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Bioengineered ; 13(5): 11564-11578, 2022 May.
Article En | MEDLINE | ID: mdl-35510394

Cerebral ischemia-reperfusion damages local brain tissue and impairs brain function, but its specific pathogenesis is still uncertain. Recent studies have clarified circPUM1 is aberrantly elevated in cerebral ischemia-reperfusion injury; however, circPUM1's function in cerebral ischemia-reperfusion-induced neuronal injury remains ambiguous. The results illustrated circPUM1 and DEAD-box helicase 5 were decreased, but microRNA-340-5p was elevated in transient middle cerebral artery occlusion mice and oxygen glucose deprivation/reoxygenation-treated SH-SY5Y cells. Knockdown of circPUM1 aggravated the neuronal injury in transient middle cerebral artery occlusion mice and motivated glial cell activation, neuronal apoptosis and inflammation. Enhancing circPUM1 restrained oxygen glucose deprivation/reoxygenation-induced SH-SY5Y cell apoptosis, the release of lactate dehydrogenase and inflammatory factors, and activation of nuclear factor-kappaB pathway, while elevating microRNA-340-5p aggravated oxygen glucose deprivation/reoxygenation-induced cell damage. Functional rescue experiments exhibited that the impacts of knockdown or enhancement of circPUM1 were turned around by microRNA-340-5p downregulation and DEAD-box helicase 5 silencing, respectively. Moreover, it was demonstrated that circPUM1 competitively adsorbed microRNA-340-5p to mediate DEAD-box helicase 5. All in all, this study clarifies that circPUM1 mitigates cerebral ischemia-reperfusion-induced neuronal injury by targeting the microRNA-340-5p/DEAD-box helicase 5 axis.

Brain Ischemia , MicroRNAs , Reperfusion Injury , Animals , Apoptosis/genetics , Brain Ischemia/genetics , Brain Ischemia/metabolism , DEAD-box RNA Helicases/genetics , Glucose/metabolism , Infarction, Middle Cerebral Artery/genetics , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Oxygen , RNA, Circular/genetics , Reperfusion Injury/metabolism
J Exp Clin Cancer Res ; 41(1): 165, 2022 May 05.
Article En | MEDLINE | ID: mdl-35513835

BACKGROUND: Circular RNAs (circRNAs) are noncoding RNAs with stable structures with high expression and tissue-specific expression. Studies have shown that circRNA dysregulation is closely related to the progression of tumours. However, the function and regulatory mechanism of most circRNAs in cervical cancer are still unclear.   METHODS: CircRNAs related to cervical cancer were screened through the Gene Expression Omnibus (GEO) database. qRT-PCR was used to verify the expression of circ_0087429 in cervical cancer tissues and cells. Then, in vivo and in vitro experiments were conducted to evaluate the role of circ_0087429 in the progression of cervical cancer. The role of the circ_0087429/miR-5003-3p/osteoglycin (OGN) axis in the epithelial to mesenchymal transition (EMT) was confirmed by rescue experiments, fluorescence in situ hybridization, luciferase reporter assays, immunofluorescence staining and western blotting. The inhibitory effect of Eukaryotic initiation factor 4A-III (EIF4A3) on the biogenesis of circ_0087429 was verified by RNA immunoprecipitation (RIP) assays and qRT-PCR. RESULTS: circ_0087429 is significantly downregulated in cervical cancer tissues and cells and negatively correlated with International Federation of Gynecology and Obstetrics (FIGO) staging and lymphatic metastasis in cervical cancer patients. circ_0087429 can significantly inhibit the proliferation, migration, invasion and angiogenesis of cervical cancer in vitro and tumour growth and metastasis in vivo. OGN is significantly downregulated in cervical cancer tissues and cells. circ_0087429 can upregulate the expression of OGN by competitively binding with miR-5003-3p, thereby reversing EMT and inhibiting the progression of cervical cancer. EIF4A3 can inhibit circ_0087429 expression by binding to its flanking regions. CONCLUSIONS: As a tumour suppressor, circ_0087429 regulated by EIF4A3 can reverse EMT and inhibit the progression of cervical cancer through the miR-5003-3p/OGN axis. It is expected to become a potential target for the treatment of cervical cancer.

MicroRNAs , Uterine Cervical Neoplasms , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , DEAD-box RNA Helicases/genetics , Epithelial-Mesenchymal Transition/genetics , Eukaryotic Initiation Factor-4A/genetics , Eukaryotic Initiation Factor-4A/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , In Situ Hybridization, Fluorescence , Intercellular Signaling Peptides and Proteins/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , Up-Regulation , Uterine Cervical Neoplasms/genetics
Technol Cancer Res Treat ; 21: 15330338221090093, 2022.
Article En | MEDLINE | ID: mdl-35509211

Objectives: Bladder cancer is the fourth most common malignancy in men in the United States. Aberrant alternative splicing (AS) events are involved in the carcinogenesis, but the association between AS and bladder cancer remains unclear. This study aimed to construct an AS-based prognostic signature and elucidate the role of the tumor immune microenvironment (TIME) and the response to immunotherapy and chemotherapy in bladder cancer. Methods: Univariate Cox regression analysis was performed to detect prognosis-related AS events. The least absolute shrinkage and selection operator (LASSO) and multivariate Cox analyses were employed to build prognostic signatures. Kaplan-Meier survival analysis, multivariate Cox regression analysis, and receiver operating characteristic (ROC) curves were conducted to validate the prognostic signatures. Then, the Estimation of Stromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) and tumor immune estimation resource (TIMER) databases were searched and the single-sample gene set enrichment analysis (ssGSEA) algorithm and CIBERSORT method were performed to uncover the context of TIME in bladder cancer. The Tumor Immune Dysfunction and Exclusion (TIDE) web tool and pRRophetic algorithm were used to predict the response to immunotherapy and chemotherapy. Finally, we constructed a correlation network between splicing factors (SFs) and survival-related AS events. Results: A total of 4684 AS events were significantly associated with overall survival in patients with bladder cancer. Eight prognostic signatures of bladder cancer were established, and a clinical survival prediction model was built. In addition, the consolidated prognostic signature was closely related to immune infiltration and the response to immunotherapy and chemotherapy. Furthermore, the correlation identified EIF3A, DDX21, SDE2, TNPO1, and RNF40 as hub SFs, and function analysis found ubiquitin-mediated proteolysis is correlated most significantly with survival-associated AS events. Conclusion: Our findings highlight the prognostic value of AS for patients with bladder cancer and reveal pivotal players of AS events in the context of TIME and the response to immunotherapy and chemotherapy, which may be important for patient management and treatment.

Urinary Bladder Neoplasms , Alternative Splicing , DEAD-box RNA Helicases/genetics , DNA-Binding Proteins/genetics , Female , Humans , Immunotherapy , Male , Prognosis , RNA Splicing Factors/genetics , Tumor Microenvironment/genetics , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/therapy
Clin Transl Med ; 12(5): e881, 2022 May.
Article En | MEDLINE | ID: mdl-35604033

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), a difficult-to-treat cancer, is expected to become the second-largest cause of cancer-related deaths by 2030, while colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer deaths. Currently, there is no effective treatment for PDAC patients. The development of novel agents to effectively treat these cancers remains an unmet clinical need. FL118, a novel anticancer small molecule, exhibits high efficacy against cancers; however, the direct biochemical target of FL118 is unknown. METHODS: FL118 affinity purification, mass spectrometry, Nanosep centrifugal device and isothermal titration calorimetry were used for identifying and confirming FL118 binding to DDX5/p68 and its binding affinity. Immunoprecipitation (IP), western blots, real-time reverse transcription PCR, gene silencing, overexpression (OE) and knockout (KO) were used for analysing gene/protein function and expression. Chromatin IP was used for analysing protein-DNA interactions. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromid assay and human PDAC/CRC cell/tumour models were used for determining PDAC/CRC cell/tumour in vitro and in vivo growth. RESULTS: We discovered that FL118 strongly binds to dephosphorylates and degrades the DDX5 oncoprotein via the proteasome degradation pathway without decreasing DDX5 mRNA. Silencing and OE of DDX5 indicated that DDX5 is a master regulator for controlling the expression of multiple oncogenic proteins, including survivin, Mcl-1, XIAP, cIAP2, c-Myc and mutant Kras. Genetic manipulation of DDX5 in PDAC cells affects tumour growth. PDAC cells with DDX5 KO are resistant to FL118 treatment. Our human tumour animal model studies further indicated that FL118 exhibits high efficacy to eliminate human PDAC and CRC tumours that have a high expression of DDX5, while FL118 exhibits less effectiveness in PDAC and CRC tumours with low DDX5 expression. CONCLUSION: DDX5 is a bona fide FL118 direct target and can act as a biomarker for predicting PDAC and CRC tumour sensitivity to FL118. This would greatly impact FL118 precision medicine for patients with advanced PDAC or advanced CRC in the clinic. FL118 may act as a 'molecular glue degrader' to directly glue DDX5 and ubiquitination regulators together to degrade DDX5.

Carcinoma, Pancreatic Ductal , Colorectal Neoplasms , Pancreatic Neoplasms , Animals , Benzodioxoles , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Humans , Indolizines , Oncogene Proteins/metabolism , Oncogene Proteins/therapeutic use , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Survivin/genetics , Survivin/metabolism , Survivin/therapeutic use
J Exp Clin Cancer Res ; 41(1): 164, 2022 May 05.
Article En | MEDLINE | ID: mdl-35509064

BACKGROUND: Circular RNAs (circRNAs) function as crucial regulators in multiple cancers, including hepatocellular carcinoma (HCC). However, the roles of circRNAs in HCC remains largely unknown. METHODS: circTOLLIP was identified in HCC by screening of two public circRNA microarray datasets and detected in HCC cells and tissues through quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). Gain- and loss-of-function assays were performed to confirm the biological effects of circTOLLIP on HCC in vitro and in vivo. Mechanistically, bioinformatics analysis of online databases, MS2-RNA pulldown, biotin-labeled circTOLLIP/miR-516a-5p RNA pulldown, RNA immunoprecipitation (RIP), luciferase reporter assay, fluorescence in situ hybridization assay (FISH) and RNA sequencing were used to confirm the regulation of Eukaryotic initiation factor 4A3 (EIF4A3) on circTOLLIP and the interaction among circTOLLIP, miR-516a-5p and PBX homeobox 3 (PBX3). RESULTS: circTOLLIP was significantly upregulated in HCC cells and tissues. High circTOLLIP expression was correlated with poor overall survival (OS) and disease-free survival (DFS) in patients. circTOLLIP promoted the proliferation and metastasis of HCC cells in vitro and in vivo. Mechanistically, EIF4A3 promoted the biogenesis of circTOLLIP without affecting its stability. Moreover, circTOLLIP sponged miR-516a-5p to elevate the expression of PBX3, thereby activating the epithelial-to-mesenchymal transition (EMT) pathway and facilitating tumor progression in HCC. CONCLUSIONS: Our findings indicate that EIF4A3-induced circTOLLIP promotes the progression of HCC through the circTOLLIP/miR-516a-5p/PBX3/EMT axis.

Carcinoma, Hepatocellular , Liver Neoplasms , MicroRNAs , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation/genetics , DEAD-box RNA Helicases/genetics , Eukaryotic Initiation Factor-4A/genetics , Eukaryotic Initiation Factor-4A/metabolism , Gene Expression Regulation, Neoplastic , Humans , In Situ Hybridization, Fluorescence , Liver Neoplasms/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics
Int J Med Sci ; 19(3): 596-608, 2022.
Article En | MEDLINE | ID: mdl-35370459

Purpose: Mutations (K11E or E271K) of DEAD-box RNA helicase 24 (DDX24) were related to multi-organ venous lymphatic malformation syndrome (MOVLD). However, the relationship between these mutations and DDX24-function still remains unknown. Understanding whether K11E and E271K cause "loss-of-function" or "gain-of-function" for DDX24 is significant for related diseases. DDX24 was reported to be related to tumors closely, thus this study aims to explore how K11E and E271K affect DDX24-function in tumor proliferation. Methods: Cell lines stably expressing wild-type DDX24, K11E-DDX24, E271K-DDX24, along with vector only based on Chinese hamster ovary cells (CHO) and Balb/c tumor-bearing mice models were constructed. Then immunofluorescence staining, proliferation assay and colony formation assay in vitro and 18F-FDG PET/CT-scan were performed. Finally, the tumor tissues were collected to perform transcriptome sequencing to predict the potential mechanism. Results: Contrasted with CHO-WT-DDX24, CHO-K11E-DDX24 or CHO-E271K-DDX24 showed a decreased number of nucleoli, a slower proliferation rate and a lower colony formation rate significantly. Moreover, mice, inoculated with CHO-K11E-DDX24 or CHO-E271K-DDX24 cells, showed lower tumor formation rate, slower tumor growth rate, better prognosis, reduced standard uptake value and Ki of glucose in subcutaneous tumors. Sequencing indicated CHO-K11E-DDX24 or CHO-E271K-DDX24 caused increasing expression of TNF or chemokines and alteration in immune-related signal pathways. Conclusion: K11E or E271K mutation could lead to "loss-of-function" of DDX24 in cell proliferation and tumor bearing mice, which may be acted by non-specific immune killing to inhibit tumor growth.

DEAD-box RNA Helicases , Neoplasms , Animals , CHO Cells , Cricetinae , Cricetulus , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Mice , Mutation , Positron Emission Tomography Computed Tomography
Ren Fail ; 44(1): 137-145, 2022 Dec.
Article En | MEDLINE | ID: mdl-35392757

BACKGROUND: Sustained type I interferon (IFN) activation via Toll-like receptor (TLR) 3, 7 and 9 signaling has been reported to play a pivotal role in the development of lupus nephritis (LN). Although type I IFN activation has been shown to induce interferon-stimulated genes (ISGs) expression in systemic lupus erythematosus, the implication of ISGs expression in intrinsic glomerular cells remains largely unknown. METHODS: We treated cultured human glomerular endothelial cells (GECs) with polyinosinic-polycytidylic acid (poly IC), R848, and CpG (TLR3, TLR7, and TLR9 agonists, respectively) and analyzed the expression of DExD/H-Box Helicase 60 (DDX60), a representative ISG, using quantitative reverse transcription-polymerase chain reaction and western blotting. Additionally, RNA interference against IFN-ß or DDX60 was performed. Furthermore, cleavage of caspase 9 and poly (ADP-ribose) polymerase (PARP), markers of cells undergoing apoptosis, was examined using western blotting. We conducted an immunofluorescence study to examine endothelial DDX60 expression in biopsy specimens from patients with LN. RESULTS: We observed that endothelial expression of DDX60 was induced by poly IC but not by R848 or CpG, and RNA interference against IFN-ß inhibited poly IC-induced DDX60 expression. DDX60 knockdown induced cleavage of caspase 9 and PARP. Intense endothelial DDX60 expression was observed in biopsy specimens from patients with diffuse proliferative LN. CONCLUSION: Glomerular endothelial DDX60 expression may prevent apoptosis, which is involved in the pathogenesis of LN. Modulating the upregulation of the regional innate immune system via TLR3 signaling may be a promising treatment target for LN.

DEAD-box RNA Helicases , Lupus Nephritis , Toll-Like Receptor 3 , Antiviral Agents , Caspase 9/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Endothelial Cells/metabolism , Humans , Interferon-beta/pharmacology , Poly I-C/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/metabolism , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism
Cell Rep ; 39(1): 110640, 2022 04 05.
Article En | MEDLINE | ID: mdl-35385737

Synthesis of ribosomes begins in the nucleolus with formation of the 90S pre-ribosome, during which the pre-40S and pre-60S pathways diverge by pre-rRNA cleavage. However, it remains unclear how, after this uncoupling, the earliest pre-60S subunit continues to develop. Here, we reveal a large-subunit intermediate at the beginning of its construction when still linked to the 90S, the precursor to the 40S subunit. This primordial pre-60S is characterized by the SPOUT domain methyltransferase Upa1-Upa2, large α-solenoid scaffolds, Mak5, one of several RNA helicases, and two small nucleolar RNA (snoRNAs), C/D box snR190 and H/ACA box snR37. The emerging pre-60S does not efficiently disconnect from the 90S pre-ribosome in a dominant mak5 helicase mutant, allowing a 70-nm 90S-pre-60S bipartite particle to be visualized by electron microscopy. Our study provides insight into the assembly pathway when the still-connected nascent 40S and 60S subunits are beginning to separate.

Ribosome Subunits, Large, Eukaryotic , Ribosomes , Saccharomyces cerevisiae Proteins , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Ribosomal/metabolism , RNA, Small Nucleolar/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
PLoS One ; 17(4): e0265742, 2022.
Article En | MEDLINE | ID: mdl-35385551

Retinitis pigmentosa (RP) is a hereditary disease affecting tens of thousands of people world-wide. Here we analyzed the effect of an amino acid substitution in the RNA helicase DHX38 (Prp16) causing RP. DHX38 has been proposed as the helicase important for the 2nd step of splicing. We showed that DHX38 associates with key splicing factors involved in both splicing steps but did not find any evidence that the RP mutations changes DHX38 interaction profile with the spliceosome. We further downregulated DHX38 and monitored changes in splicing. We observed only minor perturbations of general splicing but detected modulation of ~70 alternative splicing events. Next, we probed DHX38 function in splicing of retina specific genes and found that FSCN2 splicing is dependent on DHX38. In addition, RHO splicing was inhibited specifically by expression of DHX38 RP variant. Finally, we showed that overexpression of DHX38 promotes usage of canonical as well as cryptic 5' splice sites in HBB splicing reporter. Together, our data show that DHX38 is a splicing factor that promotes splicing of cryptic splice sites and regulate alternative splicing. We further provide evidence that the RP-linked substitution G332D modulates DHX38 splicing activity.

DEAD-box RNA Helicases , RNA Splicing Factors , Retinitis Pigmentosa , DEAD-box RNA Helicases/genetics , Humans , Mutation , RNA Splice Sites , RNA Splicing , RNA Splicing Factors/genetics , Retinitis Pigmentosa/genetics , Spliceosomes/metabolism
Bioengineered ; 13(4): 10123-10140, 2022 Apr.
Article En | MEDLINE | ID: mdl-35435126

Increasing evidence suggests that plasmacytoma variant translocation 1 (PVT1) plays a vital role in the development of multiple tumors including lung adenocarcinoma (LUAD). Eukaryotic initiation factor 4A-3 (EIF4A3) is considered a key factor in human cancers. However, the role and potential mechanism of PVT1 combined with EIF4A3 in LUAD remain unclear. This study investigated the effects and regulatory mechanisms of PVT1, EIF4A3, and circLMNB2 on the growth, migration, invasion, and epithelial-mesenchymal transition (EMT) of LUAD cells (H1299 and HCC827 cells) The expression level, diagnostic value and prognostic significance of PVT1, EIF4A3, and circLMNB2 were assessed, and enrichment analysis was performed using R package. Rescue experiments and a xenograft model were used to validate the PVT1/EIF4A3/circLMNB2 axis in LUAD. PVT1 and EIF4A3 were upregulated and indicated poor prognosis in LUAD. Knockdown of PVT1 and EIF4A3 suppressed LUAD cell proliferation, migration, invasion, and EMT. Mechanistically, PVT1 was stabilized by EIF4A3. PVT1 could recruit EIF4A3 to promote circLMNB2 expression. Rescue experiments indicated that circLMNB2 overexpression could reverse the reduced behavior caused by PVT1 or EIF4A3 knockdown. Enrichment analysis showed that PVT1/EIF4A3/circLMNB2 may regulate LUAD development by participating in ribosome biogenesis and spliceosome formation. Our findings demonstrate that PVT1/EIF4A3/circLMNB2 enhances the malignant behaviors of LUAD cells, providing a novel perspective for the clinical treatment of LUAD.

Adenocarcinoma of Lung , Lung Neoplasms , Adenocarcinoma of Lung/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Eukaryotic Initiation Factor-3/genetics , Eukaryotic Initiation Factor-3/metabolism , Eukaryotic Initiation Factor-4A/genetics , Eukaryotic Initiation Factor-4A/metabolism , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/pathology , RNA, Circular , RNA, Long Noncoding
J Neurooncol ; 157(3): 499-510, 2022 May.
Article En | MEDLINE | ID: mdl-35384518

PURPOSE: We sought to characterize clinical outcomes for adult and pediatric patients with primary CNS tumors harboring DICER1 mutations or loss of DICER1. METHODS: We conducted a retrospective cohort study of 98 patients who were treated between 1995 and 2020 for primary CNS tumors containing DICER1 mutations or loss of DICER1 on chromosome 14q, identified by targeted next generation sequencing. Kaplan-Meier plots and log rank tests were used to analyze survival. Cox proportional-hazards model was used for univariate and multivariable analyses for all-cause mortality (ACM). RESULTS: Within our cohort, the most common malignancies were grade 3/4 glioma (61%), grade 1/2 glioma (17%), and CNS sarcoma (6%). Sarcoma and non-glioma histologies, and tumors with biallelic DICER1 mutations or deletions were common in the pediatric population. Mutations occurred throughout DICER1, including missense mutations in the DexD/H-box helicase, DUF283, RNaseIIIa, and RNaseIIIb domains. For patients with grade 3/4 glioma, MGMT methylation (Hazard ratio [HR] 0.35, 95% Confidence Interval [CI] 0.16-0.73, p = 0.005), IDH1 R132 mutation (HR 0.11, 95% CI 0.03-0.41, p = 0.001), and missense mutation in the DexD/H-box helicase domain (HR 0.06, 95% CI 0.01-0.38, p = 0.003) were independently associated with longer time to ACM on multivariable analyses. CONCLUSION: DICER1 mutations or loss of DICER1 occur in diverse primary CNS tumors, including previously unrecognized grade 3/4 gliomas as the most common histology. While prior studies have described RNaseIIIb hotspot mutations, we document novel mutations in additional DICER1 functional domains. Within the grade 3/4 glioma cohort, missense mutation in the DexD/H-box helicase domain was associated with prolonged survival.

Central Nervous System Neoplasms , Glioma , Sarcoma , Adult , Central Nervous System Neoplasms/genetics , Child , DEAD-box RNA Helicases/genetics , Glioma/pathology , Humans , Mutation , Prognosis , Retrospective Studies , Ribonuclease III/genetics , Sarcoma/pathology
Stem Cell Reports ; 17(4): 879-893, 2022 Apr 12.
Article En | MEDLINE | ID: mdl-35303436

DDX41 is a tumor suppressor frequently mutated in human myeloid neoplasms, but whether it affects hematopoiesis is unknown. Using a knockout mouse, we demonstrate that DDX41 is required for mouse hematopoietic stem and progenitor cell (HSPC) survival and differentiation, particularly of myeloid lineage cells. Transplantation of Ddx41 knockout fetal liver and adult bone marrow (BM) cells was unable to rescue mice from lethal irradiation, and knockout stem cells were also defective in colony formation assays. RNA-seq analysis of Lin-/cKit+/Sca1+Ddx41 knockout cells from fetal liver demonstrated that the expression of many genes associated with hematopoietic differentiation were altered. Furthermore, differential splicing of genes involved in key biological processes was observed. Our data reveal a critical role for DDX41 in HSPC differentiation and myeloid progenitor development, likely through regulating gene expression programs and splicing.

Hematopoiesis , Hematopoietic Stem Cells , Animals , Bone Marrow Cells/metabolism , Cell Differentiation/genetics , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , Mice , Mice, Knockout
Head Neck Pathol ; 16(1): 190-199, 2022 Mar.
Article En | MEDLINE | ID: mdl-35307774

DICER1 syndrome is an autosomal dominant tumor predisposition syndrome caused by germline DICER1 mutations. In the thyroid, DICER1 syndrome is associated with early-onset multinodular goiter and thyroid carcinomas. Subsequent studies have shown that somatic DICER1 mutations, though rare, can occur in follicular-patterned thyroid tumors, such as follicular adenomas and follicular thyroid carcinomas, with a higher rate seen in pediatric follicular thyroid carcinomas and in follicular thyroid carcinomas with a macrofollicular architecture. Somatic DICER1 mutations have also been reported in pediatric papillary thyroid carcinomas lacking other alterations typically associated with thyroid tumorigenesis. Although thyroid carcinomas with underlying DICER1 mutations are usually indolent, recent studies have shown that pediatric poorly differentiated thyroid carcinoma and thyroblastoma, both aggressive tumors, also harbor DICER1 mutations. This review will discuss mechanisms of DICER1 tumorigenesis and describe thyroid tumors associated with germline and somatic DICER1 mutations.

Adenocarcinoma, Follicular , Neoplastic Syndromes, Hereditary , Thyroid Neoplasms , Adenocarcinoma, Follicular/pathology , Carcinogenesis , DEAD-box RNA Helicases/genetics , Humans , Los Angeles , Mutation , Ribonuclease III/genetics , Thyroid Cancer, Papillary , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathology
Hum Pathol ; 123: 46-58, 2022 May.
Article En | MEDLINE | ID: mdl-35245609

Many sarcomas with DICER1 pathogenic variants (PVs) exhibit a characteristic morphology, including a subepithelial layer of malignant mesenchymal cells, areas of rhabdomyoblastic differentiation and cartilaginous and/or osseous elements. We report 5 DICER1-associated neoplasms (1 moderately to poorly differentiated Sertoli Leydig cell tumour and 4 sarcomas) containing variable amounts of neuroectodermal elements. The neoplasms predominantly involved or were in close proximity to the female genital tract (ovary, uterine corpus, abdominal and pelvic cavity) and occurred in females aged 14 months to 54 years. The neuroectodermal elements were characterised by solid and tubular/rosette-like patterns and variable immunoreactivity with SALL4 and neuroendocrine markers. In some cases, the neuroectodermal component was focal while in others it was exclusive. In one case, the focal neuroectodermal component within an ovarian Sertoli Leydig cell tumour resulted in extraovarian metastasis. In reporting these cases, we suggest that neuroectodermal elements, including pure neuroectodermal tumours, are part of the morphological spectrum of DICER1-associated neoplasms. It is important that pathologists recognize that a neuroectodermal component (often admixed with other elements) may be a feature of such neoplasms. This will facilitate appropriate tumour and/or germline testing which could lead to the identification of germline DICER1 PVs (DICER1 syndrome). Three of the patients we report were subsequently shown to have a germline DICER1 PV.

Sarcoma , Sertoli-Leydig Cell Tumor , Sex Cord-Gonadal Stromal Tumors , DEAD-box RNA Helicases/genetics , Female , Humans , Male , Mutation , Ribonuclease III/genetics , Sertoli-Leydig Cell Tumor/genetics , Sertoli-Leydig Cell Tumor/pathology , Sex Cord-Gonadal Stromal Tumors/pathology
Viruses ; 14(3)2022 02 24.
Article En | MEDLINE | ID: mdl-35336874

The porcine reproductive and respiratory syndrome virus (PRRSV) remains a persistent hazard in the global pig industry. DEAD (Glu-Asp-Ala-Glu) box helicase 21 (DDX21) is a member of the DDX family. In addition to its function of regulating cellular RNA metabolism, DDX21 also regulates innate immunity and is involved in the replication cycle of some viruses. However, the relationship between DDX21 and PRRSV has not yet been explored. Here, we found that a DDX21 overexpression promoted PRRSV replication, whereas knockdown of DDX21 reduced PRRSV proliferation. Mechanistically, DDX21 promoted PRRSV replication independently of its ATPase, RNA helicase, and foldase activities. Furthermore, overexpression of DDX21 stabilized the expressions of PRRSV nsp1α, nsp1ß, and nucleocapsid proteins, three known antagonists of interferon ß (IFN-ß). Knockdown of DDX21 activated the IFN-ß signaling pathway in PRRSV-infected cells, suggesting that the effect of DDX21 on PRRSV-encoded IFN-ß antagonists may be a driving factor for its contribution to viral proliferation. We also found that PRRSV infection enhanced DDX21 expression and promoted its nucleus-to-cytoplasm translocation. Screening PRRSV-encoded proteins showed that nsp1ß interacted with the C-terminus of DDX21 and enhanced the expression of DDX21. Taken together, these findings reveal that DDX21 plays an important role in regulating PRRSV proliferation through multiple mechanisms.

Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Animals , Cell Line , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Interferon-beta/genetics , Porcine Reproductive and Respiratory Syndrome/genetics , Porcine respiratory and reproductive syndrome virus/metabolism , Swine , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/physiology
Sci Rep ; 12(1): 3967, 2022 03 10.
Article En | MEDLINE | ID: mdl-35273248

The human DEAD-box protein 3 (DDX3) has been reported as a positive regulator and functions in the induction of type I interferon signaling. We elucidated the function of DDX3 in the positive regulation of IFNB production in non-pDC cells. We found that DDX3 regulates virus-induced activation of IFNB at the level of IRF-3. However, it does not affect conventional innate signaling, including IRF-3 phosphorylation, dimerization, or nuclear translocation of IRF-3, but has some downstream events after IRF-3 phosphorylation. Co-immunoprecipitation analyses revealed that DDX3 interacts with IRF-3 through its DNA-binding domain and promotes IRF-3-mediated IFNB promoter activation. DDX3 does not affect the formation of the IRF-3/p300/CBP complex. Instead, ChIP and EMSA assay revealed that DDX3 promotes the recruitment of IRF-3 and transcriptional co-activator p300/CBP to the IFNB promoter. The ATP binding pocket of DDX3 is involved in this association and is essential for the transcriptional activation. Taken together, our study demonstrates that DDX3 plays an important role in guiding a transcription factor complex formed by antiviral signaling to the target gene promoter.

DEAD-box RNA Helicases , Interferon Regulatory Factor-3 , Cell Nucleus/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Humans , Immunoprecipitation , Interferon Regulatory Factor-3/metabolism , Promoter Regions, Genetic , Transcriptional Activation
BMC Med Genomics ; 15(1): 72, 2022 03 27.
Article En | MEDLINE | ID: mdl-35346191

BACKGROUND: Heart failure (HF) is the most common potential cause of death, causing a huge health and economic burden all over the world. So far, some impressive progress has been made in the study of pathogenesis. However, the underlying molecular mechanisms leading to this disease remain to be fully elucidated. METHODS: The microarray data sets of GSE76701, GSE21610 and GSE8331 were retrieved from the gene expression comprehensive database (GEO). After merging all microarray data and adjusting batch effects, differentially expressed genes (DEG) were determined. Functional enrichment analysis was performed based on Gene Ontology (GO) resources, Kyoto Encyclopedia of Genes and Genomes (KEGG) resources, gene set enrichment analysis (GSEA), response pathway database and Disease Ontology (DO). Protein protein interaction (PPI) network was constructed using string database. Combined with the above important bioinformatics information, the potential key genes were selected. The comparative toxicological genomics database (CTD) is used to explore the interaction between potential key genes and HF. RESULTS: We identified 38 patients with heart failure and 16 normal controls. There were 315 DEGs among HF samples, including 278 up-regulated genes and 37 down-regulated genes. Pathway enrichment analysis showed that most DEGs were significantly enriched in BMP signal pathway, transmembrane receptor protein serine/threonine kinase signal pathway, extracellular matrix, basement membrane, glycosaminoglycan binding, sulfur compound binding and so on. Similarly, GSEA enrichment analysis showed that DEGs were mainly enriched in extracellular matrix and extracellular matrix related proteins. BBS9, CHRD, BMP4, MYH6, NPPA and CCL5 are central genes in PPI networks and modules. CONCLUSIONS: The enrichment pathway of DEGs and GO may reveal the molecular mechanism of HF. Among them, target genes EIF1AY, RPS4Y1, USP9Y, KDM5D, DDX3Y, NPPA, HBB, TSIX, LOC28556 and XIST are expected to become new targets for heart failure. Our findings provide potential biomarkers or therapeutic targets for the further study of heart failure and contribute to the development of advanced prediction, diagnosis and treatment strategies.

Gene Expression Profiling , Heart Failure , Biomarkers/metabolism , Computational Biology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Gene Ontology , Gene Regulatory Networks , Heart Failure/genetics , Histone Demethylases/genetics , Histone Demethylases/metabolism , Humans , Minor Histocompatibility Antigens , Protein Interaction Maps/genetics
BMB Rep ; 55(3): 125-135, 2022 Mar.
Article En | MEDLINE | ID: mdl-35236544

Continuously renewing the proteome, translation is exquisitely controlled by a number of dedicated factors that interact with the ribosome. The RNA helicase DDX3 belonging to the DEAD box family has emerged as one of the critical regulators of translation, the failure of which is frequently observed in a wide range of proliferative, degenerative, and infectious diseases in humans. DDX3 unwinds double-stranded RNA molecules with coupled ATP hydrolysis and thereby remodels complex RNA structures present in various protein-coding and noncoding RNAs. By interacting with specific features on messenger RNAs (mRNAs) and 18S ribosomal RNA (rRNA), DDX3 facilitates translation, while repressing it under certain conditions. We review recent findings underlying these properties of DDX3 in diverse modes of translation, such as cap-dependent and cap-independent translation initiation, usage of upstream open reading frames, and stress-induced ribonucleoprotein granule formation. We further discuss how disease-associated DDX3 variants alter the translation landscape in the cell. [BMB Reports 2022; 55(3): 125-135].

DEAD-box RNA Helicases , Ribosomes , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Humans , RNA, Messenger , Ribonucleoproteins/genetics , Ribosomes/metabolism
Nucleic Acids Res ; 50(7): 3998-4011, 2022 04 22.
Article En | MEDLINE | ID: mdl-35286399

The DEAD-box protein Dbp5 is essential for RNA export, which involves regulation by the nucleoporins Gle1 and Nup159 at the cytoplasmic face of the nuclear pore complex (NPC). Mechanistic understanding of how these nucleoporins regulate RNA export requires analyses of the intrinsic and activated Dbp5 ATPase cycle. Here, kinetic and equilibrium analyses of the Saccharomyces cerevisiae Gle1-activated Dbp5 ATPase cycle are presented, indicating that Gle1 and ATP, but not ADP-Pi or ADP, binding to Dbp5 are thermodynamically coupled. As a result, Gle1 binds Dbp5-ATP > 100-fold more tightly than Dbp5 in other nucleotide states and Gle1 equilibrium binding of ATP to Dbp5 increases >150-fold via slowed ATP dissociation. Second, Gle1 accelerated Dbp5 ATPase activity by increasing the rate-limiting Pi release rate constant ∼20-fold, which remains rate limiting. These data show that Gle1 activates Dbp5 by modulating ATP binding and Pi release. These Gle1 activities are expected to facilitate ATPase cycling, ensuring a pool of ATP bound Dbp5 at NPCs to engage RNA during export. This work provides a mechanism of Gle1-activation of Dbp5 and a framework to understand the joint roles of Gle1, Nup159, and other nucleoporins in regulating Dbp5 to mediate RNA export and other Dbp5 functions in gene expression.

DEAD-box RNA Helicases , Nuclear Pore Complex Proteins/metabolism , Nucleocytoplasmic Transport Proteins , Saccharomyces cerevisiae Proteins/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Nuclear Pore Complex Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Phosphates/metabolism , RNA, Messenger/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics