Biology subjects

Chen, X.

Publications and source records attributed to Chen, X..

At least 919 records · Page 51Linked to original sources

bioRxiv2023

Directed differentiation of human hindbrain neuroepithelial stem cells recapitulates cerebellar granule neurogenesis

Cerebellar granule neurons (CGNs) are the most abundant neurons in the human brain and modulate cerebellar output to the motor cortex. Dysregulation of CGN development underlies movement disorders and medulloblastomas. It is suspected that these disorders arise in progenitor states of the CGN lineage, for which human models are lacking. Here, we have differentiated human hindbrain neuroepithelial stem (hbNES) cells to CGNs in vitro using soluble growth factors, recapitulating key progenitor states in the lineage. We show that hbNES cells are not lineage committed and retain rhombomere 1 (r1) regional identity. Upon differentiation, hbNES cells first transit through a rhombic lip (RL) progenitor state at day 7, demonstrating human specific sub-ventricular cell identities. This RL state is followed by an ATOH1+ CGN progenitor state at day 14. By the end of a 56-day differentiation procedure, we obtain mature neurons expressing CGN markers GABAAa6 and vGLUT2. These neurons generate spontaneous and evoked action potentials. A small fraction of endpoint neurons were unipolar brush cells (UBC). We noted maintenance of a RL population throughout differentiation, as is consistent with human development. We show that sonic hedgehog (SHH) promotes {gamma}-aminobutyric acid (GABA)-ergic lineage specification and is a positive regulator of CGN progenitor proliferation. Interestingly, we observed that functional neuronal maturation is impaired by either elevated or absent SHH signaling. Impaired maturation under high SHH levels represents the potential of our system to model cerebellar tumorigenesis. Further, our data suggest a potential pro-differentiation role of SHH within a certain concentration range. Our work is, to our knowledge, the first detailed temporal characterization of the complete human CGN lineage in vitro. Our system recapitulates developmentally relevant progenitor states and is a new tool to model this specific cerebellar lineage, and how it may be disrupted to cause human disease.

developmental biology↗

bioRxiv2023

Enhanced transmissibility of XBB.1.5 is contributed by both strong ACE2 binding and antibody evasion

SARS-CoV-2 recombinant subvariant XBB.1.5 is growing rapidly in the United States, carrying an additional Ser486Pro substitution compared to XBB.1 and outcompeting BQ.1.1 and other XBB sublineages. The underlying mechanism for such high transmissibility remains unclear. Here we show that XBB.1.5 exhibits a substantially higher hACE2-binding affinity compared to BQ.1.1 and XBB/XBB.1. Convalescent plasma samples from BA.1, BA.5, and BF.7 breakthrough infection are significantly evaded by both XBB.1 and XBB.1.5, with XBB.1.5 displaying slightly weaker immune evasion capability than XBB.1. Evusheld and Bebtelovimab could not neutralize XBB.1/XBB.1.5, while Sotrovimab remains weakly reactive and notably, SA55 is still highly effective. The fact that XBB.1 and XBB.1.5 showed comparable antibody evasion but distinct transmissibility suggests enhanced receptor-binding affinity would indeed lead to higher growth advantages. The strong hACE2 binding of XBB.1.5 could also enable its tolerance of further immune escape mutations, which should be closely monitored.

immunology↗

bioRxiv2023

Cooperative NF-κB and Notch1 signaling promotes macrophage-mediated MenaINV expression in breast cancer

Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and frequently patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin-regulatory protein Mena encoded by the ENAH gene that endows tumor cells with transendothelial migration activity allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. In this study, we discovered the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-{kappa}B transcription factor binding site. Using live imaging of an NF-{kappa}B activity reporter and staining of fixed tissues from mouse and human breast cancer we further determined that for maximal induction of MenaINV in cancer cell NF-{kappa}B needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but it enhances and sustains NF-{kappa}B signaling through retention of p65, an NF-{kappa}B transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-{kappa}B signaling and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease.

cancer biology↗

bioRxiv2022

Development and characterization of a recombinant silk network for 3D culture of immortalized and fresh tumor-derived breast cancer cells

Traditional cancer models rely on 2D cell cultures or 3D spheroids, which fail to recapitulate cell-extracellular matrix (ECM) interactions, a key element of tumor development. Existing hydrogel-based 3D alternatives lack mechanical support for cell growth and often suffer from low reproducibility. Here we report a novel strategy to make 3D models of breast cancer using a tissue-like, well-defined network environment based on recombinant spider silk, functionalized with a cell adhesion motif from fibronectin (FN-silk). With this approach, the canonical cancer cells SK-BR-3, MCF-7, and MDA-MB-231, maintain their characteristic expression of markers (i.e., ER, HER2, and PGR) while developing distinct morphology. Transcriptomic analyses demonstrate how culture in the FN-silk networks modulates the biological processes of cell adhesion and migration while affecting physiological events involved in malignancy, such as inflammation, remodeling of the ECM, and resistance to anticancer drugs. Finally, we show that integration in FN-silk networks promotes the viability of cells obtained from the superficial scraping of patients breast tumors.

cancer biology↗

bioRxiv2022

Hypothalamic supramammillary neurons that project to the medial septum control wakefulness

The hypothalamic supramammillary nucleus (SuM) plays a key role in controlling wakefulness, but the downstream target regions participating in this control process remain unknown. Here, using circuit-specific fiber photometry and single-neuron electrophysiology together with electroencephalogram, electromyogram and behavioral recordings, we find approximately half of SuM neurons that project to the medial septum (MS) are wake-active. Optogenetic stimulation of axonal terminals of SuM-MS projection induces a rapid and reliable transition to wakefulness from NREM or REM sleep, and chemogenetic activation of SuMMS projecting neurons significantly increases wakefulness time and prolongs latency to sleep. Consistently, chemogenetically inhibiting these neurons significantly reduces wakefulness time and latency to sleep. Therefore, these results identify the MS as a functional downstream target of SuM and provide evidence for a causal role for this hypothalamic-septal projection in wakefulness control.

neuroscience↗

bioRxiv2022

Coevolutionary dynamics via adaptive feedback in collective-risk social dilemma game

Human society and natural environment form a complex giant ecosystem, where human activities not only lead to the change of environmental states, but also react to them. By using collective-risk social dilemma game, some studies have already revealed that individual contributions and the risk of future losses are inextricably linked. These works, however, often use an idealistic assumption that the risk is constant and not affected by individual behaviors. We here develop a coevolutionary game approach that captures the coupled dynamics of cooperation and risk. In particular, the level of contributions in a population affects the state of risk, while the risk in turn influences individuals behavioral decision-making. Importantly, we explore two representative feedback forms describing the possible effect of strategy on risk, namely, linear and exponential feedbacks. We find that cooperation can be maintained in the population by keeping at a certain fraction or forming an evolutionary oscillation with risk, independently of the feedback type. However, such evolutionary outcome depends on the initial state. Taken together, a two-way coupling between collective actions and risk is essential to avoid the tragedy of the commons. More importantly, a critical starting portion of cooperators and risk level is what we really need for guiding the evolution toward a desired direction.

evolutionary biology↗

bioRxiv2022

Open-3DSIM: an Open-source three-dimensional structured illumination microscopy reconstruction platform

With optical section and defocus removal effect, three-dimensional structured illumination microscopy (3DSIM) can get a whole sight of intracellular organelle. Here, Open-3DSIM is reported as an open-source reconstruction platform with double improvement on lateral and axial resolution. MATLAB code, ImageJ version and Exe application are provided for biologists and engineers to maximize its user-friendliness and prompt its further development. Through adaptive parameter estimation and spectrum filter optimization, we demonstrate its superior performance of artifact suppression and defocus elimination over other algorithms on various specimens, under gradient signal-to-noise levels. Moreover, with the capacity to extract the dipole orientation, Open-3DSIM paves a new avenue for interpreting the subcellular structures in six dimensions (xyz{theta}{lambda}t).

bioengineering↗

bioRxiv2022

Non-coding autoimmune risk variant accelerates T peripheral helper cell development via ICOS

Fine-mapping and functional studies implicate rs117701653, a common non-coding variant in the CD28/CTLA4/ICOS locus, as a contributor to risk for rheumatoid arthritis and type 1 diabetes. Using DNA pulldown, mass spectrometry, genome editing and eQTL analysis, we establish that the disease-associated allele reduces affinity for the inhibitory chromosomal regulator SMCHD1 to drive expression of inducible T-cell costimulator (ICOS), enhancing memory CD4+ T cell ICOS expression in individuals bearing the risk allele. Higher ICOS expression is paralleled by an increase in circulating T peripheral helper (Tph) cells, and in rheumatoid arthritis patients, of blood and joint fluid Tph cells and circulating plasmablasts, suggesting a causal link. Indeed, ICOS ligation accelerates T cell differentiation into CXCR5-PD-1high Tph cells producing IL-21 and CXCL13, as does carriage of the rs117701653 risk allele. Thus, mechanistic dissection of a causal non-coding variant in human autoimmunity discloses a new pathway through which ICOS regulates Tph abundance.

genetics↗

bioRxiv2022

Identification of a regulatory pathway governing TRAF1 via an arthritis-associated non-coding variant

TRAF1/C5 was among the first loci shown to confer risk for inflammatory arthritis in the absence of an associated coding variant, but its genetic mechanism remains undefined. Using ImmunoChip data from 3,939 juvenile idiopathic arthritis (JIA) patients and 14,412 controls, we identified 132 plausible common non-coding variants, reduced serially by SNP-seq, electrophoretic mobility shift, and luciferase studies to the single variant rs7034653 in the third intron of TRAF1. Genetically manipulated experimental cells and primary monocytes from genotyped donors establish that the risk G allele reduces binding of Fos-Related Antigen 2 (FRA2), resulting in reduced TRAF1 expression and enhanced TNF production. Conditioning on this variant eliminates attributable risk for rheumatoid arthritis, implicating a mechanism shared across the arthritis spectrum. These findings reveal that rs7034653, FRA2, and TRAF1 mediate a pathway through which a non-coding causal variant drives risk of inflammatory arthritis in children and adults.

genetics↗

bioRxiv2022

Transcriptomic landscape and potential therapeutic targets for human testicular aging revealed by single-cell RNA sequencing

BackgroundTesticular aging is known to cause male age-related fertility decline and hypogonadism, but the underlying molecular mechanisms remain unclear. MethodsWe survey the single-cell transcriptomic landscape of testes from young and old men and examine age-related changes in germline and somatic niche cells. ResultsIn-depth evaluation of the gene expression dynamics of germline cells reveals that disturbance of base-excision repair pathway is a major feature of aging spermatogonial stem cells (SSCs), suggesting that defective DNA repair of SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of aging-associated transcriptional changes shows that stress-related changes and apoptotic signaling pathway accumulate in aged somatic cells. We identify age-related impairment of redox homeostasis in aged Leydig cells and find that pharmacological treatment with antioxidants alleviate this cellular dysfunction of Leydig cells and promote testosterone production. Lastly, our results reveal that decreased pleiotrophin (PTN) signaling is a contributing factor for testicular aging. ConclusionsThese findings provide a comprehensive understanding of the cell-type-specific mechanisms underlying human testicular aging at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism. FundingThis work was supported by the National Key Research and Development Program of China (2018YFA0107200, 2018YFA0801404), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 81871110, 81971759), the Key Research and Development Program of Guangdong Province (2019B020234001), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (Family Planning Research Institute of Guangdong Province) (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921), the China Postdoctoral Science Foundation (2021M703736).

developmental biology↗

bioRxiv2022

Identification of Barley Enhancers across Genome via STARR-seq

Enhancers are DNA sequences that can strengthen transcription initiation. However, the global identification of plant enhancers is complicated due to uncertainty in the distance and orientation of enhancers, especially in species with large genomes. In this study, we performed self-transcribing active regulatory region sequencing (STARR-seq) for the first time to identify enhancers across the barley genome. A total of 7323 enhancers were successfully identified, and among 45 randomly selected enhancers, over 75% were effective as validated by a dual-luciferase reporter assay system in the lower epidermis of tobacco leaves. Interestingly, up to 53.5% of the barley enhancers were repetitive sequences, especially transposable elements (TEs), thus reinforcing the vital role of repetitive enhancers in gene expression. Both the common active transcription marker H3K4me3 and repressive histone marker H3K27me3 were abundant among the barley STARR-seq enhancers. In addition, the functional range of barley STARR-seq enhancers seemed much broader than that of rice or maize and extended to {+/-} 100 KB of the gene body, and this finding was consistent with the high expression levels of genes in the genome. This work specifically depicts the unique features of barley enhancers and provides available barley enhancers for further utilization.

genomics↗

bioRxiv2022

Genetically engineered mesenchymal stem cells as a nitric oxide reservoir for acute kidney injury therapy

Nitric oxide (NO), as a gaseous therapeutic agent, shows great potential for the treatment of many kinds of diseases. Although various NO delivery systems have emerged, the immunogenicity and long-term toxicity of artificial carriers hinder the potential clinical translation of this gas therapeutics. Mesenchymal stem cells (MSCs), with the capacities of self-renewal, differentiation, and low immunogenicity, have been used as living carriers. However, MSCs as gaseous signaling molecule (GSM) carriers have not been reported. In this study, MSCs were genetically modified to produce mutant {beta}-galactosidase ({beta}-GALH363A). Furthermore, a new NO prodrug, 6-methyl-galactose-benzyl-oxy NONOate (MGP), was designed. MGP can entrance into cell and selectively trigger the NO release from genetically engineered MSCs (eMSCs) in the presence of {beta}-GALH363A. Moreover, our results revealed that eMSCs can release NO when MGP is systemically administered in a mouse model of acute kidney injury (AKI), which can achieve NO release in a precise spatiotemporal manner and augment the therapeutic efficiency of MSCs. This eMSC and NO prodrug system provides a unique and tunable platform for GSM delivery and holds promise for regenerative therapy by enhancing the therapeutic efficiency of stem cells. SignificanceGaseous signaling molecules (GSMs), such as nitric oxide (NO), hold tremendous potential in regenerative medicine and tissue engineering. However, the delivery of GSMs with stem cells to target tissues or organs in response to demand has never been reported. This study designed engineered mesenchymal stem cells (eMSCs) that produce mutant {beta}-galactosidase ({beta}-GALH363A) and trigger NO release when the NO prodrug is systemically administered, which can achieve NO release in a precise spatiotemporal manner and augment the therapeutic efficiency of MSCs in a mouse model of acute kidney injury (AKI). This eMSC and NO prodrug system provides a unique and tunable platform for GSM delivery and holds promise for regenerative therapy by enhancing the therapeutic efficiency of stem cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/519950v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1d8f4e4org.highwire.dtl.DTLVardef@b5e548org.highwire.dtl.DTLVardef@1dff1edorg.highwire.dtl.DTLVardef@184130e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Discovery and biological evaluation of a potent small molecule CRM1 inhibitor for its selective ablation of extranodal NK/T cell lymphoma

BackgroundThe overactivation of NF-{kappa}B signaling is a key hallmark for the pathogenesis of extranodal natural killer/T cell lymphoma (ENKTL), a very aggressive subtype of non-Hodgkins lymphoma yet with rather limited control strategies. Previously, we found that the dysregulated exportin-1 (also known as CRM1) is mainly responsible for tumor cells to evade apoptosis and promote tumor-associated pathways such as NF-{kappa}B signaling. MethodsHerein we reported the discovery and biological evaluation of a potent small molecule CRM1 inhibitor, LFS-1107. We validated that CRM1 is a major cellular target of LFS-1107 by biolayer interferometry assay (BLI) and the knockdown of CRM1 conferred tumor cells with resistance to LFS-1107. ResultsWe found that LFS-1107 can strongly suppresses the growth of ENKTL cells at low-range nanomolar concentration yet with minimal effects on human platelets and healthy peripheral blood mononuclear cells. Treatment of ENKTL cells with LFS-1107 resulted in the nuclear retention of IkB and consequent strong suppression of NF-{kappa}B transcriptional activities, NF-{kappa}B target genes downregulation and attenuated tumor cell growth and proliferation. Furthermore, LFS-1107 exhibited potent activities when administered to immunodeficient mice engrafted with human ENKTL cells. ConclusionsTherefore, LFS-1107 holds great promise for the treatment of ENKTL and may warrant translation for use in clinical trials.

pharmacology and toxicology↗

bioRxiv2022

Small Molecule Screen Identifies Pyrimethamine as an Inhibitor of NRF2-driven Esophageal Hyperplasia

ObjectiveNRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2high ESCC. DesignWe performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays. ResultsUsing high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30mg/kg/day, p.o.) suppressed the NRF2high esophageal phenotype with no observed toxicity. ConclusionWe have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic as a radiation and chemotherapy sensitizer for NRF2high ESCC. SummaryO_ST_ABSWhat is already known on this topic - summarise the state of scientific knowledge on this subject before you did your study and why this study needed to be doneC_ST_ABSO_LIMutational activation of the NRF2 transcription factor drives ESCC progression and therapeutic resistance. Targeted therapies to block NRF2 have not yet been realized, despite great needs. C_LI What this study adds - summarise what we now know as a result of this study that we did not know beforeO_LIA screen of >35,000 small molecules identified eleven potential NRF2 inhibitors. Pyrimethamine and mitoxantrone were validated to inhibit the expression of NRF2 and NQO1 in human ESCC cells in both dose- and time-dependent manners. C_LIO_LIPyrimethamine enhanced NRF2 protein ubiquitination and degradation, resulting a decreased half-life. C_LIO_LIA genetically modified mouse model was established to express the Nrf2E79Q mutant allele in the mouse esophageal epithelium upon tamoxifen induction. Pyrimethamine suppressed the NRF2high esophageal phenotype induced by the mutant allele. C_LI How this study might affect research, practice or policy - summarise the implications of this studyO_LIAs an FDA-approved drug, Pyrimethamine has the potential for immediate translation to a clinical trial on NRF2high ESCC in humans. C_LIO_LIFurther exploration of its mechanisms of action may lead to more potent NRF2 inhibitors for future use. C_LI

cancer biology↗

bioRxiv2022

VPS35 and α-Synuclein Fail to Interact to Modulate Neurodegeneration in Rodent Models of Parkinson's Disease

Mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene cause late-onset, autosomal dominant Parkinsons disease (PD), with a single missense mutation (Asp620Asn, D620N) known to segregate with disease in families with PD. The VPS35 gene encodes a core component of the retromer complex, involved in the endosomal sorting and recycling of transmembrane cargo proteins. VPS35-linked PD is clinically indistinguishable from sporadic PD, although it is not yet known whether VPS35-PD brains exhibit -synuclein-positive brainstem Lewy pathology that is characteristic of sporadic cases. Prior studies have suggested a functional interaction between VPS35 and the PD-linked gene product -synuclein in lower organisms, where VPS35 deletion enhances -synuclein-induced toxicity. In mice, VPS35 overexpression is reported to rescue hippocampal neuronal loss in human -synuclein transgenic mice, potentially suggesting a retromer deficiency in these mice. Here, we employ multiple well-established genetic rodent models to explore a functional or pathological interaction between VPS35 and -synuclein in vivo. We find that endogenous -synuclein is dispensable for nigrostriatal pathway dopaminergic neurodegeneration induced by the viral-mediated delivery of human D620N VPS35 in mice, suggesting that -synuclein does not operate downstream of VPS35. We next evaluated retromer levels in affected brain regions from human A53T--synuclein transgenic mice, but find normal levels of the core subunits VPS35, VPS26 or VPS29. We further find that heterozygous VPS35 deletion fails to alter the lethal neurodegenerative phenotype of these A53T--synuclein transgenic mice, suggesting the absence of retromer deficiency in this PD model. Finally, we explored the neuroprotective capacity of increasing VPS35 expression in a viral-based human wild-type -synuclein rat model of PD. However, we find that the overexpression of wild-type VPS35 is not sufficient for protection against -synuclein-induced nigral dopaminergic neurodegeneration, -synuclein pathology and reactive gliosis. Collectively, our data suggest a limited interaction of VPS35 and -synuclein in neurodegenerative models of PD, and do not provide support for their interaction within a common pathophysiological pathway.

neuroscience↗

bioRxiv2022

Phase separation-mediated actin bundling by the postsynaptic density condensates

The volume and the electric strength of an excitatory synapse is near linearly correlated with the area of its postsynaptic density (PSD). Extensive research in the past have revealed that the PSD assembly directly communicates with actin cytoskeleton in the spine to coordinate activity-induced spine volume enlargement as well as long-term stable spine structure maintenance. However, the molecular mechanism underlying the communication between the PSD assembly and spine actin cytoskeleton is poorly understood. In this study, we discover that in vitro reconstituted PSD condensates can promote actin polymerization and filamentous actin bundling without help of any actin regulatory proteins. The Homer scaffold within the PSD condensates and a positively charged actin binding surface of the Homer EVH1 domain are essential for the PSD condensate-induced actin bundle formation in vitro and for spine growth in neurons. Homer-induced actin bundling can only occur when Homer forms condensates with other PSD scaffold proteins such as Shank and SAPAP. The PSD-induced actin bundle formation is sensitively regulated by CaMKII or by the product of the immediate early gene Homer1a. Thus, the communication between PSD and spine cytoskeleton may be modulated by targeting the phase separation of the PSD condensates.

biochemistry↗

bioRxiv2022

The RNA binding proteins hnRNP H and F regulate splicing of a MYC dependent HRAS exon in Prostate Cancer Cells

The Myc proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by Myc transcriptionally upregulates the core pre-mRNA splicing machinery and causes mis-regulation of alternative splicing. However, our understanding of how splicing changes are directed by Myc is limited. We performed a signaling pathway-guided splicing analysis to identify Myc dependent splicing events. These included an HRAS cassette exon repressed by Myc across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of hnRNP H gene expression with Myc hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, hnRNP F expression showed a positive correlation with Myc hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal new mechanisms for Myc-dependent regulation of splicing, and point to new possible therapeutic targets in prostate cancers. SIGNIFICANCE STATMENTMyc Transformation by the proto-oncogene c-Myc causes dysregulation of the pre-mRNA splicing reaction in cancer, but it is not known how mRNA isoform changes are directed by Myc. Here, we use bioinformatics to identify a splicing event in another proto-oncogene, HRAS, that is regulated by Myc across multiple tumor types. We identify new splicing regulators, hnRNPs H and F, that control this HRAS exon by binding to enhancer elements within its downstream intron. Additional pan-cancer bioinformatic analyses show hnRNP H expression to be anti- correlated with Myc hallmarks, consistent with the reduced splicing of the HRAS exon in Myc driven cancer. These findings uncover new mechanisms by which Myc can alter splicing in cancer cells and provide new molecular targets for potential therapeutics.

molecular biology↗

bioRxiv2022

ExposomeX: Integrative Exposomic Platform Expediates Discovery of "Exposure-Biology-Disease" Nexus

Exposome has become the hotspot of next-generation health studies. To date, there is no available effective platform to standardize the analysis of exposomic data. In this study, we aim to propose one new framework of exposomic analysis and build up one novel integrated platform "ExposomeX" to expediate the discovery of the "Exposure-Biology-Disease" nexus. We have developed 13 standardized modules to accomplish six major functions including statistical learning (E-STAT), exposome database search (E-DB), mass spectrometry data processing (E-MS), meta-analysis (E-META), biological link via pathway integration and protein-protein interaction (E-BIO) and data visualization (E-VIZ). Using ExposomeX, we can effectively analyze the multiple-dimensional exposomics data and investigate the "Exposure-Biology-Disease" nexus by exploring mediation and interaction effects, understanding statistical and biological mechanisms, strengthening prediction performance, and automatically conducting meta-analysis based on well-established literature databases. The performance of ExposomeX has been well validated by re-analyzing two previous multi-omics studies. Additionally, ExposomeX can efficiently help discover new associations, as well as relevant in-depth biological pathways via protein-protein interaction and gene ontology network analysis. In sum, we have proposed a novel framework for standardized exposomic analysis, which can be accessed using both R and online interactive platform (http://www.exposomex.cn/).

bioinformatics↗