Phage immunoprecipitation sequencing (PhIP-Seq) is a high-throughput and low-cost method for analyzing the specific binding of target proteins to peptide libraries. The method uses oligonucleotide library synthesis (OLS) to encode proteome-scale peptide libraries for display on phages, and then immunoprecipitates these library phages with target proteins (such as antibodies) for subsequent analysis by high-throughput DNA sequencing. PhIP-Seq enables the screening of peptide targets that react specifically with hundreds of proteins or pathogens. PhIP-Seq has been successfully applied in various fields such as disease detection, screening of autoimmune disease biomarkers, vaccine development, and allergen detection, becoming a high-throughput diagnostic technology. This article systematically describes the development, applications, and result evaluation of PhIP-Seq, in order to gain a more comprehensive understanding of the application and future development prospects of this technology in various fields.
Peptide Library ; Humans ; Immunoprecipitation/methods* ; High-Throughput Nucleotide Sequencing/methods* ; Bacteriophages/genetics*

Objective To investigate the effects of folate deficiency on changes in histone H3 lysine 4 (H3K4) mono-methylation (me1)-marked enhancers and the molecular mechanism underpinning the folate deficiency-induced neural tube defects (NTD). Methods Mouse embryonic stem cells (mESCs) were cultured in the folate-free DMEM medium (folate-deficient group) and the DMEM medium containing 4 mg/L folate (normal control group),respectively.Chromatin immunoprecipitation sequencing (ChIP-seq) was performed for H3K4me1. The mouse model of folate-induced NTD was established,and transcriptome sequencing (RNA-seq) was performed for the brain tissue of fetal mice to reveal the differential expression profiles.The results were validated through real-time quantitative polymerase chain reaction (RT-qPCR).The activity of the differential peak regions of H3K4me1 was verified through the luciferase reporter assay. Results The folate content in the mESCs cultured in the folate-free medium reduced compared with that in the normal control group (P=0.008).The H3K4me1-maked enhancers in the mESCs cultured in the folate-free medium induced significant changes in intronic regions,and these changes were concentrated in metabolic and energy metabolism processes (q=9.56×10^-48,P=1.28×10^-47).The differentially expressed genes harboring H3K4me1-marked enhancers in mESCs were mainly enriched in the Wnt signaling pathway (q=0.004,P=0.004 7).ChIP-qPCR results confirmed that H3K4me1 binding decreased in the differential peak regions of the Ldlrap1 gene (P=0.008),Camta1 gene (P=0.002),and Apc2 gene (P=0.012).The H3K4 demethylase inhibitor T-448 effectively reversed the H3K4me1 binding in the differential peak regions of the aforementioned genes (P=0.01).The results of RNA-seq for the brain tissue of NTD fetal mice showed significant enrichment of the differentially expressed genes in the Wnt signaling pathway (P=1.52×10^-5).The enrichment of differential peak regions of H3K4me1-marked enhancers in Apc2,Ldlrap1,and Camta1 genes in the brain tissue also showed significant changes.The differential peak region in Apc2 exhibited transcription factor activity (P=0.020). Conclusion Folate deficiency may affect changes in H3K4me1-marked enhancers to participate in the regulation of neural tube closure genes,thereby inducing the occurrence of NTD.
Neural Tube Defects/genetics* ; Animals ; Mice ; Folic Acid Deficiency/complications* ; Histones/metabolism* ; Folic Acid/metabolism* ; Methylation ; Mouse Embryonic Stem Cells/metabolism* ; Wnt Signaling Pathway ; Lysine/metabolism* ; Chromatin Immunoprecipitation Sequencing

assay was used for miR-21 expression visualization in lymphoma tissues. Western blot was used for determination of VHL protein, Ki-67, caspase-3, and cleaved caspase-3 levels. Dual-luciferase reporter assay and RNA immunoprecipitation assay were employed to confirm the direct target of miR-21. MTT assay, flow cytometric analysis, and transwell assay were used to evaluate cell proliferation, apoptosis, and migration and invasion capacities, respectively.RESULTS: Curcumin repressed the proliferation, migration, and invasion abilities and promoted apoptosis in SU-DHL-8 cells. Curcumin inhibited miR-21 expression and curcumin exerted its anti-proliferation, anti-migration, anti-invasion, and pro-apoptosis effects by miR-21 in SU-DHL-8 cells. VHL was a direct target of miR-21. Moreover, curcumin exerted its regulatory effects on SU-DHL-8 cells by VHL.CONCLUSION: Curcumin exerted its anti-proliferation, anti-migration, anti-invasion, and pro-apoptosis functions, at least partly, by repressing miR-21 and regulating VHL expression in DLBCL cell line. Our findings provided a possible molecular mechanism of curcumin-mediated anti-cancer effect.]]>
Apoptosis ; B-Lymphocytes ; Blotting, Western ; Carcinogenesis ; Caspase 3 ; Cell Line ; Cell Proliferation ; Curcumin ; Humans ; Immunoprecipitation ; In Situ Hybridization ; Lymphoma ; Lymphoma, B-Cell ; MicroRNAs ; Real-Time Polymerase Chain Reaction ; RNA ; RNA, Messenger

PURPOSE: Reduction-oxidation reaction homeostasis is vital for regulating inflammatory conditions and its dysregulation may affect the pathogenesis of chronic airway inflammatory diseases such as asthma. Peroxiredoxin-6, an important intracellular anti-oxidant molecule, is reported to be highly expressed in the airways and lungs. The aim of this study was to analyze the expression pattern of peroxiredoxin-6 in the peripheral blood mononuclear cells (PBMCs) of asthmatic patients and in bronchial epithelial cells (BECs).METHODS: The expression levels and modifications of peroxiredoxin-6 were evaluated in PBMCs from 22 asthmatic patients. Phosphorylated and acetylated peroxiredoxin-6 in hydrogen peroxide-treated human BECs was detected using immunoprecipitation analysis. The expression level of peroxiredoxin-6 was also investigated in BECs treated with hydrogen peroxide. Cycloheximide and proteasome inhibitors were used to determine whether peroxiredoxin-6 is degraded by proteasomes.RESULTS: Peroxiredoxin-6 expression was significantly reduced in the PBMCs of asthmatic patients compared to control subjects. Distinct modification patterns for peroxiredoxin-6 were observed in the PBMCs of asthmatic patients using 2-dimensional-electrophoresis. The levels of phosphorylated serine and acetylated lysine in peroxiredoxin-6 were significantly increased in the BECs following hydrogen peroxide treatment. The level of peroxiredoxin-6 expression was reduced in hydrogen peroxide-stimulated BECs, presumably due to proteasomes.CONCLUSIONS: The expression of peroxiredoxin-6, which is down-regulated in the immune cells of asthmatic patients and BECs, can be modified by oxidative stress. This phenomenon may have an effect on asthmatic airway inflammation.
Asthma ; Cycloheximide ; Epithelial Cells ; Homeostasis ; Humans ; Hydrogen ; Hydrogen Peroxide ; Immunoprecipitation ; Inflammation ; Lung ; Lysine ; Oxidative Stress ; Proteasome Inhibitors ; Protein Processing, Post-Translational ; Serine

PURPOSE: Liver fibrosis is a major cause of morbidity and mortality and the outcome of various chronic liver diseases. Activation of hepatic stellate cells (HSCs) is the key event in liver fibrosis. Studies have confirmed that miR-140-3p plays a potential regulatory effect on HSC activation. However, whether miR-140-3p mediates the liver fibrosis remains unknown. MATERIALS AND METHODS: Expression of miR-140-3p was detected by real-time quantitative PCR (qPCR). Cell proliferation was measured by MTT, while cell apoptosis rate was determined via flow cytometry. Western blot assay was used to detect the expression of cleaved PARP. The fibrogenic effect was evaluated by expression of α-smooth muscle actin and desmin. Functional experiments were performed in transforming growth factor β1 (TGF-β1)-induced HSC-T6 cells with transfection of anti-miR-140-3p and/or siPTEN. Target binding between miR-140-3p and PTEN was predicted by the TargetScan database and identified using luciferase reporter assay and RNA immunoprecipitation. RESULTS: TGF-β1 induced the activation of HSC-T6 cells, and miR-140-3p expression varied according to HSC-T6 cell activation status. Knockdown of miR-140-3p reduced cell proliferation and the expressions of α-SMA and desmin, as well as increased apoptosis, in TGF-β1-induced HSC-T6 cells, which could be blocked by PTEN silencing. Additionally, inactivation of the AKT/mTOR signaling pathway stimulated by miR-140-3p knockdown was abolished when silencing PTEN expression. PTEN was negatively regulated by miR-140-3p via direct binding in HSC-T6 cells. CONCLUSION: miR-140-3p is an important mediator in HSC-T6 cell activation, and miR-140-3p knockdown suppresses cell proliferation and fibrogenesis in TGF-β1-induced HSC-T6 cells, indicating that miR-140-3p may be a potential novel molecular target for liver fibrosis.
Actins ; Apoptosis ; Blotting, Western ; Cell Proliferation ; Desmin ; Flow Cytometry ; Hepatic Stellate Cells ; Immunoprecipitation ; Liver Cirrhosis ; Liver Diseases ; Luciferases ; Mortality ; Polymerase Chain Reaction ; RNA ; Transfection ; Transforming Growth Factors

PURPOSE: Alzheimer's disease (AD) is the most common neurodegenerative disease, with a rising prevalence worldwide. Long noncoding RNAs (lncRNAs) have been found to play important roles in the development and treatment of AD. However, the exact role of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in neuronal damage in AD is largely unknown. MATERIALS AND METHODS: The AD model was established in SH-SY5Y and SK-N-SH cells via treatment with amyloid β1−42 (Aβ). The expression of NEAT1 and microRNA-107 (miR-107) was measured by quantitative real-time polymerase chain reaction. Cell viability and apoptosis were detected by MTT assay, immunocytochemistry, and flow cytometry. The expression of phosphorylated tau protein (p-Tau) was measured by Western blot. The interaction between NEAT1 and miR-107 was explored by bioinformatics analysis, luciferase activity, and RNA immunoprecipitation assays. RESULTS: NEAT1 expression was enhanced in Aβ-treated SH-SY5Y and SK-N-SH cells, and its knockdown attenuated Aβ-induced inhibition of viability and promotion of apoptosis and p-Tau levels. NEAT1 was indicated as a decoy of miR-107. miR-107 abundance was reduced in Aβ-treated cells, and its overexpression reversed Aβ-induced injury. Moreover, interference of miR-107 abated silencing of NEAT1-mediated inhibition of neuronal damage in Aβ-treated SH-SY5Y and SK-N-SH cells. CONCLUSION: LncRNA NEAT1 aggravated Aβ-induced neuronal damage by sponging miR-107, indicating a novel avenue for treatment of AD.
Alzheimer Disease ; Amyloid ; Apoptosis ; Blotting, Western ; Cell Survival ; Computational Biology ; Flow Cytometry ; Immunohistochemistry ; Immunoprecipitation ; Luciferases ; Neurodegenerative Diseases ; Neurons ; Prevalence ; Real-Time Polymerase Chain Reaction ; RNA ; RNA, Long Noncoding ; tau Proteins

PURPOSE: Long intergenic non-protein coding RNA 665 (LINC00665) plays a vital role in the development of cancer. Its function in hepatocellular carcinoma (HCC), however, remains largely unknown. MATERIALS AND METHODS: The expressions of LINC00665, miR-186-5p, and MAP4K3 were determined by qRT-PCR. Cell viability and apoptosis were evaluated by MTT and flow cytometry, respectively. Autophagic puncta formation was observed by fluorescence microscopy. Bioinformatics analysis, luciferase reporter assay, RNA immunoprecipitation, and RNA pulldown were performed to identify associations among LINC00665, miR-186-5p, and MAP4K3. Western blot was utilized to examine the expressions of MAP4K3, Beclin-1, and LC3. Tumor growth was evaluated in a xenograft model. RESULTS: Elevations in LINC00665 were observed in HCC tissues and cells. The overall survival of HCC patients with high levels of LINC00665 was shorter than those with low levels. In vitro, LINC00665 depletion inhibited viability and induced apoptosis and autophagy. miR-186-5p interacted with LINC00665 and was downregulated in HCC tissues and cells. Upregulation of miR-186-5p inhibited viability and induced apoptosis and autophagy, which were attenuated by upregulation of LINC00665. MAP4K3 was found to possess binding sites with miR-186-5p and was upregulated in HCC tissues and cells. MAP4K3 depletion inhibited viability and induced apoptosis and autophagy, which were attenuated by miR-186-5p inhibitor. In vivo, miR-186-5p expression was negatively correlated with LINC00665 or MAP4K3 in HCC tissues, while LINC00665 was positively correlated with MAP4K3. LINC00665 knockdown suppressed tumor growth. CONCLUSION: LINC00665 was involved in cell viability, apoptosis, and autophagy in HCC via miR-186-5p/MAP4K3 axis, which may provide a new approach for HCC treatment.
Apoptosis ; Autophagy ; Binding Sites ; Blotting, Western ; Carcinoma, Hepatocellular ; Cell Survival ; Computational Biology ; Flow Cytometry ; Heterografts ; Humans ; Immunoprecipitation ; In Vitro Techniques ; Luciferases ; Microscopy, Fluorescence ; RNA ; RNA, Long Noncoding ; Up-Regulation

PURPOSE: Long noncoding RNA 00703 (LINC00703) was found originating from a region downstream of Kruppel-like factor 6 (KLF6) gene, having 2 binding sites for miR-181a. Since KLF6 has been reported as a target of miR-181a in gastric cancer (GC), this study aims to investigate whether LINC00703 regulates the miR-181a/KLF6 axis and plays a functional role in GC pathogenesis.MATERIALS AND METHODS: GC tissues, cell lines, and nude mice were included in this study. RNA binding protein immunoprecipitation (RIP) and pull-down assays were used to evaluate interaction between LINC00703 and miR-181a. Quantitative real-time polymerase chain reaction and western blot were applied for analysis of gene expression at the transcriptional and protein levels. A nude xenograft mouse model was used to determine LINC00703 function in vivo.RESULTS: We revealed that LINC00703 competitively interacts with miR-181a to regulate KLF6. Overexpression of LINC00703 inhibited cell proliferation, migration/invasion, but promoted apoptosis in vitro, and arrested tumor growth in vivo. LINC00703 expression was found to be decreased in GC tissues, which was positively correlated with KLF6, but negatively with the miR-181a levels.CONCLUSIONS: LINC00703 may have an anti-cancer function via modulation of the miR-181a/KLF6 axis. This study also provides a new potential diagnostic marker and therapeutic target for GC treatment.
Animals ; Apoptosis ; Binding Sites ; Blotting, Western ; Cell Line ; Cell Proliferation ; Gene Expression ; Heterografts ; Immunoprecipitation ; In Vitro Techniques ; Mice ; Mice, Nude ; Real-Time Polymerase Chain Reaction ; RNA, Long Noncoding ; RNA-Binding Proteins ; Stomach Neoplasms

OBJECTIVE: To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data. METHODS: Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis. RESULTS: Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%. CONCLUSIONS Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.
Chromatin Immunoprecipitation ; DNA ; Gene Library ; High-Throughput Nucleotide Sequencing ; Humans ; Reproducibility of Results ; Sequence Analysis, DNA

5-HT₆ receptor (5-HT₆R) is implicated in cognitive dysfunction, mood disorder, psychosis, and eating disorders. However, despite its significant role in regulating the brain functions, regulation of 5-HT₆R at the molecular level is poorly understood. Here, using yeast two-hybrid assay, we found that human 5-HT₆R directly binds to neuro-oncological ventral antigen 1 (Nova-1), a brain-enriched splicing regulator. The interaction between 5-HT₆R and Nova-1 was confirmed using GST pull-down and co-immunoprecipitation assays in cell lines and rat brain. The splicing activity of Nova-1 was decreased upon overexpression of 5-HT₆R, which was examined by detecting the spliced intermediates of gonadotropin-releasing hormone (GnRH), a known pre-mRNA target of Nova-1, using RT-PCR. In addition, overexpression of 5-HT₆R induced the translocation of Nova-1 from the nucleus to cytoplasm, resulting in the reduced splicing activity of Nova-1. In contrast, overexpression of Nova-1 reduced the activity and the total protein levels of 5-HT₆R. Taken together, these results indicate that when the expression levels of 5-HT₆R or Nova-1 protein are not properly regulated, it may also deteriorate the function of the other.
Animals ; Brain ; Cell Line ; Cytoplasm ; Eating ; Gonadotropin-Releasing Hormone ; Humans ; Immunoprecipitation ; Mood Disorders ; Psychotic Disorders ; Rats ; RNA Precursors ; RNA-Binding Proteins ; Serotonin ; Two-Hybrid System Techniques