技术资料

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner,thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation,differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10,IRF8,KLF4,MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML. View Publication

Despite the key role that antibodies play in protection,the cellular processes mediating the acquisition of humoral immunity against malaria are not fully understood. Using an infection model of severe malaria,we find that germinal center (GC) B cells upregulate the transcription factor T-bet during infection. Molecular and cellular analyses reveal that T-bet in B cells is required not only for IgG2c switching but also favors commitment of B cells to the dark zone of the GC. T-bet was found to regulate the expression of Rgs13 and CXCR3,both of which contribute to the impaired GC polarization observed in the absence of T-bet,resulting in reduced IghV gene mutations and lower antibody avidity. These results demonstrate that T-bet modulates GC dynamics,thereby promoting the differentiation of B cells with increased affinity for antigen. View Publication

When transmitted through the oral route,Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication,as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However,due to a lack of tractable infection models,we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However,the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here,we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular,we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization,and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with,and support replication of,T. gondii. Using quantitative label-free mass spectrometry,we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism,extracellular exosomes,intermicrovillar adhesion,and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses,indicating that de novo synthesis may support,but is not required for,parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection. View Publication

Apoptosis and autophagy are dynamic processes that determine the fate of cells. Vitamin D receptor (VDR) deficiency in the intestine leads to abnormal Paneth cells and impaired autophagy function. Here,we will elucidate the mechanisms of the intestinal epithelial VDR regulation of autophagy and apoptosis. We used in vivo VDRlox and VDR∆IEC mice and ex vivo organoids generated from small intestine and colon tissues. We found that VDR deficiency induced more apoptotic cells and significantly increased cell death in the small intestine and colon of VDR∆IEC mice. The proapoptotic protein B-cell lymphoma 2 (BCL-2) associated X protein (Bax) was enhanced,whereas autophagy related 16 like 1 (ATG16L1) and Beclin-1 were decreased in the intestines of VDR$\Delta$IEC mice. Apoptosis induced by Bax reduced autophagy by decreasing Beclin-1. Physical interactions between Beclin-1 and Bcl-2 were increased in the VDR-deficient epithelia from mice. The growth of VDR∆IEC organoids was significantly slower with fewer Paneth cells than that of VDR+/+ organoids. The expression levels of Beclin-1 and lysozyme were decreased in VDR∆IEC organoids. Bacterial endotoxin levels were high in the serum from VDR∆IEC mice and made mice susceptible to colitis. In the organoids and colitis IL-10-/- mice,vitamin D3 treatment increased VDR and ATG16L1 protein expression levels,which activated autophagic responses. In summary,intestinal epithelial VDR regulates autophagy and apoptosis through ATG16L1 and Beclin-1. Our studies provide fundamental insights into the tissue-specific function of VDR in modulating the balance between autophagy and apoptosis.-Lu,R.,Zhang,Y.-G.,Xia,Y.,Sun,J. Imbalance of autophagy and apoptosis in intestinal epithelium lacking the vitamin D receptor. View Publication

BACKGROUND MiR-199a-3p (miR-199a) can enhance the chemosensitivity of hepatocellular carcinoma (HCC). Because of the easy degradation of miRNA by direct infusion,effective vehicle-mediated delivery of miR-199a may represent a new strategy for improving HCC chemotherapy. Considering mesenchymal stem cell (MSC)-derived exosomes as promising natural nanovectors for drug and molecule delivery,we aimed to determine whether exosomes from adipose tissue-derived MSCs (AMSCs) could be used to deliver miR-199a and improve HCC chemosensitivity. METHODS MiR-199a-modified AMSCs (AMSC-199a) were constructed by miR-199a lentivirus infection and puromycin selection. MiR-199-modified exosomes (AMSC-Exo-199a) were isolated from the supernatant of AMSC-199a and were assessed by transmission electron microscopy,nanoparticle tracking analysis,and flow cytometry analysis. The expression levels of miR-199a in HCC samples,AMSCs,exosomes,and HCC cells were quantified by real-time PCR. The effects of AMSC-Exo-199a on HCC chemosensitivity were determined by cell proliferation and apoptosis assays and by i.v. injection into orthotopic HCC mouse models with doxorubicin treatment. MTOR,p-4EBP1 and p-70S6K levels in HCC cells and tissues were quantified by Western blot. RESULTS AMSC-Exo-199a had the classic characteristics of exosomes and could effectively mediate miR-199a delivery to HCC cells. Additionally,AMSC-Exo-199a significantly sensitized HCC cells to doxorubicin by targeting mTOR and subsequently inhibiting the mTOR pathway. Moreover,i.v.-injected AMSC-Exo-199a could distribute to tumor tissue and markedly increased the effect of Dox against HCC in vivo. CONCLUSIONS AMSC-Exo-199a can be an effective vehicle for miR-199a delivery,and they effectively sensitized HCC to chemotherapeutic agents by targeting mTOR pathway. AMSC-Exo-199a administration may provide a new strategy for improving HCC chemosensitivity. View Publication

The gut microbiome contributes to inflammatory bowel disease (IBD),in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD,and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here,we treated human colonic biopsies,epithelial colonoids,and epithelial cells with an uncoupler of oxidative phosphorylation,dinitrophenol (DNP),with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6,an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis,induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather,specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note,epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria,thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation. View Publication

Autoimmune diseases are a physiological state wherein immune responses are directed against and damage the body's own tissues. Cytokines secreted by infiltrated inflammatory cells contribute to the pathogenesis of autoimmune diseases. TIPE2,one of the four family members of Tumor necrosis factor-$\alpha$ induced protein-8 (TNFAIP8),is a negative regulator of innate and adaptive immunity and plays essential roles in the maintenance of immune tolerance. However,studies on the role of TIPE2 during the development of autoimmune diseases have generated contradictory results. In the current study,we sought to determine the role of TIPE2 during the development of IMQ-induced psoriasis and Experimental Autoimmune Uveitis (EAU) in mice. Our study revealed that,while TIPE2-deficiency alleviates psoriasis,it exacerbates the development of EAU. Further studies demonstrated that,although TIPE2-deficient T cells produced more IL-17A,they do not migrate efficiently to the local inflammatory site,i.e.,the skin. This in turn led to the decreased IL-17A production in the skin and consequently reduced the severity of psoriasis in TIPE2-deficient mice. However,although TIPE2-deficient T cells still produced more IL-17A in EAU model,they migrate into the inflamed eye as efficient as TIPE2-sufficient T cells,and consequently exacerbates the development of EAU in TIPE2-deficient mice. Taken together,these results indicate that TIPE2 may either promote or suppress autoimmunity depending on the specific inflammatory microenvironment in different types of autoimmune diseases. View Publication

Our previous studies have demonstrated that a previously unrecognized role of CFTR in hematopoiesis and acute leukemia. Here,we show that CFTR inhibitor CFTR-inh172 possesses ability to inhibit human T-cell acute lymphoblastic leukemia cells. In detail,CFTR-inh172 inhibited cell proliferation,promoted apoptosis and arrested the cell cycle in human T-cell acute lymphoblastic leukemia cell CCRF-CEM,JURKAT and MOLT-4. Furthermore,transcriptome analysis reveals that CFTR-inh172 induces significant alteration of gene expression related to apoptosis and proliferation. These findings demonstrate the potential of CFTR inhibitor CFTR-inh172 in human T-cell acute lymphoblastic leukemia treatment. View Publication

Rapidly evolving RNA viruses,such as the GII.4 strain of human norovirus (HuNoV),and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection,moreover,remain poorly understood. Here,we report the GII.4-serological antibody repertoire-pre- and post-vaccination-and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However,one antibody,A1431,showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes,which were occluded on the virion or partially exposed,allowing for broad blockade with neutralizing activity. Overall,our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen. View Publication

BACKGROUND Upregulation of RNA polymerase (Pol) III products,including tRNAs and 5S rRNA,in tumor cells leads to enhanced protein synthesis and tumor formation,making it a potential target for cancer treatment. In this study,we evaluated the inhibition of Pol III transcription by triptolide and the anti-cancer effect of this drug in colorectal tumorigenesis. METHODS The effect of triptolide on colorectal cancer development was assessed in colorectal cancer mouse models,3D organoids,and cultured cells. Colorectal cancer cells were treated with triptolide. Pol III transcription was measured by real-time quantitative polymerase chain reaction (PCR). The formation of TFIIIB,a multi-subunit transcription factor for Pol III,was determined by chromatin immunoprecipitation (ChIP),co-immunoprecipitation (Co-IP),and fluorescence resonance energy transfer (FRET). RESULTS Triptolide reduced both tumor number and tumor size in adenomatous polyposis coli (Apc) mutated (ApcMin/+) mice as well as AOM/DSS-induced mice. Moreover,triptolide effectively inhibited colorectal cancer cell proliferation,colony formation,and organoid growth in vitro,which was associated with decreased Pol III target genes. Mechanistically,triptolide treatment blocked TBP/Brf1interaction,leading to the reduced formation of TFIIIB at the promoters of tRNAs and 5S rRNA. CONCLUSIONS Together,our data suggest that inhibition of Pol III transcription with existing drugs such as triptolide provides a new avenue for developing novel therapies for colorectal cancer. View Publication

Cytotoxic chemotherapies could cause the dysregulation of hematopoiesis and even put patients at increased risk of hematopoietic malignancy. Therapy-related leukemia is mainly caused by cytotoxic chemotherapy-induced genetic mutations in hematopoietic stem/progenitor cells (HSPCs). In addition to the intrinsic mechanism,some extrinsic events occurring in the bone marrow (BM) microenvironment are also possible mechanisms involved in genetic alteration. In the present study,we investigated the damage to BM stromal cells induced by a chemotherapy drug,daunorubicin (DNR) and further identified the DNA damage in hematopoietic cells caused by drug-treated stromal cells. It was found that treatment with DNR in mice caused a temporary reduction in cell number in each BM stromal cell subpopulation and the impairment of clonal growth potential in BM stromal cells. DNR treatment led to a tendency of senescence,generation of intracellular reactive oxygen species,production of cytokines and chemokines,and dysfunction of mitochondrial in stromal cells. Transcriptome microarray data and gene ontology (GO) or gene set enrichment analysis (GSEA) showed that differentially expressed genes that were down-regulated in response to DNR treatment were significantly enriched in mitochondrion function,and negative regulators of reactive oxygen species. Surprisingly,it was found that DNR-treated stromal cells secreted high levels of H2O2 into the culture supernatant. Furthermore,coculture of hematopoietic cells with DNR-treated stromal cells led to the accumulation of DNA damage as determined by the levels of histone H2AX phosphorylation and 8-oxo-2'-deoxyguanosine in hematopoietic cells. Overall,our results suggest that DNR-induced BM stromal cell damage can lead to genomic instability in hematopoietic cells. View Publication

The intestine is a highly radiosensitive tissue that is susceptible to structural and functional damage due to systemic as well as localized radiation exposure. Unfortunately,no effective prophylactic or therapeutic agents are available at present to manage radiation-induced intestinal injuries. We observed that the vanillin derivative VND3207 improved the survival of lethally irradiated mice by promoting intestinal regeneration and increasing the number of surviving crypts. Pre-treatment with VND3207 significantly increased the number of Lgr5+ intestinal stem cells (ISCs) and their daughter cells,the transient Ki67+ proliferating cells. Mechanistically,VND3207 decreased oxidative DNA damage and lipid peroxidation and maintained endogenous antioxidant status by increasing the level of superoxide dismutase and total antioxidant capacity. In addition,VND3207 maintained appropriate levels of activated p53 that triggered cell cycle arrest but were not sufficient to induce NOXA-mediated apoptosis,thus ensuring DNA damage repair in the irradiated small intestinal crypt cells. Furthermore,VND3207 treatment restores the intestinal bacterial flora structures altered by TBI exposure. In conclusion,VND3207 promoted intestinal repair following radiation injury by reducing reactive oxygen species-induced DNA damage and modulating appropriate levels of activated p53 in intestinal epithelial cells. View Publication