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OBJECTIVES@#A study was conducted to investigate the molecular mechanism of chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) influencing the invasion and metastasis of tongue squamous cell carcinoma and to provide a new target for clinical inhibition of invasion and metastasis of tongue squamous cell carcinoma.@*METHODS@#Ualcan website was used to analyze the expression of CHD1L in normal epithelial tissue and primary head and neck squamous cell carcinoma and to analyze the effect of lymph node metastasis on the expression of CHD1L in tissues with head and neck squamous cell carcinoma. The relationship between CHD1L expression and the survival rate of patients with head and neck squamous cell carcinoma was tested by the GEPIA website. Western blot was used to quantify the levels of CHD1L protein in human tongue squamous cell carcinoma CAL27 and immortalized human skin keratinocyte cell HaCaT. After knocking down CAL27 in human tongue squamous cell carcinoma cells with an RNA interference plasmid, the cells were designated as SiCHD1L/CAL27 and Scr/CAL27. Western blot was utilized to detect the expression of CHD1L in each group of cells. The change in CAL27 cell proliferation ability was tested by EdU proliferation test after CHD1L knockdown. The change of cell migration ability of each group cells was tested through the wound healing assay. Western blot was used to detect epithelial-mesenchymal transition (EMT) marker E-cadherin and Vimentin protein expression levels.@*RESULTS@#Ualcan database showed that the expression of CHD1L in primary head and neck squamous cell carcinoma tissues was higher than in normal epithelial tissues and in head and neck squamous cell carcinoma tissues with lymph node metastasis. GEPIA website analysis showed that the overall survival rate of patients with head and neck squamous cell carcinoma with high expression of CHD1L was significantly lower than that of patients with low expression. Western blot results showed that CHD1L expression in human tongue squamous carcinoma cells CAL27 was higher than that of human normal skin cells HaCaT. CHD1L expression in SiCHD1L/CAL27 cells was much lower than that in Scr/CAL27 cells. Results of EdU proliferation experiments showed the significant reduction in the cell proliferation ability of the SiCHD1L/CAL27 cells. Results of the wound healing experiments showed the reduction in the migration capacity of the SiCHD1L/CAL27 cells. The expression of E-cadherin increased, whereas that of Vimentin decreased, in SiCHD1L/CAL27 cells.@*CONCLUSIONS@#CHD1L promoted the EMT, proliferation, migration, and invasion ability of tongue squamous cell carcinoma cells.
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Humans , Adenosine Triphosphatases , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , DNA Helicases , DNA-Binding Proteins , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms , Neoplasm Invasiveness/genetics , Tongue , Tongue Neoplasms/geneticsABSTRACT
RNA helicases, the largest family of proteins that participate in RNA metabolism, stabilize the intracellular environment through various processes, such as translation and pre-RNA splicing. These proteins are also involved in some diseases, such as cancers and viral diseases. Autophagy, a self-digestive and cytoprotective trafficking process in which superfluous organelles and cellular garbage are degraded to stabilize the internal environment or maintain basic cellular survival, is associated with human diseases. Interestingly, similar to autophagy, RNA helicases play important roles in maintaining cellular homeostasis and are related to many types of diseases. According to recent studies, RNA helicases are closely related to autophagy, participate in regulating autophagy, or serve as a bridge between autophagy and other cellular activities that widely regulate some pathophysiological processes or the development and progression of diseases. Here, we summarize the most recent studies to understand how RNA helicases function as regulatory proteins and determine their association with autophagy in various diseases.
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Recently emerged SARS-CoV-2 caused a major outbreak of coronavirus disease 2019 (COVID-19) and instigated a widespread fear, threatening global health safety. To date, no licensed antiviral drugs or vaccines are available against COVID-19 although several clinical trials are under way to test possible therapies. During this urgent situation, computational drug discovery methods provide an alternative to tiresome high-throughput screening, particularly in the hit-to-lead-optimization stage. Identification of small molecules that specifically target viral replication apparatus has indicated the highest potential towards antiviral drug discovery. In this work, we present potential compounds that specifically target SARS-CoV-2 vital proteins, including the main protease, Nsp12 RNA polymerase and Nsp13 helicase. An integrative virtual screening and molecular dynamics simulations approach has facilitated the identifi-cation of potential binding modes and favourable molecular interaction profile of corresponding com-pounds. Moreover, the identification of structurally important binding site residues in conserved motifs located inside the active site highlights relative importance of ligand binding based on residual energy decomposition analysis. Although the current study lacks experimental validation, the structural infor-mation obtained from this computational study has paved way for the design of targeted inhibitors to combat COVID-19 outbreak.
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Background: We assessed the frequency of epigenetic lesions, including lymphoid-specific helicase (LSH), 5-hydroxymethylcytosine (5-hmC) and E2F1, and the possible correlations among molecular findings, phenotype, clinical features, and outcome. Methods: We investigated 181 paraffin-embedded B-cell lymphoma samples using immunohistochemistry and in situ hybridization. Results: The levels of Ki67, LSH, 5-hmC, and E2F1 were all increased in germinal center B-cell lymphomas when compared with those in normal lymph nodes, and LSH was highly expressed in diffuse large B-cell lymphomas (DLBCLs) and Burkitt lymphomas (BLs) that were positive for Epstein-Barr virus (EBV) infection, indicating that LSH is linked to EBV infection in DLBCL and BL. Interestingly, LSH was mainly localized in the germinal centers of lymph nodes whereas 5-hmC staining localized to areas surrounding the germinal centers. Conclusions: These findings indicate a critical role for LSH as a biomarker and therapeutic target in follicular germinal center B-cell lymphoma.
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Objective To investigate the expression and prognostic significance of DEAH-box helicase (DHX16) by pan-cancer analysis. Methods The expression and prognostic significance of DHX16 were analyzed using the UALCAN web-portal. Gene ontology and kyoto encyclopedia of genes and genomes analyses of proteins interacting with DHX16 were performed using DAVID 6.8 functional annotation software. Results DHX16 was highly expressed in bladder urothelial carcinoma, head and neck squamous cell carcinoma, esophageal carcinoma, liver hepatocellular carcinoma, and cholangiocarcinoma (all P ＜ 0.001). Proteins interacting with DHX16 were located mainly in catalytic step 2 spliceosome, nucleoplasm, and cell membrane, and participated in mRNA splicing and processing, binding to poly (A) RNA and nucleic acids, and RNA helicase activity. Spliceosome, mRNA surveillance, RNA degradation, and transport pathways were implicated. Conclusion The high expression of DHX16 is helpful for the prognosis of bladder urothelial carcinoma prognosis, and unfavorable for prognoses of adrenocortical carcinoma, sarcoma, brain lower grade glioma, liver hepatocellular carcinoma, and acute myeloid leukemia. Thus, DHX16 may have value as a prognostic marker.
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; Aim To explore the anti-tumor mechanism of dihydromyricetin (DMY), a kind of flavonoid compound with anti-inflammatory and anti-tumor effects, via studying the effect of DMY on biological activities of Bloom helicase. Methods The effect of DMY on the biological activities of BLM helicase was studied by ultraviolet spectrum (UV), circular dichroism (CD), fluorescence polarization and free phosphorus detection. Results The results of CD and UV showed that DMY could bind to a site of the BLM helicase. In the concentration of DMY in 0 ~ 25 μmol . L 1 range, DMY showed a positive correlation with the interference ability of BLM helicase secondary structure with the increase of concentration, while in the concentration of DMY in 25 ~ 75 μmol . L 1 range, DMY showed a negative correlation. Fluorescence polarization and free phosphorus detection experiments showed that DMY could bind to BLM helicase, thus inhibiting the helicase activity of BLM helicase. Conclusions DMY can competitively bind to the DNA binding site of BLM helicase and change the spatial structure of BLM helicase, inhibiting the binding of BLM helicase to DNA and the biological activity of BLM helicase accordingly.
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The helicase genes and hypervariable regions (HVRs) of three avian hepatitis E viruses (HEVs) detected at three different farms were sequenced and characterized. Two isolates (DW-L and GI-B2) were classified as genotype 2 and one isolate (GR-B) was classified as genotype 1. A phylogenetic tree, based on the helicase gene and HVR nucleotide sequences, revealed the newly detected viruses and other avian HEVs were classified similarly. Unlike previously reported avian HEVs, the DW-L isolate detected in broiler breeders with characteristic lesions of avian HEV had no prolinerich motif in its HVR, suggesting that the proline-rich motif is non-essential for viral replication and infection.
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Agriculture , Base Sequence , Genotype , Hepevirus , Korea , TreesABSTRACT
Objective To investigate whether Zika virus NS3 has ATP hydrolase and DNA helicase activity. Methods Zika NS3 with His tag was expressed in E.coli BL21 and purified by Nicke lagarose beads.ATP hy-drolysis assay was performed to examinethe ATPase activity of NS3.The dsDNA unwinding activity of NS3 was detected according to the principle of fluorescence resonance energy transfer(FRET).NS3 G198A mutant was generated by site-directed mutagenesis and its enzymatic activity was measured subsequently.Results Zika NS3 was capable of hydrolyzing ATP in vitro[Vmax=2.76 μmol/(L· min),Km=0.11 mmol/L].Moreover, Zika NS3 could unwind dsDNA in vitro as that proved by the fluorescence was enhancement in FRET system.G198A mutation impaired NS3 enzymatic activities, including ATP hydrolysis and dsDNA unwinding activities.Conclu-sions Zika NS3 has DNA helicase activity in vitro depending on ATP hydrolysis,and the glycine 198 is impor-tant for its enzymatic activity.
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Aim To investigate the effects of bisbenzyl-isoquinoline alkaloid tetrandrine derivative HL-27 on the biological properties of the BLM642-1290 helicase. Methods Fluorescence polarization technique was used to investigate the effects of bisbenzylisoquinoline alkaloid tetrandrine derivative HL-27 on the DNA bind-ing activity and unwinding activity of the BLM642-1290 helicase. Malachite green-phosphate ammonium molyb-date colorimetry was used to investigate the effects of HL-27 on the ATPase activity of the BLM642-1290 heli-case. Ultraviolet spectral scanning was used to investi-gate the effects of HL-27 on the conformation of the BLM642-1290 helicase. Results When the concentra-tion of HL-27 reached 33.34 μmol·L-1, the inhibi-tion ratio of dsDNA and ssDNA binding activity of the BLM642-1290 helicase was 41.35% and 59.54% , re-spectively. When the concentration of HL-27 reached 50 μmol·L-1, the inhibition ratio of DNA unwinding activity of the BLM642-1290 helicase was 78.68% . When the concentration of HL-27 reached 100 μmol· L-1, the inhibition ratio of ATPase activity of the BLM642-1290 helicase was 43.8% . Conclusion The DNA binding activity, ATPase activity and unwinding activity of the BLM642-1290 helicase can be inhibited by bisbenzylisoquinoline alkaloid tetrandrine derivative HL-27.
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Objective To investigate the regulatory role of host restriction factor Moloney leukemia virus 10 (MOV10) protein in HBV replication. Methods Firstly, a HBV replication-expression plasmid was transfected into Huh7 cells to investigate the effect of HBV replication on MOV10 expression. Secondly, HBV DNA was extracted and measured by quantitative PCR ( qPCR) after knocking down the expression of endogenous MOV10 or enhancing the expression of exogenous MOV10. Furthermore, MOV10 conserved do-mainⅡenzyme active mutants (D645A, E646Q and G648A) were constructed and analyzed regarding their antiviral activities. The HBV replication plasmid and MOV10 expression plasmid were co-transfected into hu-man renal epithelial cells (HEK293) to investigate whether MOV10 could bind to HBV mRNA using RNA binding protein immunoprecipitation ( RIP) . Results The expression of MOV10 was increased after trans-fection of HBV replication plasmid into Huh7 cells. After knocking down the expression of endogenous MOV10 by siRNA in Huh7 cells, HBV replication was increased about 1. 5 times compared with control group, while the viral DNA level was significantly decreased in Huh7 cells that overexpressed MOV10. MOV10 domain Ⅱ mutants also significantly inhibited HBV replication. MOV10 could bind to 3. 5 kb HBV RNA. Conclusion In liver cancer cells, the expression of the host restriction factor MOV10 was associated with HBV replication. Its inhibitory effect against HBV replication was independent of its helicase activity, but might be associated with its binding activity with 3. 5kb HBV RNA.
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Previous studies suggested that chromodomain helicase DNA-binding proteins (CHDs), including CHD 1-8, were associated with several human diseases and cancers including lymphoma, liver cancer, colorectal cancer, stomach cancer, etc. To date, little research on CHD 9 in human cancers has been reported. In this study, we assessed the prognostic value of CHD 9 in patients with colorectal cancer (CRC). We screened for CHD 9 expression using immunohistochemical analysis in 87 surgical CRC specimens and found that the expression was upregulated in 81.5% of the cases, while 7.4% were decreased; in the remaining 11.1% of the cases, levels were not altered. Kaplan-Meier analysis showed that patients with high CHD 9 expression had better prognosis than those with low CHD 9 expression (54.5 vs 32.1%, P=0.034). Subsequently, Cox multi-factor survival regression analysis revealed that expression of CHD 9 protein was an independent predictor for CRC, with a hazard ratio of 0.503 (P=0.028). In addition, we found that CHD 9 expression was positively correlated with MSH2 (rs=0.232, P=0.036). We speculated that CHD9 might be a putative tumor suppressor gene, and could inhibit the development of CRC by participating in DNA repair processes. Our findings suggest that CHD 9 could be a novel prognostic biomarker and a therapeutic target for CRC. Further studies are needed to detect the effect of CHD 9 on cellular function and the expression of mismatch repair genes.
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Humans , Male , Adult , Middle Aged , Aged , Aged, 80 and over , Young Adult , Transcription Factors/metabolism , Colorectal Neoplasms/metabolism , Biomarkers, Tumor/metabolism , DNA-Binding Proteins/metabolism , Prognosis , Transcription Factors/genetics , Immunohistochemistry , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Biomarkers, Tumor/genetics , Gene Expression Regulation, Neoplastic , Trans-Activators , DNA Helicases , DNA-Binding Proteins/genetics , Kaplan-Meier Estimate , Neoplasm StagingABSTRACT
OBJECTIVE: To explore the effects of glycyrrhetinic acid on the proliferation, invasion, migration and apoptosis of PC3 cells and the expression of BLM and Caspase-3. METHODS: The cell proliferation, invasion, migration and apoptosis were monitored by MTT assay, Transwell, scratch and Hoechst/PI double staining method. The transcription levels of BLM and Caspase-3 gene were tested by Real-time quantitative PCR, as well as their expression levels of protein were detected by Western blot. RESULTS: MTT results revealed that the effect on cell proliferation was not significant when the concentration of glycyrrhetic acid under 0.12 mol•L-1. The proliferation of the cell was gradually decreased when the concentration of glycyrrhetic acid is greater than 0.12 mol•L-1. The results of invasion and migration of the cells showed that it is decreased obviously when the concentration of glycyrrhetinic acid was above 0.16 mol•L-1. Hoechst/PI double staining showed that the cell apoptosis was obvious when the concentration of glycyrrhetinic acid was above 0.12 mol•L-1. Fluorescence quantitative PCR results showed that the expression of the BLM gene decreased significantly and the expression of the Caspase-3 gene increased significantly in PC3 cells compared with the control group when the concentration of glycyrrhetinic acid was above 0.12 mol•L-1. Western-blot results showed that BLM protein expression was significantly reduced while the expression of the Caspase-3 protein was significantly increased when the concentration of glycyrrhetinic acid in 0.12 mol•L-1. CONCLUSION: Glycyrrhetinic acid inhibited the proliferation, invasion and migration of the PC3 cells is related to the low expression of the BLM, whereas promoted apoptosis of the PC3 cells and is related to the high expression of Caspase-3.
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Autism spectrum disorder (ASD) is a series of neurodevelopmental disorder with a large genetic component. However, the pathogenic genes and molecular mechanisms of ASD have not been clearly defined. Recent technological advancements, such as next-generation sequencing, have led to the identification of certain loci that is responsible for the pathophysiology of ASD. Three functional pathways, such as chromatin remodeling, Wnt signaling and mitochondrial dysfunction are potentially involved in ASD. In this review, we will focus on recent studies of the involvement of Wnt signaling pathway components in ASD pathophysiology and related drugs used in ASD treatment.
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Autism Spectrum Disorder , Autistic Disorder , beta Catenin , Chromatin Assembly and Disassembly , Neurodevelopmental Disorders , Wnt Signaling PathwayABSTRACT
DEAD-box family protein is a kind of ATP dependent RNA helicase,which plays a critical role in RNA metabolism.The DEAD-box family proteins can affect cell proliferation,differentiation,and apoptosis through regulating the expression of oncogenes,tumor suppressor genes and tumor related signaling pathways.It plays the role in promoting or suppressing cancer.
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Objective To observe the effect of PIF1 knockdown on cell growth and cell cycle arrest induced by ionizing radiation.Methods HeLa cell lines that consistently down-regulated PIF1 were prepared by the lentivirus granules interfering technology and confirmed by real-time PCR and Western blotting.The effect of down-regulation of PIF1 on cell growth and cell cycle arrest induced by ionizing radiation was evaluated by cell counting and flow cytometry.Results HeLa cell lines consistently down-regulating PIF1 were established.The growth of HeLa that down-regulated PIF1 was inhibited greatly after 4 Gy of γ-ray irradiation.There was little cell proliferation until the 5th day post 4 Gy γ-ray.Moreover, the S phase block and G2/M phase block of PIF1 knock-downed cell lines were significantly delayed after 8 Gy γ-ray irradiation.Conclusion Knockdown of PIF1 can significantly enhance the radiation sensitivity and delayes the S phase block and G 2 /M phase block induced by ionizing radiation.
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<p><b>PURPOSE</b>RNA helicase p68 plays an important role in organ development and maturation through tuning cell proliferation. However, the character and role of p68 in the whole wound healing process need more study.</p><p><b>METHODS</b>First, we characterize expression of p68 in normal rat skin development postnatal. Then, we assayed dynamic change of p68 in rat skin from different stage after injury, and explored the role of p68 in proliferation and migration of three types of wound healing related cells.</p><p><b>RESULTS</b>p68 was down-regulated during skin developmental and maturation process, up-regulated after wound, peaked on day 14 and then significantly decreased. Wound fluid enhanced wound healing related cell proliferation and up-regulated expression of p68. Conversely, reducing p68 expression by RNA interference resulted in significantly slower proliferation and migration.</p><p><b>CONCLUSION</b>Our results define an important role of RNA helicase p68 in skin wound healing process.</p>
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Objective To explore the inhibitory effect of down-regulating Bloom helicase gene using RNA interference (RNAi) vectors on prostate cancer PC3 cells. Methods The BLM-RNAi vectors constructed previously in our lab were transfected into prostate cancer PC3 cells, and the proliferation ability of PC3 cells was detected with MTT at 24 h, 48 h and 72 h after transfection of recombinant vectors. At 48 h, the invasion and migration ability was detected with Transwell chamber assay, migration distance was examined with cell scratch test, and cell apoptosis was detected with Hoechst/PI. Results The proliferation ability of cells after transfection was significantly weaker than that in the blank control group (P<0.05), the number of cells across Transwell membrane after transfection was significantly less than that in the blank control group (invasion ability: 119±24, 118±30 vs 227±38, P<0.05; migration ability: 122±13, 121±47 vs 277±32, P<0.05), cell scratch test also showed that the wound healig rate and cell migration distance in the experimental group were significantly lower than those in the blank control group (P<0.05), and cell apoptosis in the experimental group was more notable compared with the blank control group. Conclusion RNAi vectors targeting Bloom helicase gene can inhibit the proliferation, migration and invasion ability of prostate cancer PC3 cells, and increase the cell apoptosis, which may cast new lights on the gene therapy of prostate cancer.
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Objective To explore the associations between interferon induced helicase C domain 1 (IFIH1) gene single nucleotide polymorphism (SNP) rs1990760 C >T and polymyositis/dermatomyositis (PM/DM) in Chinese Han patients from Jiangsu area. Methods A total of 183 PM/DM patients and 400 healthy controls were included. SNP typing was performed by Taqman MGB probe method. The distributions of genotypes and alleles and Hardy-Weinberg equilibrium (HWE) were examined by χ2 tests or Fisherˊs exact tests. Results The distribution of IFIH1 rs1990760 genotypes of all patients group and the control group were consistent with HWE. The frequencies of T and C alleles were not significantly different between the PM/DM group (20.8% and 79.2%, respectively), the DM group (20.9% and 79.1%, respectively), the PM group (20.5%and 79.5%, respectively) and control group (18.0%and 82.0%, respectively). In recessive model (TT vs CT+CC) and additive model (TT vs CC), the frequencies of TT genotype in PM/DM group (6.6% vs 2.8%; χ2=4.8 and 4.54, respectively; both P=0.03) and in DM with interstitial lung disease (ILD) group (8.3% vs 2.8%;χ2=4.84 and 4.41, respectively; P=0.03 and 0.04, respectively) were significantly higher than those in the control group.In recessive model (TT vs CT+CC), the frequency of TT genotype in PM without ILD group was significantly higher than that in the control group (9.8% vs 2.8%, χ2=5.56, P=0.04). The genotype and allele distributions of rs1990760 were not significantly different between the anti-MDA5 positive group and control group. Conclusion The rs1990760 TT genotype of IFIH1 gene may be associated with the susceptibility of PM/DM in Jiangsu Han Chinese population, and the associations may differ based on the ILD status.
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The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn(2+) and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn(2+). The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.
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Crystallography, X-Ray , Protein Domains , RNA Helicases , Chemistry , RNA, Viral , Chemistry , Viral Proteins , Chemistry , Zika VirusABSTRACT
DEAD/DExH-box RNA helicases catalyze the folding and remodeling of RNA molecules in prokaryotic and eukaryotic cells, as well as in many viruses. They are characterized by the presence of the helicase domain with conserved motifs that are essential for ATP binding and hydrolysis, RNA interaction, and unwinding activities. Large families of DEAD/DExH-box proteins have been described in different organisms, and their role in all molecular processes involving RNA, from transcriptional regulation to mRNA decay, have been described. This review aims to summarize the current knowledge about DEAD/DExH-box proteins in selected protozoan and nematode parasites of medical importance worldwide, such as Plasmodium falciparum, Leishmania spp., Trypanosoma spp., Giardia lamblia, Entamoeba histolytica, and Brugia malayi. We discuss the functional characterization of several proteins in an attempt to understand better the molecular mechanisms involving RNA in these pathogens. The current data also highlight that DEAD/DExH-box RNA helicases might represent feasible drug targets due to their vital role in parasite growth and development.