OBJECTIVES: To investigate the mechanism of DDX17 for regulating proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) during the development of pulmonary hypertension (PH). METHODS: In murine PASMCs cultured under normoxic or hypoxic conditions, the effects of transfection with si-Ddx17 and insulin treatment, alone or in combination, on cell proliferation and migration were evaluated using Ki-67 immunofluorescence staining, scratch assay and Transwell assay. Western Blotting was performed to detect the changes in protein expression levels of DDX17, 4EBP1, S6, p-4EBP1, and p-S6. In a mouse model of PH induced by intraperitoneal injection of monocrotaline (MCT), the changes in pulmonary vasculature were examined using HE staining following tail vein injection of AD-Ddx17i. RESULTS: The PASMCs in hypoxic culture exhibited significantly enhanced cell proliferation and migration and protein expressions of p-4EBP1 and p-S6, and these changes were obviously reversed by transfection with si-Ddx17. Treatment with insulin significantly attenuated the effect of si-Ddx17 against hypoxic exposure-induced changes in PASMCs. In the mouse model of MCT-induced PH, transfection with AD-Ddx17i obviously alleviated pulmonary vascular stenosis and intimal hyperplasia. CONCLUSIONS The expression of DDX17 is elevated in hypoxia-induced PASMCs and PH mice, and silencing DDX17 significantly inhibits PASMC proliferation and migration in vitro and pulmonary vascular remodeling in PH mice by reducing mTORC1 activity.
Animals ; Cell Proliferation ; Cell Movement ; DEAD-box RNA Helicases/metabolism* ; Myocytes, Smooth Muscle/metabolism* ; Mice ; Pulmonary Artery/cytology* ; Hypertension, Pulmonary/metabolism* ; Mechanistic Target of Rapamycin Complex 1 ; Cells, Cultured ; Muscle, Smooth, Vascular/cytology*

Loss of protein homeostasis is a hallmark of cellular senescence, and ribosome pausing plays a crucial role in the collapse of proteostasis. However, our understanding of ribosome pausing in senescent cells remains limited. In this study, we utilized ribosome profiling and G-quadruplex RNA immunoprecipitation sequencing techniques to explore the impact of RNA G-quadruplex (rG4) on the translation efficiency in senescent cells. Our results revealed a reduction in the translation efficiency of rG4-rich genes in senescent cells and demonstrated that rG4 structures within coding sequence can impede translation both in vivo and in vitro. Moreover, we observed a significant increase in the abundance of rG4 structures in senescent cells, and the stabilization of the rG4 structures further exacerbated cellular senescence. Mechanistically, the RNA helicase DHX9 functions as a key regulator of rG4 abundance, and its reduced expression in senescent cells contributing to increased ribosome pausing. Additionally, we also observed an increased abundance of rG4, an imbalance in protein homeostasis, and reduced DHX9 expression in aged mice. In summary, our findings reveal a novel biological role for rG4 and DHX9 in the regulation of translation and proteostasis, which may have implications for delaying cellular senescence and the aging process.
G-Quadruplexes ; Cellular Senescence ; Ribosomes/genetics* ; Humans ; Animals ; Mice ; DEAD-box RNA Helicases/genetics* ; Protein Biosynthesis ; RNA/chemistry* ; Neoplasm Proteins

OBJECTIVES: To investigate the regulatory mechanism of aurora kinase B (AURKB) for promoting malignant phenotype of osteosarcoma cells. METHODS: HA-Vector or HA-AURKB was transfected in 293T cells to identify the molecules interacting with AURKB using immunoprecipitation combined with liquid chromatography-tandem mass spectrometry followed by verification with co-immunoprecipitation and Western blotting. In cultured osteosarcoma cells with lentivirus-mediated RNA interference of AURKB or DHX9 or their overexpression, the changes in cell proliferation, migration, and invasion activities were observed with EDU and Transwell assays. Mechanistic analysis was performed using Co-IP and in vivo ubiquitination experiments to detect the interaction between AURKB and DHX9 and the phosphorylation and ubiquitination levels of DHX9. Western blotting was used to detect the effect of AURKB and DHX9 on activation of nuclear factor-κB (NF-κB) signaling. RESULTS: AURKB was highly expressed in osteosarcoma cell lines, and in osteosarcoma 143B cells, AURKB silencing significantly reduced cell proliferation, migration and invasion abilities. Interactions between AURKB and DHX9 were detected, and they were both highly expressed in osteosarcoma tissues; silencing AURKB reduced the protein expression of DHX9, and AURKB overexpression increased DHX9 phosphorylation. Silencing AURKB did not significantly affect the transcription and translation of DHX9 but accelerated its degradation and ubiquitination. Overexpression of DHX9 effectively reversed the effects of AURKB silencing on IKBα protein and phosphorylated p65, promoted nuclear translocation of p65 to activate the NF-κB signaling pathway, and enhanced the proliferation, migration, and invasion abilities of cultured osteosarcoma cells. CONCLUSIONS AURKB overexpression promotes the malignant phenotype of osteosarcoma cells by activating the NF-κB signaling pathway via regulating DHX9.
Humans ; Osteosarcoma/genetics* ; Cell Proliferation ; NF-kappa B/metabolism* ; Signal Transduction ; Cell Line, Tumor ; Cell Movement ; DEAD-box RNA Helicases/genetics* ; Aurora Kinase B/genetics* ; Phenotype ; Bone Neoplasms/genetics* ; Neoplasm Invasiveness ; Phosphorylation ; Neoplasm Proteins

We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P < 0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability (P < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.
Humans ; HEK293 Cells ; CRISPR-Cas Systems ; DEAD Box Protein 58/metabolism* ; Signal Transduction ; Receptors, Immunologic/metabolism* ; Gene Knockout Techniques ; Transfection ; DEAD-box RNA Helicases/metabolism* ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Interferon-Induced Helicase, IFIH1/metabolism* ; Transcription Factor RelA/metabolism* ; Interferon-beta/metabolism*

Humans ; Mutation ; Sarcoma/genetics* ; Ribonuclease III/genetics* ; DEAD-box RNA Helicases/genetics*

A complete proteomics study characterizing active androgen receptor (AR) complexes in prostate cancer (PCa) cells identified a diversity of protein interactors with tumorigenic annotations, including known RNA splicing factors. Thus, we chose to further investigate the functional role of AR-mediated alternative RNA splicing in PCa disease progression. We selected two AR-interacting RNA splicing factors, Src associated in mitosis of 68 kDa (SAM68) and DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5) to examine their associative roles in AR-dependent alternative RNA splicing. To assess the true physiological role of AR in alternative RNA splicing, we assessed splicing profiles of LNCaP PCa cells using exon microarrays and correlated the results to PCa clinical datasets. As a result, we were able to highlight alternative splicing events of clinical significance. Initial use of exon-mini gene cassettes illustrated hormone-dependent AR-mediated exon-inclusion splicing events with SAM68 or exon-exclusion splicing events with DDX5 overexpression. The physiological significance in PCa was investigated through the application of clinical exon array analysis, where we identified exon-gene sets that were able to delineate aggressive disease progression profiles and predict patient disease-free outcomes independently of pathological clinical criteria. Using a clinical dataset with patients categorized as prostate cancer-specific death (PCSD), these exon gene sets further identified a select group of patients with extremely poor disease-free outcomes. Overall, these results strongly suggest a nonclassical role of AR in mediating robust alternative RNA splicing in PCa. Moreover, AR-mediated alternative spicing contributes to aggressive PCa progression, where we identified a new subtype of lethal PCa defined by AR-dependent alternative splicing.
Humans ; Male ; Alternative Splicing ; Cell Line, Tumor ; DEAD-box RNA Helicases/metabolism* ; Disease Progression ; Gene Expression Regulation, Neoplastic ; Prostatic Neoplasms/pathology* ; Receptors, Androgen/metabolism* ; RNA Splicing Factors/metabolism*

Myelodysplastic syndrome (MDS) are a heterogeneous group of hematological malignancies. Currently, in addition to demethylated chemotherapy and hematopoietic stem cell transplantation, MDS patient-derived mesenchymal stem cells (MDS-MSC) play an important role in understanding the pathogenesis of MDS and related therapeutic targets. For example, abnormal expression of DICER1 gene, abnormalities of PI3K/AKT and Wnt/β-catenin signaling pathways provide new therapeutic targets for MDS. In addition, MDS-MSC is also affected by abnormal microenvironment of the body, such as inflammatory factor S100A9, as well as hypercoagulation and iron overload. In this review, genes, signaling pathways, cytokines, hematopoietic microenvironment, and the effect of therapeutic drugs for MDS-MSC were briefly summarized.
Cytokines/metabolism* ; DEAD-box RNA Helicases/metabolism* ; Hematologic Neoplasms/metabolism* ; Humans ; Mesenchymal Stem Cells ; Myelodysplastic Syndromes/genetics* ; Phosphatidylinositol 3-Kinases/metabolism* ; Ribonuclease III/metabolism* ; Tumor Microenvironment

OBJECTIVE: To analyze the clinical characteristics and genetic variant of a child featuring X-linked mental retardation. METHODS: Whole exome sequencing and Sanger sequencing were used for the detection of variant and pedigree validation, respectively. Clinical manifestation of patients with DDX3X gene variants were also reviewed. RESULTS: The child was found to harbor a heterozygous NM_001193416.3: c.1332_1333delCT (p.Leu445Serfs*19) variant of the DDX3X gene. The same variant was not found in either of her parents. CONCLUSION The child was diagnosed with X-linked mental retardation due to variant of the DDX3X gene. Above finding has enriched the spectrum of DDX3X gene variants and provided a basis for clinical diagnosis and prenatal diagnosis for this pedigrees.
Child ; DEAD-box RNA Helicases/genetics* ; Female ; Heterozygote ; Humans ; Intellectual Disability/genetics* ; Mental Retardation, X-Linked/genetics* ; Mutation ; Pedigree ; Pregnancy ; Exome Sequencing

OBJECTIVE: To develop a multiplex PCR method for a rapid detection of Y chromosome-specific sequences in patients with Turner syndrome. METHODS: Nine genes were selected from various regions of the Y chromosome for designing the primers, which included SRY, TBL1Y, TSPY on the short arm of the Y chromosome, DDX3Y, HSFY1, RPS4Y2 and CDY1 on the long arm of Y chromosome and SHOX in the short arm and SPRY3 in the long arm of the pseudoautosomal region (PAR) of X and Y chromosomes. A multiplex PCR method for the nine genes in Y chromosome was established and optimized. The sensitivity was tested by using different amounts of genomic DNA. A total of 36 patients with Turner syndrome and a patient with male dwarfism with karyotype of 46, X, +mar were examined by the multiplex PCR method for the existence of materials from the Y chromosome. RESULTS: The optimization results of the multiplex PCR reaction system (50 μL) showed that when the final concentration of upstream and downstream of each pair of primers was 0.1 μM, the multiplex PCR reaction of the 9 pairs of primers clearly amplified the target with the expected band size, and there was no non-specific amplification. The bands were clearly visible when the amount of genomic DNA in the multiple PCR reaction system was as low as 1 ng. By using the method, we have examined the 36 patients with Turner syndrome. One patient with Turner syndrome with karyotype of 45,X[40]/47XYY[21] amplified specific seven genes on Y chromosome, 35 patients with Turner syndrome amplified only two target genes SHOX and SPRY3, but not the other seven specific genes on the Y chromosome, which was in keeping with the clinical manifestations of such patients. CONCLUSION This study established a multiplex PCR reaction system with nine genes, which can quickly and accurately screen Y chromosome materials in patients with Turner syndrome. It has the advantages of low cost, simple operation, high specificity and rapid turn-around time, and can be used to detect Turner syndrome patients with Y chromosome material in time. The method has provided a diagnostic basis for preventive gonad resection to prevent malignant gonadal tumors.
Humans ; Male ; Turner Syndrome/genetics* ; Multiplex Polymerase Chain Reaction ; Y Chromosome ; Karyotyping ; DNA Primers ; DNA ; Chromosomes, Human, Y/genetics* ; Transducin/genetics* ; Minor Histocompatibility Antigens ; DEAD-box RNA Helicases/genetics*

OBJECTIVE: To explore the effective components of Yiqi Jiedu recipe and the main biological processes and signal pathways involved in the therapeutic mechanism of the recipe in treatment of primary liver cancer through network pharmacology and molecular docking approaches. METHODS: TCMSP, Uniport, Genecards and String databases were searched to obtain the target genes of drugs and disease using Cytoscape 3.8.2 software. GO and KEGG enrichment analyses were performed to identify the common genes in the target genes of the drugs and disease. Using Pubcham, RCSB and Autoduck, the effective components of the drugs were connected with the final core genes. The effects of different concentrations of Yiqi Jiedu recipe on the expressions of the core genes DHX9, HNRNPK, NCL and PABPC1 in HepG2 cells were analyzed with Western blotting and real- time fluorescence quantitative PCR. RESULTS: We finally identified 8 core genes from the drug and disease targets, including DDX5, HNRNPK, PABPC1, DHX9, RPS3A, RPS3, RPL13, and NCL. GO analysis showed that these core genes were involved mainly in the biological processes of adrenaline receptor signal communication, movement of cellular or subcellular components, blood particles, adhesion class and iron ion binding. KEGG analysis showed that the Ras signaling pathway had the greatest gene enrichment. The results of molecular docking suggested that the effective components of the recipe were capable of docking with the core genes under natural conditions, and PABPC1 and stigmasterol had the highest binding energy. In HepG2 cells, treatment with 10% medicated serum for 48 h had the strongest effect on the expression of DHX9, HNRNPK, NCL and PABPC1 (P < 0.05). CONCLUSION Yiqi Jiedu recipe is capable of regulating viral expression of primary liver cancer multiple effective components that bind to DHX9, HNRNPK, NCL and PABPC1.
DEAD-box RNA Helicases ; Drugs, Chinese Herbal/therapeutic use* ; Humans ; Liver Neoplasms/drug therapy* ; Molecular Docking Simulation ; Neoplasm Proteins ; Network Pharmacology ; Ribosomal Proteins ; Signal Transduction