea as well as giving insight into how the gene knockdown response progresses.
Dicer
RNA Silencing
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