Objective To investigate the effect of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) on the proliferation, migration and tumor immune microenvironment of colorectal cancer cells and its possible molecular mechanism. Methods The Cancer Genome Atlas (TCGA) database was used to analyze the expression levels of IGF2BP2 and MYC in colorectal cancer and adjacent tissues. The expression of IGF2BP2 in HCT-116 and SW480 human colorectal cancer cells was silenced by RNA interference (RNAi), and the silencing effect was detected by quantitative real-time PCR. After knocking down IGF2BP2, colony formation assay, CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were employed to detect cell colony formation and proliferation ability. Transwell^TM assay was used to detect cell migration ability. Quantitative real-time PCR was used to detect the mRNA expression of IGF2BP2, MYC, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β) and interleukin-10 (IL-10). The protein expression of IGF2BP2 and MYC was detected by western blot. The binding ability of IGF2BP2 and MYC in HCT-116 cells was detected by quantitative real-time PCR after RNA immunoprecipitation. Results The results of TCGA database showed that the expression of IGF2BP2 and MYC in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the survival time of colorectal cancer patients with high expression of IGF2BP2 was shorter. After silencing IGF2BP2, the viability, proliferation and migration of HCT-116 and SW480 cells were decreased. The mRNA expression of MYC, TGF-β and IL-10 in IGF2BP2 knockdown group was significantly decreased, while the expression of TNF-α mRNA was increased. The expression of MYC protein and the stability of MYC mRNA were significantly decreased. RIP-qPCR results showed that IGF2BP2 could bind to MYC mRNA. Conclusion Knockdown of IGF2BP2 inhibits colorectal cancer cell proliferation, migration and promotes tumor immunity by down-regulating MYC expression.
Humans ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Colorectal Neoplasms/metabolism* ; Gene Expression Regulation, Neoplastic ; Interleukin-10/metabolism* ; RNA, Messenger ; RNA-Binding Proteins/metabolism* ; Transforming Growth Factor beta/genetics* ; Tumor Microenvironment/immunology* ; Tumor Necrosis Factor-alpha/metabolism* ; Proto-Oncogene Proteins c-myc/metabolism*

Objective To screen the potential key genes of osteosarcoma by bioinformatics methods and analyze their immune infiltration patterns. Methods The gene expression profiles GSE16088 and GSE12865 associated with osteosarcoma were obtained from the Gene Expression Omnibus(GEO),and the differentially expressed genes(DEGs)related to osteosarcoma were screened by bioinformatics tools.Gene Ontology(GO)annotation,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment,and analysis of immune cell infiltration were then carried out for the DEGs.The potential Hub genes of osteosarcoma were identified by protein-protein interaction network,and the expression of Hub genes in osteosarcoma and normal tissue samples was verified via the Cancer Genome Atlas(TCGA). Results A total of 108 DEGs were screened out.GO annotation and KEGG pathway enrichment revealed that the DEGs were mainly involved in integrin binding,extracellular matrix (ECM) structural components,ECM receptor interactions,and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway.Macrophages were the predominant infiltrating immune cells in osteosarcoma.Secreted phosphoprotein 1(SPP1),matrix metallopeptidase 2(MMP2),lysyl oxidase(LOX),collagen type V alpha(II)chain(COL5A2),and melanoma cell adhesion molecule(MCAM)presented differential expression between osteosarcoma and normal tissue samples(all P<0.05). Conclusions SPP1,MMP2,LOX,COL5A2,and MCAM are all up-regulated in osteosarcoma,which may serve as potential biomarkers of osteosarcoma.Macrophages are the key infiltrating immune cells in osteosarcoma,which may provide new perspectives for the treatment of osteosarcoma.
Bone Neoplasms/immunology* ; Computational Biology/methods* ; Gene Expression Profiling/methods* ; Humans ; Osteosarcoma/immunology* ; Phosphatidylinositol 3-Kinases/genetics* ; Tumor-Associated Macrophages/immunology*

Ambystoma mexicanum/immunology* ; Amputation ; Animals ; Biomarkers/metabolism* ; Blastomeres/immunology* ; Cell Lineage/immunology* ; Connective Tissue Cells/immunology* ; Epithelial Cells/immunology* ; Forelimb ; Gene Expression ; High-Throughput Nucleotide Sequencing ; Humans ; Immunity ; Peroxiredoxins/immunology* ; Regeneration/immunology* ; Regenerative Medicine/methods* ; Single-Cell Analysis/methods*

The aim of this study was to investigate the expression of MHCⅠ gene in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila (Ah), and to provide evidence for revealing the anti-infective immune response mechanism of amphibians. The experimental animal model of Aeromonas hydrophila infection was first constructed, and the pathological changes were observed by HE staining. The MHCⅠ gene α1+α2 peptide binding region of Rana dybowskii was cloned by RT-PCR and analyzed by bioinformatics. Real-time PCR was used to detect the transcription level of MHCⅠ in different tissues under Ah stress. After Ah infection, the skin, liver and muscle tissues showed signs of cell structure disappearance and texture disorder. The MHCⅠ gene α1+α2 peptide binding region fragment was 494 bp, encoding 164 amino acids, and homology with amphibians. Above 77%, the homology with mammals was as low as 14.96%, indicating that the α1+α2 region of MHC gene was less conserved among different species. The results of real-time PCR show that the liver, spleen and kidney of the experimental group were under Ah stress. The transcript levels of MHCⅠ gene in skin and muscle tissues were higher than those in the control group at 72 h, but the time to peak of each tissue was different (P<0.01), indicating that the response time of MHCⅠ gene in different tissues was different under Ah stress. This study provides a reference for further exploring the immune function of MHC molecules in anti-infection.
Aeromonas hydrophila ; Animals ; Gene Expression Profiling ; Gene Expression Regulation ; immunology ; Gram-Negative Bacterial Infections ; immunology ; Liver ; metabolism ; Ranidae ; genetics ; immunology ; microbiology ; Skin ; metabolism

In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.
Animals ; Antibodies, Viral ; genetics ; immunology ; Cricetinae ; Cytomegalovirus ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immunoglobulin M ; immunology ; Mice ; Protein Sorting Signals ; Recombinant Fusion Proteins ; immunology

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.
Adult ; Aged ; Aged, 80 and over ; Aging ; genetics ; immunology ; Betacoronavirus ; CD4-Positive T-Lymphocytes ; metabolism ; Cell Lineage ; Chromatin Assembly and Disassembly ; Coronavirus Infections ; immunology ; Cytokine Release Syndrome ; etiology ; immunology ; Cytokines ; biosynthesis ; genetics ; Disease Susceptibility ; Flow Cytometry ; methods ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Gene Rearrangement ; Humans ; Immune System ; cytology ; growth & development ; immunology ; Immunocompetence ; genetics ; Inflammation ; genetics ; immunology ; Mass Spectrometry ; methods ; Middle Aged ; Pandemics ; Pneumonia, Viral ; immunology ; Sequence Analysis, RNA ; Single-Cell Analysis ; Transcriptome ; Young Adult

Chimeric antigen receptor (CAR) T cell therapy has exhibited dramatic anti-tumor efficacy in clinical trials. In this study, we reported the transcriptome profiles of bone marrow cells in four B cell acute lymphoblastic leukemia (B-ALL) patients before and after CD19-specific CAR-T therapy. CD19-CAR-T therapy remarkably reduced the number of leukemia cells, and three patients achieved bone marrow remission (minimal residual disease negative). The efficacy of CD19-CAR-T therapy on B-ALL was positively correlated with the abundance of CAR and immune cell subpopulations, e.g., CD8 T cells and natural killer (NK) cells, in the bone marrow. Additionally, CD19-CAR-T therapy mainly influenced the expression of genes linked to cell cycle and immune response pathways, including the NK cell mediated cytotoxicity and NOD-like receptor signaling pathways. The regulatory network analyses revealed that microRNAs (e.g., miR-148a-3p and miR-375), acting as oncogenes or tumor suppressors, could regulate the crosstalk between the genes encoding transcription factors (TFs; e.g., JUN and FOS) and histones (e.g., HIST1H4A and HIST2H4A) involved in CD19-CAR-T therapy. Furthermore, many long non-coding RNAs showed a high degree of co-expression with TFs or histones (e.g., FOS and HIST1H4B) and were associated with immune processes. These transcriptome analyses provided important clues for further understanding the gene expression and related mechanisms underlying the efficacy of CAR-T immunotherapy.
Adult ; Antigens, CD19 ; metabolism ; Bone Marrow ; metabolism ; CD8-Positive T-Lymphocytes ; immunology ; Female ; Gene Expression Regulation, Leukemic ; Gene Regulatory Networks ; Humans ; Immunotherapy, Adoptive ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; therapy ; RNA, Long Noncoding ; genetics ; metabolism ; Receptors, Antigen, T-Cell ; Transcription Factors ; metabolism ; Transcriptome ; genetics

Macrophages show significant heterogeneity in function and phenotype, which could shift into different populations of cells in response to exposure to various micro-environmental signals. These changes, also termed as macrophage polarization, of which play an important role in the pathogenesis of many diseases. Numerous studies have proved that Hesperidin (HDN), a traditional Chinese medicine, extracted from fruit peels of the genus citrus, play key roles in anti-inflammation, anti-tumor, anti-oxidant and so on. However, the role of HDN in macrophage polarization has never been reported. Additional, because of its poor water solubility and bioavailability. Our laboratory had synthesized many hesperidin derivatives. Among them, hesperidin derivatives-12 (HDND-12) has better water solubility and bioavailability. So, we evaluated the role of HDND-12 in macrophage polarization in the present study. The results showed that the expression of Arginase-1 (Arg-1), interleukin-10 (IL-10), transforming growth factor β (TGF-β) were up-regulated by HDND-12, whereas the expression of inducible Nitric Oxide Synthase (iNOS) was down-regulated in LPS- and IFN-γ-treated (M1) RAW264.7 cells. Moreover, the expression of p-JAK2 and p-STAT3 were significantly decreased after stimulation with HDND-12 in M1-like macrophages. More importantly, when we taken AG490 (inhibitor of JAK2/STAT3 signaling), the protein levels of iNOS were significantly reduced in AG490 stimulation group compare with control in LPS, IFN-γ and HDND-12 stimulation cells. Taken together, these findings indicated that HDND-12 could prevent polarization toward M1-like macrophages, at least in part, through modulating JAK2/STAT3 pathway.
Animals ; Cytokines ; genetics ; metabolism ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; drug effects ; Hesperidin ; chemistry ; pharmacology ; Inflammation ; genetics ; metabolism ; Janus Kinase 2 ; antagonists & inhibitors ; metabolism ; Macrophages ; drug effects ; immunology ; metabolism ; Medicine, Chinese Traditional ; Mice ; Molecular Structure ; Phosphorylation ; drug effects ; RAW 264.7 Cells ; STAT3 Transcription Factor ; antagonists & inhibitors ; metabolism ; Signal Transduction ; drug effects

Objective To investigate the expression and regulation of programmed cell death protein 1 (PD1), B lymphocyte and T lymphocyte attenuator (BTLA) in peripheral blood of patients with non-small cell lung cancer (NSCLC); to examine the correlation of the mRNA levels between PD and BTLA in NSCLC. Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8⁺ T cells and γδ⁺ T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals. We compared the expression of PD1 and BTLA on the surfaces of γδ⁺ T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid. The correlations of PD1 and BTLA, as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform. Results The frequency of PD1 on the surfaces of CD8⁺ T cells was significantly higher than that of the γδT cells in both healthy controls (t=2.324, P=0.024) and NSCLC patients（t=2.498, P=0.015). The frequency of PD1 on CD8⁺ T cells, rather than on γδ⁺ T cells, was significantly upregulated in advanced NSCLC patients compared with that in healthy controls (t=4.829, P<0.001). The PD1⁺ BTLA⁺γδT cells of the healthy controls were significantly lower than that of the NSCLC patients (t=2.422, P=0.0185). No differences in percentage of PD1⁺γδ⁺ and BTLA⁺γδ⁺ T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment. PD1 was positively correlated with BTLA in both lung adenocarcinoma (r=0.54; P<0.05) and lung squamous cell carcinoma (r=0.78; P<0.05). Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8⁺ T cells and γδT cells in advanced NSCLC, suggesting that these molecules were involved in regulating the inactivation of CD8⁺ T cells and γδ⁺ T cells, immune escape and tumor invasion.
Bone Neoplasms/secondary* ; CD8-Positive T-Lymphocytes ; Carcinoma, Non-Small-Cell Lung/immunology* ; Case-Control Studies ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Ligands ; Lung Neoplasms/immunology* ; Lymphocyte Subsets/immunology* ; Male ; Middle Aged ; Programmed Cell Death 1 Receptor/metabolism* ; RNA, Messenger/metabolism* ; Receptors, Antigen, T-Cell, gamma-delta ; Receptors, Immunologic/metabolism*

Immunosuppressive regulatory T lymphocytes (Treg) expressing the transcription factor Foxp3 play a vital role in the maintenance of tolerance of the immune-system to self and innocuous non-self. Most Treg that are critical for the maintenance of tolerance to self, develop as an independent T-cell lineage from common T cell precursors in the thymus. In this organ, their differentiation requires signals from the T cell receptor for antigen, from co-stimulatory molecules, as well as from cytokine-receptors. Here we focus on the cytokines implicated in thymic development of Treg, with a particular emphasis on the roles of interleukin-2 (IL-2) and IL-15. The more recently appreciated involvement of TGF-β in thymic Treg development is also briefly discussed. Finally, we discuss how cytokine-dependence of Treg development allows for temporal, quantitative, and potentially qualitative modulation of this process.
Animals ; Cell Differentiation ; genetics ; Cytokines ; immunology ; Forkhead Transcription Factors ; genetics ; immunology ; Gene Expression Regulation ; Immune Tolerance ; genetics ; Interleukin-15 ; genetics ; immunology ; Interleukin-2 ; genetics ; immunology ; Mice ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta ; genetics ; immunology