DJ-1 interacting proteins analyzed by tandem affinity purification mass spectrometry / 中国医师杂志

Objective:RNA interference technology (siRNA) was used to inhibit the expression of DJ-1 gene in lung squamous cell carcinoma HTB-182 cells, then, tandem affinity purification mass spectrometry (TAP-MS) was performed to screen the interacting proteins of DJ-1 in lung cancer cell line of HTB-182.Methods:The siRNA lentivirus vector targeting DJ-1 gene was constructed to infect HTB-182 cells (DJ-1 siRNA group), and the lentivirus vector control group (control siRNA group) and blank control group were established. The expression level of DJ-1 protein was detected by Western blot, and the endogenous DJ-1 protein silenced si-DJ-1-HTB-182 cells were established. The specific primers of DJ-1 were designed, and the DJ-1 expression plasmid pNTAP-DJ-1 with streptomycin binding peptide label (SBP) and calmodulin binding peptide label (CBP) was constructed. The cell line DJ-1 siRNA HTB-182 was stably transfected with liposome, and the positive clones were screened by G418. The positive clones were verified by Western blot, and the interacting proteins of DJ-1 were found by TAP-MS.Results:The protein expression of DJ-1 in DJ-1 siRNA interference group was significantly lower than that in empty plasmid group and blank control group ( P<0.05); HTB182 cell line stably expressing pNTAP-DJ-1 plasmid was successfully constructed; Three proteins interacting with DJ-1 were screened by TAP-MS: cytokeratin 1 (keratin 1), cytokeratin 10 (keratin 10) and NADPH oxidase activating protein P47 (P47 Px). Conclusions:Keratin 1, Keratin l0 and P47 Px protein may be DJ-1 interactions protein.

Expression change of SH2B1, SOCS3, PTP1B and NPY in mice hypothalamus and its relation with obesity / 中南大学学报(医学版)

OBJECTIVE@#To investigate the expression pattern of adapter protein with a Src-homology 2 domain (SH2B1), the suppressor of cytokine signaling-3 (SOCS3), protein-tyrosine phosphatase 1B (PTP1B) and neturopetide Y (NPY) in obese and normal mice hypothalamus and its relation with serum leptin and insulin levels.@*METHODS@#The obesity animal model was prepared with healthy C57/bl6 mice. Lee's index and Homeostasis model assessment-insulin resistance (HOMA-IR) were calculated. The mRNA levels of SH2B1, SOCS3, PTP1B and NPY were measured by fluorescent quantitation RT-PCR. The SH2B1 and NPY protein expressions were detected by Western blot.@*RESULTS@#Compared with the normal mice of the same age, SH2B1 mRNA expression in the obese mice hypothalamus decreased. SOCS3 and PTP1B mRNA expression increased. Western blot showed that SH2B1 protein expression decreased, while NPY protein expression increased in the obese mice. Linear correlation analysis showed that the serum leptin and fasting insulin levels were negatively correlated with SH2B1mRNA expression and positively correlated with SOCS3 and PTP1B mRNA expression.@*CONCLUSION@#SH2B1, SOCS3, PTP1B and NPY are key factors for obesity development.

Effect of DJ-1 siRNA on biological behavior of human lung squamous carcinoma SK-MES-1 cells / 中南大学学报(医学版)

OBJECTIVE@#RNA interference technology (siRNA) was used to inhibit the expression of DJ-1 gene in lung squamous cell carcinoma SK-MES-1 cells, and the cell biological behaviors were investigated to explore the function of DJ-1 gene.@*METHODS@#A targeted DJ-1 siRNA lentiviral vector with a green fluorescent protein (GFP) as a reporter was constructed. The constructed DJ-1 siRNA and control-siRNA vectors were infected into SK-MES-1 cells as experimental (DJ-1 siRNA) and control (Control siRNA) groups, respectively. The DJ-1 protein expression was determined by Western blot. The cell proliferation capability was measured with methyl thiazolyl tetrazolium (MTT). The cell cycle was analyzed by flow cytometry. The capability of cell migration was determined by Transwell method.@*RESULTS@#Compared with control-siRNA and blank-control groups, the protein expression of DJ-1 gene was down-regulated, the capability of cell proliferation was obviously inhibited (P<0.01), the cell cycle was arrested with increased number of G1- and G2-phase cells and reduced number of S-phase cells, and the capability of cell migration was significantly decreased (P<0.01) in the DJ-1 siRNA-infected cells.@*CONCLUSION@#DJ-1 gene might play a role in promoting cell proliferation and cell migration capability in vitro in lung cancer SK-MES-1 cells.

Preliminary study of the prohibitin protein and paclitaxel resistance in ovarian cancer / 中南大学学报(医学版)

OBJECTIVE@#To determine the effect of RNA interference with transferred pshRNA/PHB on the biological characteristics of paclitaxel-resistant ovarian cancer cell lines.@*METHODS@#Western blot and real time-PCR were used to assay the expression of PHB protein and mRNA in SKOV3/Taxol-25 and SKOV3 cell lines. The SKOV3/Taxol-25 cell lines were transiently transfected by 3 target-specific small hairpin RNA (shRNA) interference fragments with fluorescent protein named the pshRNA427/PHB1, pshRNA248/PHB2, and pshRNA136/PHB3. The empty plasmid transfection via vehicle Lipofectamine2000 served as a negative control. The expression levels of PHB protein and mRNA were detected by Western blot and real time-PCR after the transfection for 48 h. The silence effect of PHB1 and PHB3 groups was obvious. PHB1, PHB3, and the negative control groups were used for the following experiments. MTT and flow cytometry assay were used to test the cell proliferation, IC50 of paclitaxel, and cell apoptosis in the 3 groups.@*RESULTS@#The expression levels of PHB protein and mRNA (2(-ΔΔCt)) were significantly higher in SKOV3/Taxol-25 cell line than those in SKOV3 cell line (P<0.05). The expression levels of PHB protein and mRNA were significantly lower in the PHB1 and PHB3 groups than those in the negative control group (P<0.05). The cell proliferations in the PHB1 and PHB3 groups were obviously slower than those in the negative control group after transfection for 48 h and 72 h (P<0.05). The IC50 of paclitaxel in the PHB1 and PHB3 groups significantly decreased after transfection for 72 h compared with the negative control group(P<0.05). The cell apoptotic rate in the PHB1 and PHB3 groups significantly increased after transfection for 48 h compared with the negative control group (P<0.05).@*CONCLUSION@#The shRNA/PHB can effectively suppress the expression of PHB gene in paclitaxel-resistant ovarian cancer cell lines. The cell proliferation in paclitaxel-resistant cell lines with removed PHB gene is significantly reduced. The apoptotic rate and the paclitaxel sensitivity of resistant cell lines with removed PHB gene are significantly increased. PHB gene is related to paclitaxel-resistance and interfering PHB gene expression may reduce paclitaxel resistance in ovarian cancer.

Screening for methylation-silenced genes in acute myeloid leukemia HL-60 cell line by a quantitative proteomic approach / 中南大学学报(医学版)

Objective To screen for new methylation association genes in HL-60 to reveal the pathogenesis of leukemia, and provide important theoretical and scientific basis for the prevention and cure of leukemia. Methods Two-dimensional fluorescence difference gel electrophoresis (F-2D-DIGE) was performed to separate the total proteins from acute myelogenous leukemia (AML) cell line HL-60 cells with or without 5-aza-2-deoxycytidine (5-aza-2-dC) treatment. Imaging software Decyder 6.5 and PDQuest were used to detect the differential expression protein spots, and matrix-assisted laser desorption/ionizaion time-of-flight mas spectrometer (MALDI-TOF MS) was adopted to identify the differential expression proteins. Results F-2D-DIGE maps of 5-aza-2-dC-untreated HL-60 and-treated HL-60 cells were established. A total of 53 differential protein spots were detected, and 35 differential proteins were successfully identified. Of the identified proteins, 32 proteins were up-regulated, and 3 proteins were down-regulated in HL-60 cells after 5-aza-2-dC treatment.Conclusion Thirty-five differential proteins may be associated with methylation in HL-60 cell line, which provides the important clues for epigenetic study of leukemia.

Establishment of protein expression profile of laser capture microdissection-purified nasopharyngeal carcinoma tissue / 中南大学学报(医学版)

OBJECTIVE@#To establish the protein expression map of nasopharyngeal carcinoma (NPC), and provide a basis for proteomic study of NPC.@*METHODS@#Laser capture microdissection (LCM) was used to isolate cancer cells from NPC tissues. Two-dimensional gel electrophoresis(2-DE) was used to separate the total proteins of LCM purified NPC cells. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was performed to identify the proteins, and bioinformatics was used to construct the 2-DE database of NPC proteome.@*RESULTS@#A 2-DE reference map of NPC was established. On the 2-DE map, a total of (1 312+/-30) protein spots were detected, and 427 protein spots representing 241 non-redundant proteins were identified. The 2-DE database of NPC proteome was constructed. These data could be accessed at our website (http://www.xyproteomics.org).@*CONCLUSION@#A protein expression profile of LCM purified NPC tissues has been established for the first time, which provides useful information and source for proteomic study of NPC.

Effects of Tongxinluo on cell viability and tissue factor in AngII induced vascular endothelial cells / 中南大学学报(医学版)

OBJECTIVE@#To determine the effects of Tongxinluo on cell viability and tissue factor (TF) in AngII induced vascular endothelial cells and to investigate its mechanism.@*METHODS@#AngII(10(-6)mol/L) was added to human vascular endothelial cells (HUVECs) culture media alone or with various concentration of Tongxinluo drug containing plasma (5%,10%, and 20%) added 30 minutes before AngII. Cell viability was evaluated after 24-hour incubation with AngII in a dose manner. TF, AngII type 1 receptor (AT(1)) mRNA, NO synthase (NOS) and NO were observed after 24-hour incubation with AngII. In addition, NOS inhibitor nomega-nitro-larginine (L-NAME) was added 30 minutes before Tongxinluo and AngII. Cell viability, TF, AT(1)mRNA, the level of NOS and NO were evaluated after 24-hour incubation with Tongxinluo and AngII.@*RESULTS@#Tongxinluo significantly improved AngII induced endothelial cell viability and the effect was the most obvious at 10%. Tongxinluo (10%) decreased the TF and AT(1) mRNA while increased the NOS and NO levels. L-NAME obviously inhibited the effects of Tongxinluo on cell viability, TF, AT(1) mRNA, and NOS and NO levels.@*CONCLUSION@#Up-regulating NOS-NO signaling may be the mechanism of Tongxinluo on cell viability and TF in AngII induced vacular endothelial cells.

Effects of insulin like growth factor-1 on cell viability and tissue factor in vascular endothelial cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effects of insulin like growth factor-1 (IGF-1) on cell viability and tissue factor (TF) in angiotensin II (Ang II) induced vascular endothelial cells and to investigate its mechanisms.</p><p><b>METHODS</b>10(-6) mol/L Ang II was added to human vascular endothelial cells (HUVECs) culture media alone or 30 min after pretreatment with IGF-1 (0.1 microg/ml , 0.5 microg/ml, 2.5 microg/ml). Cell viability and AngII type 1 receptor (AT1-R) mRNA were evaluated after 24 h incubation with AngII. At the optimum concentration of IGF-1 affecting cell viability, the time dependent manner for 12 - 48 h incubation with Ang II was evaluated. TF, NOS and NO were investigated after 24 h incubation with Ang II. In addition, NO synthase inhibitor Nomega-nitro-1-arginine methylester(L-NAME) was added 30 min before addition of IGF-1 and Ang II, and cell viability, TF, AT1-R mRNA, NOS and NO were evaluated after 24 h incubation.</p><p><b>RESULTS</b>(1) Ang II induced a decrease in cell vitality, an upregulation of AT1-R mRNA, an increase in TF, and a decrease in the activity of NOS and content of NO. (2) Pretreatment with IGF-1 significantly inhibited the decreased cell viability and upregulation of AT1-R mRNA. IGF-1 at 0.5 microg/ml showed the most obvious effects. This effect of cell viability recovery was in a time dependent manner during 12 -48 h. (3) IGF-1 also inhibited the increased content of TF, the decreased activity of NOS and the decreased content of NO. (4) The beneficial effects of IGF-1 on cultured endothelial cells were completely abolished by L-NAME.</p><p><b>CONCLUSION</b>IGF-1 pretreatment could enhance the ang II injured cell viability and anti-thrombosis capacity, and the protective effects may be related to activation of NOS-NO signaling pathway which inhibited AT1-R.</p>

Comparative proteome analysis of human lung squamous cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>This study was designed to establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous cell carcinoma tissue and paired tumor-adjacent normal bronchial epithelial tissue, and to identify differential expression of tumor-associated proteins by using proteome analysis.</p><p><b>METHODS</b>Comparative proteome analysis of human lung squamous carcinoma and paired normal bronchial mucosa adjacent to tumors from 20 cases were carried out. Total proteins of the carcinoma tissue and normal bronchial mucosa were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).</p><p><b>RESULTS</b>(1) Seventy-six differentially expressed proteins were screened by analyzing the electrophoretic maps of the 20 carcinoma and control mucosa tissues. (2) Sixty-eight differential proteins were identified by peptide mass fingerprinting (PMF). Some proteins were products of oncogenes and others were involved in the regulation of cell cycle and signal transduction. (3) The expression of three proteins mdm2, c-Jun and EGFR, correlated with lung squamous carcinoma, were detected by immunohistochemical staining and Western blot analysis. The results showed that the expression of mdm2, c-Jun and EGFR were up-regulated in lung squamous carcinomas, whereas down-regulated in control normal mucosa. It was consistent with our proteome analysis results. Those results suggested that those proteins may play roles in the carcinogenesis of lung squamous carcinoma.</p><p><b>CONCLUSION</b>sixty-eight differentially expressed proteins were successfully characterized by comparative proteome analysis. Those results may provide scientific foundation for screening the molecular biomarkers which can be used in diagnosis and treatment of lung squamous carcinoma, as well as to improve patients' prognosis and provide a new clue for carcinogenesis research of lung squamous cell carcinoma.</p>

Sorcin overexpression and multidrug resistance in human gastric cancer cells / 中南大学学报(医学版)

OBJECTIVE@#To screen multidrug resistance (MDR) related proteins in human gastric cancer using proteomics technique.@*METHODS@#Two-dimensional electrophoresis (2-DE) was used to separate the total proteins of vincristine-resistant human gastric cancer cell line SGC7901/VCR and its counterpart SGC7901. PDQuest software was used to analyze 2-DE images, and the differential expression proteins between the 2 cell lines were identified by both MALDI-TOF-MS and ESI-Q-TOF. The differential expression level of sorcin, one of the identified proteins, was confirmed by western blot analysis. The effect of sorcin on the development of MDR of SGC7901/VCR was determined by antisense oligonucleotides (ASO) technique.@*RESULTS@#Sorcin as a high expression protein in SGC7901/VCR was identified and the suppression of sorcin expression by sorcin ASO could enhance the vincristine chemosensitivity in SGC7901/VCR.@*CONCLUSION@#Sorcin overexpression is related to MDR in human gastric cancer cell line SGC7901/VCR.