Identification of miRNA-mRNA Pairs in Relation to TNF-α/IL-1ß Induced Inflammatory Response in Intervertebral Disc Degeneration.

Objective: The determination of miRNA-mRNA pairs for intervertebral disc degeneration (IVDD) regulated by pro-inflammatory cytokines were investigated. Methods: Two dataset (accession number GSE27494 and GSE41883 from platform GPL1352) of expression profiling was downloaded from Gene Expression Omnibus (GEO). The annulus cells were isolated from annulus fibrosus in patients with degenerative disc disease. The cells were then cultured in a three-dimensional (3D) collagen containing with/without proinflammatory cytokines (tumor necrosis factor alpha (TNF-α) or interleukin beta (IL-1ß)). After being cultured for 14 days, the isolated total RNA was analyzed via microarray, and the expression array data were obtained using BRB-Array Tools followed by analyzing the differentially expressed genes (DEGs) and the prediction of potential miRNA targets of hub genes through online database. Results: Firstly, 52 and 296 DEGs were found in IL-1ß- and TNF-α-induced annulus cells, respectively, of these there had 42 common DEGs (co-DEGs) with 34 increased transcripts and 8 reduced ones. Based on the GO and KEGG software, these co-DEGs were mainly enriched in the response to lipopolysaccharide (LPS) and molecule of bacterial origin, the regulation of receptor ligand activity and signaling receptor activator activity, as well as the following signaling pathways, including TNF signaling pathway, IL-17 signaling pathway, and NF-κB signaling pathway. Top hub genes (CXCL1, CXCL2, CXCL8, IL1Β and PTGS2) regulated by several potential microRNAs were involved in TNF-α/IL-1ß treated annulus cells. Conclusions: Several candidate genes regulated by miRNAs caused by TNF-α/IL-1ß in the annulus cells were found, which will guide diagnosis and treatment for degenerative disc disease.

The cytotoxicity study of praziquantel enantiomers.

Praziquantel (PZQ) is prescribed as a racemic mixture (racemic-PZQ, rac-PZQ), which is composed of (R)-PZQ and (S)-PZQ. In this work, the cytotoxicity of rac-PZQ and its two enantiomers (R)-PZQ and (S)-PZQ on eight cell lines (L-02, HepG2, prf-plc-5, SH-SY5Y, HUVEC, A549, HCT-15, Raw264.7) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide and lactate dehydrogenase assays. The morphology of apoptotic cells was studied by fluorescence microscope using Hoechst 33342 staining, and the cytotoxicity of the compounds was also tested by lactate dehydrogenase assay. Results revealed that (R)-PZQ had negligible cytotoxicity against L-02, SH-SY5Y, HUVEC, A549, HCT-15, and Raw264.7 cells but selectively inhibited tumor cell lines (prf-plc-5 and HepG2). However, in contrast to (R)-PZQ, the (S)-isomer showed higher cytotoxicity against L-02 cells and lower inhibition on prf-plc-5 and HepG2 cells. Besides, (R)-PZQ showed lower cytotoxicity on SH-SY5Y cells than (S)-PZQ. Meanwhile, (R)-PZQ at <80 µM concentration could promote proliferation of macrophage cells (Raw264.7). Our research revealed that (R)-PZQ has lower cytotoxicity than (S)-PZQ and has similar cytotoxicity with rac-PZQ. (S)-PZQ is the principal enantiomer to cause side effects on human definitive hosts. These findings gave the reasonable reasons for World Health Organization to produce (R)-PZQ as a replacement for rac-PZQ for the treatment of schistosomiasis.

Fluoroalkane thioheterocyclic derivatives and their antitumor activity.

Two series of novel trifluorobutenyl derivatives of heterocyclic with convenient and efficient synthesis methods and their antitumor activity on three cell lines have been reported for the first time. The derivatives were synthesized by the nucleophilic substitution between 4-bromo-1,1,2-trifluorobutene-1-ene and commercially available nitrogen-containing heterocycles with sulfydryl or monosubstituted malononitrile. The twenty-four new compounds were characterized by (1)HNMR, (13)CNMR and HR-MS. Totally, thirty-seven compounds were evaluated for the antitumor activity on three cancer cell lines (SH-SY5Y, MCF-7 and HepG2) using conventional MTT assay. The pharmacological results indicated that the compounds 3c, 3h, 4c, 8, 9, 10 and 11 showed potent to moderate antitumor activity against three cancer cell lines, with IC50 values ranging between 0.4 µM and 41.5 µM. Even though they had less active than the reference compound taxol against MCF-7 and HepG2 lines, but they were better than the reference compound noscapine against SH-SY5Y cells, especially the compound 3h with a IC50 value of 0.4 µM.

LAP3 promotes glioma progression by regulating proliferation, migration and invasion of glioma cells.

Leucine aminopeptidase 3 (LAP3), belonging to the M1 family, has been proved to catalyze the hydrolysis of leucine residues. Leucine aminopeptidases are involved in many pathological disorders and regulate cell proliferation, invasion and/or angiogenesis of tumor. Recent study showed that LAP3 is highly expressed in several malignant and affects tumor angiogenesis. We aimed at investigating the clinical significance of LAP3 expression in human gliomas and its biological function in glioma cells. RT-PCR, Western blot and immunohistochemistry analysis were used to detect the expression levels of LAP3 in 121 glioma tissues, high LAP3 expression was correlated with the grade of malignancy and poor prognosis of glioma patients. In vitro, after increasing of LAP3 by myc-LAP3 transfection and knockdown of LAP3 by siRNA transfection in glioma cells, cell viability, cell cycle, migration and invasion were determined with CCK8 assay, flow cytometry, wound healing and transwell invasion assays. The results indicated that increasing LAP3 could promte cell viability, cell cycle, migration and invasion, knockdown LAP3 could decrease cell viability, suppress cell proliferation, migration and invasion. Our findings uncover that LAP3 might be a new prognostic factor and be close correlation with glioma cell growth, migration, invasion.

Expression of NF45 correlates with malignant grade in gliomas and plays a pivotal role in tumor growth.

Nuclear factor of activated T cells 45 kDa (NF45), is a transcription factor that interacts with NF90 to regulate gene expression. It has been proved to be associated with tumor proliferation in various human malignancies. However, the role of NF45 in glioma is poorly understood. The aim of our study was to examine the relationship between NF45 expression and pathological grade in glioma and the impact of NF45 on the proliferation of glioma cells. Expression levels of NF45 are significantly elevated in high-grade human tissue samples compared with low-grade human glioma tissues samples (P < 0.0001) in Western blot analysis. The result of immunohistochemical also revealed that the expression of NF45 was overexpressed in 121 resected gliomas of different pathologic grades and associated with Ki-67. To investigate the role of NF45 in glioma carcinogenesis, we reduced the expression of NF45 by small interfering RNAs, and results showed suppression of cell proliferation, arrest of the cell cycle, and reduction in clone in vitro. Importantly, we show that SiNF45 can induce the expression of p21 and reduce the expression of proliferating cell nuclear antigen (PCNA) and cyclin E. These findings indicate that NF45 plays an important role in the growth regulation of glioma cells, suggesting that NF45 maybe a molecular marker for pathology and a novel therapeutic target for malignant glioma.

Innate immune responses of pulmonary epithelial cells to Burkholderia pseudomallei infection.

BACKGROUND: Burkholderia pseudomallei, a facultative intracellular pathogen, causes systemic infection in humans with high mortality especially when infection occurs through an infectious aerosol. Previous studies indicated that the epithelial cells in the lung are an active participant in host immunity. In this study, we aimed to investigate the innate immune responses of lung epithelial cells against B. pseudomallei. METHODOLOGY AND PRINCIPAL FINDINGS: Using a murine lung epithelial cell line, primary lung epithelial cells and an inhalational murine infection model, we characterized the types of innate immunity proteins and peptides produced upon B. pseudomallei infection. Among a wide panel of immune components studied, increased levels of major pro-inflammatory cytokines IL-6 and TNFalpha, chemokine MCP-1, and up-regulation of secretory leukocyte protease inhibitor (SLPI) and chemokine (C-C motif) ligand 20 (CCL20) were observed. Inhibition assays using specific inhibitors suggested that NF-kappaB and p38 MAPK pathways were responsible for these B. pseudomallei-induced antimicrobial peptides. CONCLUSIONS: Our findings indicate that the respiratory epithelial cells, which form the majority of the cells lining the epithelial tract and the lung, have important roles in the innate immune response against B. pseudomallei infection.

Structural and biological diversity of lipopolysaccharides from Burkholderia pseudomallei and Burkholderia thailandensis.

Burkholderia pseudomallei, the etiological agent of melioidosis, is a facultative intracellular pathogen. As B. pseudomallei is a gram-negative bacterium, its outer membrane contains lipopolysaccharide (LPS) molecules, which have been shown to have low-level immunological activities in vitro. In this study, the biological activities of B. pseudomallei LPS were compared to those of Burkholderia thailandensis LPS, and it was found that both murine and human macrophages produced levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-10 in response to B. pseudomallei LPS that were lower than those in response to B. thailandensis LPS in vitro. In order to elucidate the molecular mechanisms underlying the low-level immunological activities of B. pseudomallei LPS, its lipid A moiety was characterized using mass spectrometry. The major lipid A species identified in B. pseudomallei consists of a biphosphorylated disaccharide backbone, which is modified with 4-amino-4-deoxy-arabinose (Ara4N) at both phosphates and penta-acylated with fatty acids (FA) C(14:0)(3-OH), C(16:0)(3-OH), and either C(14:0) or C(14:0)(2-OH). In contrast, the major lipid A species identified in B. thailandensis was a mixture of tetra- and penta-acylated structures with differing amounts of Ara4N and FA C(14:0)(3-OH). Lipid A species acylated with FA C(14:0)(2-OH) were unique to B. pseudomallei and not found in B. thailandensis. Our data thus indicate that B. pseudomallei synthesizes lipid A species with long-chain FA C(14:0)(2-OH) and Ara4N-modified phosphate groups, allowing it to evade innate immune recognition.

Identification of HLA-A*0201-restricted cytotoxic T lymphocyte epitope from TRAG-3 antigen.

PURPOSE: Identification of tumor antigen and subsequent identification of T-cell epitope from these antigens make specific immunotherapy for malignant tumor applicable. Because TRAG-3 antigen is expressed in most melanomas and 54% of non-small cell lung carcinomas and HLA-A2.1-expressing individuals cover >50% in the population of China, we aim at identifying TRAG-3-encoded peptide presented by HLA-A2.1. EXPERIMENTAL DESIGN: In our study, a HLA-A2.1-restricted CTL epitope was identified by using the following four-step procedure: (a) computer-based epitope prediction from the amino acid sequence of TRAG-3 antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward LB373-MEL cells expressing TRAG-3 antigen and HLA-A2.1. RESULTS: Of the four tested peptides, effectors induced by a peptide of TRAG-3 at residue position 58-66 lysed LB373-MEL cells expressing both TRAG-3 and HLA-A2.1. Our results indicate that peptide TRAG-3(58 approximately 66) (ILLRDAGLV) is a new HLA-A2.1-restricted CTL epitope capable of inducing TRAG-3 specific CTLs in vitro. CONCLUSIONS: Because TRAG-3 is a cancer/testis antigen expressed in most melanomas and half of non-small cell lung carcinomas, identification of the TRAG-3/HLA-A2.1 peptide ILLRDAGLV may facilitate peptide-based specific immunotherapy for various histological tumors.