Up-regulated SAMD9L modulated by TLR2 and HIF-1α as a promising biomarker in tuberculosis.

The aim of this study was to identify potential biomarkers of TB in blood and determine their function in Mtb-infected macrophages. First of all, WGCNA was used to analyse 9451 genes with significant changes in TB patients' whole blood. The 220 interferon-γ-related genes were identified, and then 30 key genes were screened using Cytoscape. Then, the AUC values of key genes were calculated to further narrow the gene range. Finally, we identified 9 genes from GSE19444. ROC analysis showed that SAMD9L, among 9 genes, had a high diagnostic value (AUC = 0.925) and a differential diagnostic value (AUC>0.865). To further narrow down the range of DEGs, the top 10 hub-connecting genes were screened from monocytes (GSE19443). Finally, we obtained 4 genes (SAMD9L, GBP1, GBP5 and STAT1) by intersections of genes from monocytes and whole blood. Among them, it was found that the function of SAMD9L was unknown after data review, so this paper studied this gene. Our results showed that SAMD9L is up-regulated and suppresses cell necrosis, and might be regulated by TLR2 and HIF-1α during Mtb infection. In addition, miR-181b-5p is significantly up-regulated in the peripheral blood plasma of tuberculosis patients, which has a high diagnostic value (AUC = 0.969).

STAT1 and its related molecules as potential biomarkers in Mycobacterium tuberculosis infection.

Tuberculosis (TB) is a severe infectious disease that seriously endangers human health. The immune defence mechanism of the body against TB is still unclear. The purpose of this study was to find the key molecules involved in the immune defence response during TB infection, and provide reference for the treatment of TB and further understanding of the immune defence mechanism of the body. Data from GSE83456 were downloaded from GEO data sets for analysis, and a total of 192 differentially expressed genes were screened out. Most of these genes are enriched in the interferon signalling pathway and are defence response-related. We also found that STAT1 plays an important role in the immune defence of TB infection and it is one of the key genes related to interferon signalling pathway. STAT1-related molecules including hsa-miR-448, hsa-miR-223-3p, SAMD8_hsa_circRNA 994 and TWF1_hsa_circRNA 9897 were therefore screened out. Furthermore, expression levels of hsa-miR-448 and hsa-miR-223-3p were then verified by qRT-PCR. Results showed that both hsa-miR-448 and hsa-miR-223-3p were down-regulated in plasma from patients with pulmonary TB. Taken together, our data indicate that an mRNA-miRNA-circRNA interaction chain may play an important role in the infection of MTB, and STAT1 and related molecules including hsa-miR-223-3p, has-miR-448, SAMD8_hsa_circRNA994 and TWF1_hsa_circRNA9897 were identified as potential biomarkers in the development of active TB.

Differential transcriptional response in macrophages infected with cell wall deficient versus normal Mycobacterium Tuberculosis.

Host-pathogen interactions determine the outcome following infection by mycobacterium tuberculosis (Mtb). Under adverse circumstances, normal Mtb can form cell-wall deficient (CWD) variants within macrophages, which have been considered an adaptive strategy for facilitating bacterial survival inside macrophages. However, the molecular mechanism by which infection of macrophages with different phenotypic Mtb elicits distinct responses of macrophages is not fully understood. To explore the molecular events triggered upon Mtb infection of macrophages, differential transcriptional responses of RAW264.7 cells infected with two forms of Mtb, CWD-Mtb and normal Mtb, were studied by microarray analysis. Some of the differentially regulated genes were confirmed by RT-qPCR in both RAW264.7 cells and primary macrophages. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was used to analyze functions of differentially expressed genes. Distinct gene expression patterns were observed between CWD-Mtb and normal Mtb group. Mapt was up-regulated, while NOS2 and IL-11 were down-regulated in CWD-Mtb infected RAW264.7 cells and primary macrophages compared with normal Mtb infected ones. Many deregulated genes were found to be related to macrophages activation, immune response, phagosome maturation, autophagy and lipid metabolism. KEGG analysis showed that the differentially expressed genes were mainly involved in MAPK signaling pathway, nitrogen metabolism, cytokine-cytokine receptor interaction and focal adhesion. Taken together, the present study showed that differential macrophage responses were induced by intracellular CWD-Mtb an normal Mtb infection, which suggested that interactions between macrophages and different phenotypic Mtb are very complex. The results provide evidence for further understanding of pathogenesis of CWD-Mtb and may help in improving strategies to eliminate intracellular CWD-Mtb.

[Expression and bioinformatic analysis of miR-29 family, target gene IFN-γ in CD4(+) T cells from subjects with latent tuberculosis infection].

OBJECTIVE: To investigate the effect of latent and active pulmonary tuberculosis(TB) on expression of miR-29 family and target gene IFN-γ in CD4(+)T cells. METHODS: Subjects from two hospitals of Weifang were enrolled from March 2012 to December 2012 and divided into three groups: active TB group(30 cases), latent tuberculosis infection(LTBI) group(25 cases) and healthy control group(30 cases). CD4(+) T cells in blood were collected from the three groups.Levels of miR-29a, miR-29b and miR-29c were measured by nucleic acid hybridization and RT-qPCR.Expression of IFN-γ was analyzed by RT-qPCR. Target genes of miR-29 family were predicted with both TargetScan and PicTar.GO annotation and pathway overrepresentation were further analyzed with David database and Cytoscape. RESULTS: Levels of miR-29a, miR-29b and miR-29c showed significant differences among the three groups(P < 0.05): levels of miR-29b and miR-29c in the active TB group(561.63 ± 65.36, 281.85 ± 42.78) were higher than the healthy controls(260.74 ± 38.69, 128.21 ± 19.98), but lower than the LTBI group(2030.29 ± 321.68, 620.93 ± 79.14); expression of miR-29a in the healthy control group(913.95 ± 104.73) were higher than the active TB group(323.37 ± 54.38), but lower than the LTBI group(4782.13 ± 567.81).Level of IFN-γ showed significant differences among the three groups(P < 0.05): level of IFN-γ in the LTBI group(0.45 ± 0.09) were lower than the healthy controls(1.00), but higher than the active TB group(0.11 ± 0.03). The target genes of miR-29 family mainly existed in molecular function such as extracellular matrix structural constituent and transcription regulator activity.In KEGG pathway, the gene set mostly existed in signaling pathway such as Focal adhesion,ECM-receptor interaction and mTOR signaling pathway. CONCLUSION: The expression of miR-29 family was increased and target gene IFN-γ in CD4(+) T cells was decreased by latent and active pulmonary TB, which might play important role in alteration of signal pathway.

[Expression of miR-29a in serum of patients with pulmonary tuberculosis and prediction of its function with bioinformatics analysis].

OBJECTIVE: To analyze the expression of miR-29a in serum of patients with pulmonary tuberculosis, and to predict and analyze function of its target genes for further studying of its biological function and regulatory mechanism in tuberculosis (TB). METHODS: Fasting venous blood samples were collected from 65 untreated patients with active pulmonary TB (case group) and 45 healthy controls (control group) in the morning, respectively, and then sera were isolated. Total RNA was extracted with TRIzol reagent and was further purified. RNA quality was measured by gel electrophoresis and ultraviolet spectrophotometer. Nucleic acid hybridization and real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to investigate expression of miR-29a in serum. Both TargetSean and PicTar software were used to predict comprehensively target genes of miR-29a and their intersection was regarded as target genes of miR-29a. Gene ontology of target genes was analyzed with DAVID database and three category annotations were extracted, respectively. GO overrepresentation was further analyzed by BINGO of Cytoscape. Enriched pathways of target genes were analyzed. RESULTS: The hybridization result showed that miR-29a was increased in serum of the case group (854 ± 93) compared to the control group (80 ± 22) (t = 3.541, P < 0.05). The PCR result showed that compared to the control group (0.18 ± 0.07), miR-29a was increased in serum of the case group (1.35 ± 0.62) (t = 2.987, P < 0.05). The target genes were mainly involved in biological processes including regulation of transcription, molecular function including metal ion binding, and cellular components including extracellular matrix, respectively. In the KEGG pathway, the target gene set was significantly enriched in the 7 signaling pathways including adhesion, ECM-receptor interaction and so on. In the PANTHER pathway, the gene set mostly existed in integrin signaling pathway, cadherin signaling pathway and Wnt signaling pathway. CONCLUSIONS: Level of miR-29a was increased significantly in serum of patients with active pulmonary tuberculosis. Target genes of miR-29a were mainly involved in biological processes including cell adhesion, regulation of transcription and so on.

[Preparation of polyclonal antibody and prokaryotic expression of recombinant protein RelA from Mycobacterium tuberculosis].

AIM: To prepare polyclonal antibodies against RelA protein of Mycobacterium tuberculosis. METHODS: RelA gene segment was inserted into pET-32a(+) and the recombinant protein RelA was expressed in E.coli under IPTG induction.The protein was purified and identified by SDS-PAGE and Western blot.Polyclonal antibody to RelA was got by immunizing rabbits with the protein. Quality and quantity of the antibody was identified. RESULTS: RelA gene segment was successfully inserted into pET-32a(+) and recombinant protein RelA was obtained.The polyclonal antibody to RelA had a good specificity, and the titer reached more than 1:6 400. CONCLUSION: RelA recombinant protein and rabbit anti-RelA polyclonal antibody with high specificity were obtained, which provided good tools for further studying functional characterization of RelA.

Effects of Bifidobacterium bifidum on adaptive immune senescence in aging mice.

Bifidobacteria are a natural component of the bacterial flora of the human body and have a symbiotic bacteria-host relationship with human beings. Aging is associated with reduced numbers of beneficial colonic Bifidobacteria and impaired immunity. The possible anti-senescence effects of Bifidobacteria are presently unknown. The aims of the present study were to investigate possible anti-senescence effects of B. bifidum on naturally senescent mice and to explore their mechanisms. After treatment with B. bifidum, mice were killed and samples collected. Cytokine production in serum and lymphocyte culture supernatant, anti-oxidation activity and gene expression were measured. B. bifidum significantly increased cytokine IL-2 and IFN-γ levels but decreased proinflammatory cytokines IL-6 and TNF-α concentrations. Moreover, B. bifidum improved anti-oxidation activity and reduced lipid peroxidation in thymus and spleen. In addition, B. bifidum down-regulated p16 expression in thymus and spleen. Taken together, the results indicate, for the first time, that B. bifidum delays senescence by several mechanisms, including enhancement of anti-oxidation activity in thymus and spleen, alteration of gene expression and improvement in immune function.

Effect of LTA isolated from bifidobacteria on D-galactose-induced aging.

BACKGROUND: Bifidobacteria are a natural part of the bacterial flora in the human body and have a symbiotic bacteria-host relationship with human beings. Aging is associated with reduced number of beneficial colonic bifidobacteria and impaired immunity. Lipoteichoic acid is a major constituent of the cell wall of bifidobacteria which is important for bacterial survival, growth, and function. The possible anti-aging effects of lipoteichoic acid isolated from bifidobacteria is presently unknown. OBJECTIVE: The aim of the present study was to investigate possible anti-aging effects of lipoteichoic acid isolated from bifidobacteria on senescent mice artificially induced by chronic injection of d-galactose and explore potential anti-aging's mechanisms. METHODS: Mice were artificially induced senescence by consecutive injection of d-galactose (100mg/kg) once daily for 7weeks and lipoteichoic acid from bifidobacterium bifidum, was simultaneously administered to them once a week by intraperitoneal infusion. Mice were sacrificed, blood and other samples were collected at the indicated time. Anti-oxidation activity in brain, histology of tissue, gene expression, lymphocyte's DNA damage and cytokine production of lymphocytes in vitro and in vivo were measured. RESULTS: Lipoteichoic acid could significantly improve general appearance of the aging model mice, improve anti-oxidation activity in brain, increase IL-2 level and decrease TNF-alpha level in vitro and in vivo, respectively. Besides, LTA remarkably inhibited DNA damage in the both splenic lymphocytes and circulating lymphocytes. Moreover, LTA could decrease p16 expression while increase c-fos expression in the d-galactose treated mice. CONCLUSION: Taken together, the results indicated, for the first time, that LTA could suppress the aging process via the following several mechanisms, including enhancement of anti-oxidation activity in brain, improvement of immune function and alteration of gene expression.

[Effect of human granulysin on apoptosis, mitochondrial transmembrane potential and cytochrome C release of SMMC-7721 cells].

OBJECTIVE: To construct a plasmid carrying granulysin (GLS) and to study the effect of the GLS on apoptosis, mitochondrial transmembrane potential and cytochrome C release of SMMC-7721 cells. METHODS: The coding sequence of the GLS was amplified from the total RNA of human CTL cells, and it was inserted into pBudCE4.1 plasmid and then it was used to transfect SMMC-7721 cells. The expression of GLS was detected by RT-PCR and confirmed by immunocytochemistry method. Cell apoptosis was ascertained by Hoechst staining and electron microscopy; mitochondrial transmembrane potential was detected using Mitocapture and cytochrome C release was studied using Western blot. RESULTS: Recombinant pBudCE4.1/GLS plasmid was successfully constructed. GLS protein was successfully expressed in the SMMC-7721 cells and it induced apoptosis of the SMMC-7721 cells, and at the same time, mitochondrial transmembrane potential was reduced and cytochrome C was released from mitochondria into the cytosol. CONCLUSIONS: GLS gene carried by recombinant plasmid could express in SMMC-7721 cells and induce cells apoptosis. The change of mitochondrial transmembrane potential and the release of cytochrome C might be one of the key factors of apoptosis induced by GLS.

[Quantitative analysis of nuclear proteome in apoptosis hepatoma cells induced by hydroxycamptothecin].

The experimental foundation for investigating the pharmacological action of hydroxycamptothecin at subcellular quantitative proteomic level can be obtained depending on the information of differentially expressed nuclear proteins in hydroxycamptothecin-treated cells and control cells. The apoptosis was induced by hydroxycamptothecin in hepatoma cells, the nucleus of cells were isolated and verified with western blot. Nuclear proteins labelled with cleavable isotope-coded affinity tag (c-ICAT) reagent were digested and purified. The expression ratio of the identical nuclear protein derived from apoptosis cell and control cell can be gained using shotgun proteomic method based on multiple dimensional liquid chromatography-linear ion trap/orbitrap mass spectrometer combined with c-ICAT strategy. A total of 42 nuclear proteins were significantly (P<0.05) altered in hydroxycamptothecin-treated cells, among them, 12 proteins showed significantly down-regulation, and 30 proteins showed up-regulation compared with control cells. The function of these proteins were likely involved in life processes of cells such as proliferation, apoptosis, differentiation, energy metabolism, nucleic acid synthesis and metabolism, structure of cell skeleton.

[A quantitative analysis of mitochondrial protein differential expressions in hydroxycamptothecin-treated hepatoma cells].

OBJECTIVES: To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by using quantitative proteome. METHODS: SMMC-7721 cell apoptosis was induced by HCPT and the mitochondria were isolated with a mitochondria isolation kit. Mitochondrial proteins labeled with a cleavable isotope-coded affinity tag were identified and quantified using two-dimensional liquid chromatography/tandem mass spectrometry. RESULTS: Highly purified mitochondria were obtained. Seventy-four mitochondrial proteins, which were statistically significantly altered (P less than 0.05) in HCPT-treated cells, were identified and analyzed. A total of 42 proteins were significantly down-regulated, and 32 were up-regulated in the cells that responded to apoptosis. The functions of these proteins were likely involved in cell energy metabolism, nucleic acid translation and transcription, cytoskeleton, etc. CONCLUSION: Our results about the information of differentially expressed mitochondrial proteins in HCPT-treated cells and the control cells will help to understand the mechanism by which HCPT induces cell apoptosis. The integrated techniques we used in this study will be helpful to the investigation of subcellular quantitative proteomics.

[Proteomic analysis of mitochondrial proteins in hydroxycamptothecin-treated SMMC-7721 cells].

OBJECTIVE: To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by comparative proteomic analysis. METHODS: Apoptosis of SMMC-7721 cells were induced by using HCPT and their mitochondria were isolated with a mitochondria isolation kit for cultured cells. Three different solubility protein fractions were extracted with ReadyPrep Sequential Extraction Kit and were separated by two-dimensional gel electrophoresis (2-DE). PDQuest software was used to differentiate mitochondrial proteins between control cells and HCPT-treated cells. Matrix assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) was used to identify some of the different proteins. RESULTS: Highly purified mitochondria and high resolution 2-DE patterns of the proteins were obtained. Forty-four mitochondrial protein spots from the HCPT-treated cells showed different expressions compared to those of the control cells. Twenty of the different protein spots were analyzed by MALDI-TOF-MS. CONCLUSION: Differently expressed mitochondrial proteins in HCPT-treated cells and control cells were obtained in this study. This will be of help to understand the mechanism by which HCPT induces cell apoptosis.

[Cloning and expression of recombinant mitochondrial Delta1-120 apoptosis-inducing factor and its enhancive effect on apoptosis of hepatocellular carcinoma cell line SMMC-7721].

BACKGROUND & OBJECTIVE: Mitochondria play a key role in cell apoptosis, and apoptosis-inducing factor (AIF) is a kind of apoptotic protein located in mitochondria. The research on mitochondrial protein can be helpful for elucidating the role of mitochondria in apoptosis. This study was to clone and express recombinant human Delta1-120 AIF and validate its biological activities of binding DNA and inducing nuclear apoptosis. METHODS: A human AIF gene fragment of 1515 bp(mitochondrial localization sequence was deleted) was amplified from SMMC-7721 cells by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pET32a(+) vector to construct recombinant plasmid pET32a-AIF. The recombinant plasmid was transfected into E.coli BL2l (DE3). AIF expression was induced by isoprophylthio-beta-D-galactoside (IPTG), and detected by SDS-PAGE and Western blot. AIF protein was purified by Ni afinity chromatography and then renatured. The biological activity of renatured AIF protein was detected by electrophoretic mobility shift assay (EMSA) and Hoechst staining. RESULTS: The 1.5 kb AIF gene was successfully isolated, and cloned into pET32a(+) vector. Plasmid pAIF was identified by restrictive enzyme analysis and sequencing. Recombinant E.coli. strains expressing AIF were obtained. AIF protein amounted to 11% of the total bacterial protein when induced with IPTG at 37 degrees Celsius for 4 h. AIF was specially recognizing by anti-AIF and anti-his antibody. The purity of purified protein reached over 95%. After renaturation, AIF protein binded DNA and induced nuclear apoptosis. CONCLUSION: AIF protein with high purity and biological activity was obtained by the method described above.

Changes in the protein spectrum of mitochondria isolated from hydroxycamptothecin-treated hepatoma cells.

As one of the most potent topoisomerase inhibitors, hydroxycamptothecin is more active and less toxic than conventional camptothecin. Recently, we found that hydroxycamptothecin can induce cell apoptosis via the mitochondrial pathway. This study was designed to investigate the mitochondrial protein profile in HCPT-treated cells using high-accuracy and high-sensitivity protein-identification technology. Of the 39 mitochondrial protein spots investigated, 25 displayed elevated and 14 suppressed abundance in hydroxycamptothecin-treated cells. The 25 spots were identified by mass spectrometry and they included proteins involved in many essential cellular functions. The potential role of these proteins in hydroxycamptothecin-mediated apoptosis is also discussed. This study has produced a short list of mitochondrial proteins that might hold the key to the mechanism by which hydroxycamptothecin induces mitochondrial dysfunction and cell apoptosis. It has laid the foundation for further elucidating the role of hydroxycamptothecin during apoptosis. Successful applications of multiple techniques including two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Western blot analysis have demonstrated that proteomic analyses provide appropriate approaches for understanding of the roles of anticancer drugs.

Hydroxycamptothecin-induced apoptosis in hepatoma SMMC-7721 cells and the role of mitochondrial pathway.

The camptothecin (CPT) derivative hydroxycamptothecin (HCPT) containing 10-hydroxy represents one of the most potent topoisomerase I inhibitors described. This anticancer agent, currently undergoing clinical trials on gastric tumours, has been shown more active and less toxic than conventional camptothecins. To shed light on the mechanism of action of HCPT at the cellular level, we examined cell growth, apoptosis, changes of mitochondrial membrane potential, cytochrome c and AIF translocation in cancer cells by exposing these cells to HCPT for indicated time. The effect of HCPT on cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay and apoptosis was measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes of mitochondrial membrane potential were monitored by fluorescence microscope. Western blot analysis was used to evaluate the release of mitochondrial cytochrome c and AIF; On the other hand, translocation of cytochrome c and AIF from mitochondria to cytosol during apoptosis were confirmed by confocal microscopy. HCPT could noticeably inhibit the proliferation of SMMC-7721cells and the IC(50) dose was about 0.22 microM; SMMC-7721 cells treated with HCPT showed typical characteristics of apoptosis rather than necrotic including phosphatidylserine (PS) exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology, chromatin condensation and nuclear fragmentation; On the other hand, during process of cell apoptosis, mitochondrial transmembrane potential was reduced; Compared with the control group, the mRNA and protein expression of cytochrome c and AIF in treated and untreated SMMC-7721 cells were not significantly changed (not shown). However, when cells were treated with HCPT, the massive translocation of cytochrome c and AIF to the nucleus was evident. Our results indicate that HCPT can inhibit proliferation and induce apoptosis of human hepatoma SMMC-7721 cells. Mitochondrial pathway of apoptosis, especially for cytochrome c and AIF translocation, may play an important role in apoptosis induced by HCPT.

[Effect of hydroxycamptothecin on apoptosis-inducing factor (AIF) expression and on AIF translocation in human hepatocellular cancer cell SMMC-7721].

OBJECTIVE: To study the effect of hydroxycamptothecin (HCPT) on apoptosis-inducing factor (AIF) expression and AIF translocation from mitochondria to the nucleus in human hepatocellular cancer cell SMMC-7721 during apoptosis. METHODS: After treatment with 80 mg/ml of HCPT, the cancer cells were stained with A0/EB to monitor their apoptosis. Their mitochondria was examined with electronmicroscopy and the AIF expression of the cells was tested by RT-PCR and Western blot. The translocation of AIF from mitochondria to the nucleus during apoptosis was analyzed by confocal microscopy. RESULTS: SMMC-7721 cells treated with HCPT showed chromatin condensation, nuclear fragmentation and mitochondria swelling. The mRNA and protein expression of AIF in treated and untreated SMMC-7721 cells were not significantly different. However, cells treated with 80 mg/ml HCPT for 6 h or 12 h showed massive translocation of AIF into the nuclei. CONCLUSION: These results show the important role the mitochondrial pathway of apoptosis plays in HCPT-induced tumor cell death, at least in SMMC-7721 cells.

[Determination of catalpol in dried rehmannia root and Taohong Siwu Decoction with high performance liquid chromatography].

OBJECTIVE: To determinate the catalpol contents in dried rehmannia root and Taohong Siwu Decoction containing rehmannia root with high performance liquid chromatography (HPLC). METHODS: Catalpol was separated on a YWG-C18 column using water-acetonitrile (99.4:0.6) as mobile phase and detective wavelength at 210 nm. RESULTS: The linear curve of tested catalpol concentration within the range of 0.0536-5.3600 microg/microl was ideal (n=5, r=0.999 7). The average recovery rate of the dried rehmannia root and Taohong Siwu decoction was 98.7% (RSD=0.48%) and 98.2% (RSD=1.29%) respectively. CONCLUSION: HPLC method is accurate and valuable for the quality control of Radix Rehmanniae and Taohong Siwu Decoction.