USP9X promotes apoptosis in cholangiocarcinoma by modulation expression of KIF1Bβ via deubiquitinating EGLN3

Background Cholangiocarcinoma represents the second most common primary liver malignancy. The incidence rate has constantly increased over the last decades. Cholangiocarcinoma silent nature limits early diagnosis and prevents efficient treatment. Methods Immunoblotting and immunohistochemistry were used to assess the expression profiling of USP9X and EGLN3 in cholangiocarcinoma patients. ShRNA was used to silence gene expression. Cell apoptosis, cell cycle, CCK8, clone formation, shRNA interference and xenograft mouse model were used to explore biological function of USP9X and EGLN3. The underlying molecular mechanism of USP9X in cholangiocarcinoma was determined by immunoblotting, co-immunoprecipitation and quantitative real time PCR (qPCR). Results Here we demonstrated that USP9X is downregulated in cholangiocarcinoma which contributes to tumorigenesis. The expression of USP9X in cholangiocarcinoma inhibited cell proliferation and colony formation in vitro as well as xenograft tumorigenicity in vivo. Clinical data demonstrated that expression levels of USP9X were positively correlated with favorable clinical outcomes. Mechanistic investigations further indicated that USP9X was involved in the deubiquitination of EGLN3, a member of 2-oxoglutarate and iron-dependent dioxygenases. USP9X elicited tumor suppressor role by preventing degradation of EGLN3. Importantly, knockdown of EGLN3 impaired USP9X-mediated suppression of proliferation. USP9X positively regulated the expression level of apoptosis pathway genes de through EGLN3 thus involved in apoptosis of cholangiocarcinoma. Conclusion These findings help to understand that USP9X alleviates the malignant potential of cholangiocarcinoma through upregulation of EGLN3. Consequently, we provide novel insight into that USP9X is a potential biomarker or serves as a therapeutic or diagnostic target for cholangiocarcinoma.

CRISPR-Cas-mediated tethering recruits the yeast HMR mating-type locus to the nuclear periphery but fails to silence gene expression

To investigate the relationship between genome structure and function, we have developed a programmable CRISPR-Cas system for nuclear peripheral recruitment in yeast. We benchmarked this system at the HMR and GAL2 loci, both well-characterized model systems for localization to the nuclear periphery. Using microscopy and gene silencing assays, we demonstrate that CRISPR-Cas-mediated tethering can recruit the HMR locus but does not silence reporter gene expression. A previously reported Gal4-mediated tethering system does silence gene expression, and we demonstrate that the silencing phenotype has an unexpected dependence on the structure of the protein tether. The CRISPR-Cas system was unable to recruit GAL2 to the nuclear periphery. Our results reveal potential challenges for synthetic genome structure perturbations and suggest that distinct functional effects can arise from subtle structural differences in how genes are recruited to the periphery.

Quantification of durable CRISPR-based gene silencing activity

Development of CRISPR-based technologies for regulating gene expression stands to provide novel methods for the study and engineering of biological behavior. New tools capable of inducing long-lasting changes in gene expression will increase the utility of these techniques, providing durable effects from one-time doses of reagents. We describe here a reporter system for quantifying the ability of CRISPR-based effectors to induce stable gene repression. We observe a continuous gradation of the ability of these effectors to silence gene expression, depending on the domain composition and configuration. We also report the creation of a single CRISPR protein capable of producing durable gene silencing. This assay should allow for the continued development of enhanced gene repression tools which will be useful in a wide array of biological research and engineering applications.

The RNAi Mechanism Regulates a New Exonuclease Gene Involved in the Virulence of Mucorales

Mucormycosis is a lethal disease caused by Mucorales, which are emerging as human causes that explain the high mortality for this disease. Consequently, the research community is searching for virulence determinants that could be repurposed as targets to develop new treatments against mucormycosis. Our work explores an RNA interference (RNAi)-based approach to find targets involved in the virulence of Mucorales. A transcriptomewide analysis compared sRNAs and their target mRNAs in two Mucor lusitanicus different pathotypes, virulent and avirulent, generating a list of 75 loci selected by their differential sRNA accumulation in these strains. As a proof of concept and validity, an experimental approach characterized two loci showing opposite behavior, confirming that RNAi activity causes their differential expression in the two pathotypes. We generated deletion mutants for two loci and a knockin-strain overexpressing for one of these loci. Their functional analysis in murine virulence assays identified the gene wex1, a putative DEDDy exonuclease with RNase domains, as an essential factor for virulence. The identification of wex1 showed the potential of our approach to discover virulence factors not only in Mucorales but also in any other fungal model with an active RNAi machinery. More importantly, it adds a new layer to the biological processes controlled by RNAi in M. lusitanicus, confirming that the Dicer-dependent RNAi pathway can silence gene expression to promote virulence.

The RNAi Mechanism Regulates a New Exonuclease Gene Involved in the Virulence of Mucorales

Mucormycosis is a lethal disease caused by Mucorales, which are emerging as human pathogens with virulent behavior. Antifungal resistance and ineffective treatments are two major causes that explain the high mortality for this disease. Consequently, the research community is searching for virulence determinants that could be repurposed as targets to develop new treatments against mucormycosis. Our work explores an RNA interference (RNAi)-based approach to find targets involved in the virulence of Mucorales. A transcriptome-wide analysis compared sRNAs and their target mRNAs in two Mucor lusitanicus different pathotypes, virulent and avirulent, generating a list of 75 loci selected by their differential sRNA accumulation in these strains. As a proof of concept and validity, an experimental approach characterized two loci showing opposite behavior, confirming that RNAi activity causes their differential expression in the two pathotypes. We generated deletion mutants for two loci and a knockin-strain overexpressing for one these loci. Their functional analysis in murine virulence assays identified the gene wex1, a putative DEDDy exonuclease with RNase domains, as an essential factor for virulence. The identification of wex1 showed the potential of our approach to discover virulence factors not only in Mucorales but also in any other fungal model with an active RNAi machinery. But, more importantly, it adds a new layer to the biological processes controlled by RNAi in M. lusitanicus, confirming that the Dicer-dependent RNAi pathway can silence gene expression to promote virulence.

Stabilization of EGLN3 by USP9X Contributes to Antineoplastic Function in Cholangiocarcinoma

Background: Cholangiocarcinoma represents the second most common primary liver malignancy. The incidence rate has constantly increased over the last decades. Cholangiocarcinoma silent nature limits early diagnosis and prevents efficient treatment. Methods: Immunoblotting and immunohistochemistry were used to assess the expression profiling of USP9X and EGLN3 in cholangiocarcinoma patients. ShRNA was used to silence gene expression. Cell apoptosis, cell cycle, CCK8, clone formation, shRNA interference and xenograft mouse model were used to explore biological function of USP9X and EGLN3. The underlying molecular mechanism of USP9X in cholangiocarcinoma was determined by immunoblotting, co-immunoprecipitation and quantitative real time PCR (qPCR).Results: Here we demonstrated that USP9X is downregulated in cholangiocarcinoma which contributes to tumorigenesis. The expression of USP9X in cholangiocarcinoma inhibited cell proliferation and colony formation in vitro as well as xenograft tumorigenicity in vivo. Clinical data demonstrated that expression levels of USP9X were positively correlated with favorable clinical outcomes. Mechanistic investigations further indicated that USP9X was involved in the deubiquitination of EGLN3, a member of 2-oxoglutarate and iron-dependent dioxygenases. USP9X elicited tumor suppressor role by preventing degradation of EGLN3. Importantly, knockdown of EGLN3 impaired USP9X-mediated suppression of proliferation. USP9X positively regulated the expression level of apoptosis pathway genes KIF1Bβ through EGLN3 thus involved in apoptosis of cholangiocarcinoma. Conclusion: These findings help to understand that USP9X alleviates the malignant potential of cholangiocarcinoma through upregulation of EGLN3. Consequently, we provided novel insight into that USP9X is a potential biomarker or serves as a therapeutic or diagnostic target for cholangiocarcinoma.

Polyornithine-based polyplexes to boost effective gene silencing in CNS disorders

Novel biodegradable and biocompatible polyornithine derivatives as non-viral vectors for siRNA exhibit effectively silence gene expression in primary neural cells.

Extracellular Vesicles and their miRNA Cargo: A Means of Communication between Soma and Germline in the Mammalian Reproductive System

MicroRNAs (miRNAs) are small non-coding RNAs able to silence gene expression by RNA interference. They are present in cells but many are contained in extracellular vesicles (EVs) that can be released by cells in the circulation. Circulating EVs can encounter other cells in the body and deliver their miRNA cargo. This process enables long-range communication between different cells and has been proposed to play important physiological roles. One of these roles that remains less well studied is in the reproductive system. In ovaries and testes, constant communication between somatic cells and developing germ cells is necessary for their maturation and EVs have been proposed to contribute to this communication. EVs might also enable external factors derived from environmental exposure to reach gametes and keep a trace of exposure for the offspring.

A broad-host-range CRISPRi toolkit for silencing gene expression in Burkholderia

Genetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus Burkholderia have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit Burkholderia GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in Burkholderia, testing it in B. cenocepacia, B. multivorans and B. thailandensis. Tunable expression was provided by placing a codon-optimized dcas9 from Streptococcus pyogenes under control of a rhamnose-inducible promoter. As a proof of concept, the paaABCDE operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne sgRNAs, resulting in near complete inhibition of growth on phenylacetic acid as the sole carbon source. This was supported by reductions in paaA mRNA expression. The utility of CRISPRi to probe other functions at the single cell level was demonstrated by knocking down phbC and fliF, which dramatically reduces polyhydroxybutyrate granule accumulation and motility, respectively. As a hallmark of the mini-CTX system is the broad host-range of integration, we putatively identified 67 genera of Proteobacteria that might be amenable to modification with our CRISPRi toolkit. Our CRISPRi tool kit provides a simple and rapid way to silence gene expression to produce an observable phenotype. Linking genes to functions with CRISPRi will facilitate genome editing with the goal of enhancing biotechnological capabilities while reducing Burkholderia’s pathogenic arsenal.Author contributionsSTC conceived the idea and design of the research; AMH designed and cloned the dCas9 constructs; AMH and ASMZ designed the sgRNAs, assessed knockdown phenotypes, processed data, and wrote and edited the manuscript; TJL performed RT-qPCR analysis and edited the manuscript; STC supervised the work and provided financial support.Graphical Abstract