Immunoamphisomes in dendritic cells amplify TLR signaling and enhance exogenous antigen presentation on MHC-II

Autophagy, a specialized lysosomal degradation pathway, has proven to be a potent cell-autonomous defense mechanism against a range of intracellular microbes. In addition, autophagy emerged recently as a critical regulator of innate and adaptive immune responses. Links between autophagy and innate immunity are being progressively unveiled. For instance, several TLR (Toll-Like Receptor) agonists upregulate autophagy flux in immune cell types such as DC (dendritic cells) or macrophages. Conversely, and perhaps surprisingly, is the observation that TLR7-mediated responses might depend on autophagy in plasmacytoid DC, thus suggesting a more complex link between TLR-dependent responses and autophagy. Recently, the demonstration that NOD2 increases autophagy suggests that innate immune responses initiated via a broad range of pathogen recognition receptors can regulate autophagy. In addition to its involvement in innate immune responses, autophagy regulates adaptive immune responses via both MHC class I and class II molecules depending on the cellular context and the nature of the antigen.     

Steroids excreted in urine by neonates with 21-hydroxylase deficiency: Characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism.Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring.All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized.GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed.We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.     

microRNA-10b confers cisplatin resistance by activating AKT/mTOR/P70S6K signaling via targeting PPARγ in esophageal cancer

It is well known that the acquisition of chemoresistance is a major obstacle for the effective treatment of human cancers. It is reported that microRNAs (miRNAs) are implicated in chemotherapy resistance of various malignancies. miR-10b was previously proved as an oncogene in multiple malignancies, including esophageal cancer. However, its biological significance in regulating cisplatin (DDP) resistance in esophageal cancer is still elusive. Here, we observed that miR-10b expression was upregulated and peroxisome proliferator-activated receptor-γ (PPARγ) expression was downregulated in esophageal cancer tumor tissues and cells. PPARγ was proved as a functional target of miR-10b. Moreover, suppression of miR-10b enhanced the chemosensitivity of esophageal cancer cells to DDP in vitro and in vivo. In addition, PPARγ-mediated DDP sensitivity was weakened by miR-10b overexpression. Furthermore, miR-10b-activated AKT/mTOR/p70S6K signaling pathway through targeting PPARγ. Inactivation of AKT/mTOR/p70S6K by AKT inhibitor (GSK690693) attenuated miR-10b-induced DDP resistance in esophageal cancer cells. Taken together these observation, miRNA-10b-mediated PPARγ inhibition enhanced DDP resistance by activating the AKT/mTOR/P70S6K signaling in esophageal cancer, suggesting a potential target to improve therapeutic response of patients with esophageal cancer to DDP.     

Inhibition of Cell Proliferation and Migration by miR-509-3p That Targets CDK2, Rac1, and PIK3C2A

CDK2 is a key regulator of cell cycle progression. In this study, we screened for miRNAs targeting CDK2 using a luciferase-3′-untranslated region reporter assay. Among 11 hit miRNAs, miR-509-3p reduced CDK2 protein levels and significantly inhibited cancer cell growth. Microarray, Western blotting, and luciferase reporter analyses revealed additional targets of miR-509-3p, including Rac1 and PIK3C2A. Overexpression of miR-509-3p induced G1 cell-cycle arrest and inhibited colony formation and migration. RNAi experiments indicated that the growth-inhibitory effects of miR-509-3p may occur through down-regulation of CDK2, Rac1, and PIK3C2A. Targeting of multiple growth regulatory genes by miR-509-3p may contribute to effective anti-cancer therapy.     

27-Hydroxycholesterol enhanced osteoclastogenesis in lung adenocarcinoma microenvironment

27-Hydroxycholesterol (27-HC) has been implicated in the pathological process of estrogen receptor positive breast cancer. However, the role of 27-HC in lung adenocarcinoma is still unclear. Because bone metastasis is a main reason for the high mortality of lung adenocarcinoma, this study aimed to investigate the effect of 27-HC on osteoclastogenesis in lung adenocarcinoma microenvironment. The results showed that the conditioned media (CM) from lung adenocarcinoma cells cocultured with macrophages promoted osteoclast differentiation, which was enhanced by 27-HC. Further investigation showed that CM inhibited miR-139 expression and promoted c-Fos expression. Luciferase reporter assay identified c-Fos as a direct target of miR-139. CM also induced the expression and nuclear translocation of NFATc1 and STAT3 phosphorylation, which was enlarged by 27-HC but was attenuated by miR-139. Coimmunoprecipitation assay demonstrated that 27-HC increased the interaction between NFATc1 and phosphorylated STAT3, which was restricted by miR-139. Chromatin immunoprecipitation assay showed that pSTAT3 could bind to the promoter of c-Fos, c-Fos could bind to the promoter of NFATc1, and both pSTAT3 and NFATc1 could bind to the promoter of Oscar, which were enlarged by 27-HC but were blocked by miR-139. Knockdown of c-Fos mimicked the effect of miR-139. These results suggested that CM, especially containing 27-HC, promoted osteoclastogenesis by inhibiting miR-139 expression and activating the STAT3/c-Fos/NFATc1 pathway.     

Long noncoding RNA PTENP1 affects the recovery of spinal cord injury by regulating the expression of miR-19b and miR-21

Exosomes derived from differentiated P12 cells and MSCs were proved to suppress apoptosis of neuron cells, and phosphatase and tensin homolog pseudogene 1 (PTENP1) was reported to inhibit cell proliferation. In this study, we aimed to investigate the role of PTENP1 in the process of post-spinal cord injury (SCI) recovery, so as to evaluate the therapeutic effects of exosomes derived from MSCs transfected with PTENP1 short hairpin RNA (shRNA), as a type of novel biomarkers in the treatment of SCI. Electron microscopy was used to observe the morphology of different exosomes. Real-time polymerase chain reaction and western blot, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry, Nissl staining, immunohistochemistry assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were conducted to investigate and validate the underlying molecular signaling pathway. PTENP1-shRNA downregulated PTENP1 and PTEN while upregulating miR-21 and miR-19b. PTENP1-shRNA also accelerated cell apoptosis and reduced cell viability. In addition, PTENP1 reduced the miR-21 and miR-19b expression by directly targeting miR-21 and miR-19b. Meanwhile, both miR-21 and miR-19b reduced the expression of PTEN by directly targeting the 3′-untranslated region of PTEN. Furthermore, PTEN level and apoptosis index of neuron cells was the highest in the SCI group, while the treatment with exosomes+PTENP1-shRNA reduced the PTEN expression to a level similar to that in the sham group. Finally, PTENP1 inhibited miR-21 and miR-19b expression but upregulated PTEN expression. The upregulation of miR-21/miR-19b also suppressed the apoptosis of neuron cells by downregulating the PTEN expression. PTENP1 is involved in the recovery of SCI by regulating the expression of miR-19b and miR-21, and exosomes from PTENP1-shRNA-transfected cells may be used as a novel biomarker in SCI treatment.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

LncRNA882 regulates leukemia inhibitory factor (LIF) by sponging miR-15b in the endometrial epithelium cells of dairy goat

Despite the fact that long noncoding RNAs (lncRNAs) play roles in almost all biological processes, little is known about their biological function in the endometrium during the formation of endometrial receptivity. In this study, a comprehensive analysis of lncRNAs in goat endometrial tissues on Day 5 (prereceptive endometrium, PE) and Day 15 (receptive endometrium, RE) of pregnancy was performed by using RNA-Seq. As a result, 668 differentially expressed lncRNAs (DELs) were found between the PE and RE. Further study showed that lncRNA882, regulated by estrogen (E2) and progestin (P4), could act as competing endogenous RNAs (ceRNAs) for miR-15b, which inhibited the expression of transforming growth factor-b-activated kinase 1 binding protein 3 (TAB3) and then indirectly regulated the level of leukemia inhibitory factor (LIF). This was helpful for the formation of endometrial receptivity in dairy goats. In conclusion, we elucidated the endometrium lncRNA profiles of PE and RE in dairy goats; lncRNA882 acted as a ceRNA for miR-15b and then indirectly regulated the level of LIF in goat endometrial epithelium cells. Thus, this study helped us to better understand the molecular regulation of endometrial receptivity in dairy goats.     

Down-regulation of miR-17 family expression in response to retinoic acid induced neuronal differentiation

Whole-genome microRNA and gene expression analyses were used to monitor changes during retinoic acid induced differentiation of neuroblasts in vitro. Interestingly, the entire miR-17 family was over-represented among the down-regulated miRNA. The implications of these changes are considerable, as target gene prediction suggests that the miR-17 family is involved in the regulation of the mitogen-activated protein kinase (MAPK) signaling pathway, synaptic plasticity and other markers of neuronal differentiation. Significantly, many of the target responses predicted by changes in miRNA expression were supported by the observed changes in gene expression. As expected, markers of neuronal differentiation such as anti-apoptotic protein B-cell lymphoma 2 (BCL2), myocyte enhancer factor-2D (MEF2D) and zipper protein kinase (MAP3K12; aka ZPK/MUK/DLK) were each up-regulated in response to differentiation. The expression of these genes was also reduced in response to miR-17 and miR-20a transfection, and more specifically they were also shown to contain functional miRNA recognition elements for members of the miR-17 family by reporter gene assay. This suggests that the miR-17 family have an integral role in fine-tuning the pathways involved in the regulation of neuronal differentiation.