Serum comparative proteomic analysis of radiation-induced hepatic injury in cirrhotic rats

AIM: To investigate the differential expression of serum proteins in cirrhotic SD rats for exploring the pathogenesis and identifying the potential biomarkers of radiation-induced hepatic injury. METHODS: Liver cirrhosis was induced in 8 healthy SD rats with subcutaneous injection of carbon tetrachloride (CCl₄) for 6 weeks and then the animals were randomly divided into 2 groups (4 rats in each group): control group (CCl₄ alone) and experimental group (CCl₄ plus radiation). The latter received hemi-liver radiation with single dose of 15 Gy while the former did not receive radiation. Total serum proteins of the 2 groups were extracted 6 h after radiation. Two-dimensional gel electrophoresis (2-DE) were performed to look for differentially expressed proteins. These proteins were then analyzed and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Western bloltting was used to validate the expression of heparanase and hepatocyte growth factor receptor in all independent series of serum samples. RESULTS: Two-dimensional gel images were acquired with good resolution and repetition. Thirty-three differentially expressed proteins were selected and 12 proteins were successfully identified by MS, in which 5 were up-regulated while 7 were down-regulated. The increased level of heparanase and decreased level of hepatocyte growth factor receptor were further confirmed by Western blotting. CONCLUSION: Twelve proteins associated with radiation-induced hepatic injury are successfully characterized by serum comparative proteomics. Heparanase and hepatocyte growth factor receptor might be useful for detecting and monitoring radiation-induced hepatic injury.     

Differential proteomic analysis of human lung adenocarcinoma cell line and bronchial epithelium cell line

AIM: The comparative analysis of total proteins expressed differentially between adenocarcinoma cell line A549 (a human lung adenocarcinoma) and normal cell line HBE (a human lung bronchial epithelium) was conducted to search the proteins involved in tumorigenesis and potential biomarkers of diagnosis or prognosis. METHODS: The proteins of dramatic differential expression were screened in adenocarcinoma cell Line A549 and normal cell line HBE by using immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry. Furthermore, the differential expressed proteins were confirmed by the method of Western blotting. RESULTS: Compared with the two cell lines, 21 differential expressed proteins were found and their functions involves in cell metabolism, protein modification, cell motility, protein trafficking and signal transduction. The up-regulation of heat shock protein beta-1 (HSPB1) in A549 cells was identified and confirmed. CONCLUSION: These results suggest that dramatic differential proteomic expression exists between the two cell lines. The high level of HSPB1 might play an important role in the process of tumorigenesis.     

Differential analysis of serum proteomics in Crohn's disease treated with infliximab

AIM: To identify the serum proteins that might serve as biomarkers for predicting mucosal healing (MH) in the patients with Crohn's disease (CD) treated with infliximab (IFX). METHODS: We collected serum samples before treatment (0 week, group A) and 14 weeks after treatment (group B) from 7 CD patients with IFX treatment who had achieved MH, as well as the serum samples from 7 CD patients who had not achieved MH (0 week, group C; 14 weeks, group D). Two-dimensional fluorescence difference gel electrophoresis was applied to analyze and compare the results of serum profiles between groups A and B, C and D, A and C, B and D. Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry and bioinformatics tools were utilized to preliminarily identify and figure out the differentially expressed proteins. RESULTS: (1) In total, there were 44 differentially expressed spots, 36, 3, 10 and 31 differentially expressed spots were detected while comparing A with B, C with D, A with C and B with D, respectively. (2) Among those spots, 17, 2, 2 and 15 proteins were identified, respectively. In total, there were 19 differentially expressed proteins, including apolipoprotein E, apolipoprotein A-I, complement factor H, and so on. (3) Protein functional association networks were carried out based on STRING database. CONCLUSION: The serum protein profiles obviously change after IFX treatment in MH CD patients, and the serum protein profiles of MH patients are different from that of non-MH patients after IFX treatment. The 19 proteins we identified may serve as potential biomarkers for predicting MH in CD patients with IFX treatment.     

Proteomics analysis of sera from Crohns disease patients and healthy individuals

AIM: To explore differentially expressed proteins of sera from Crohn's disease patients with comparative proteomics technology.METHODS: Two-dimensional differential in-gel electrophoresis was used to define patterns of protein expression in serum cross-labeled with variant CyDye from 4 patients and matched health adults.Proteins that showed differential expressions were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry.RESULTS: Two-dimensional cross-labeled with variant CyDye protein maps of Crohn's disease patients and the health adults were gained successfully.Gel-analysis software identified differently expressed 29 spots Crohn's disease compared with that of health adults,22 of which were identified by using mass spectrometry,including serine/threonine-protein kinase ATR,leukocyte common antigen precursor CD45,60 kD SS-A/Ro ribonucleo-protein,etc.CONCLUSION: Proteomic analysis can identify the serum proteins with variance of Crohn's disease versus health,the different expressed proteins may providing probable new biomarkers correlated with biological behavior of Crohn's disease.     

Diagnosis of colorectal cancer by WCX magnetic bead-based serum proteomic fingerprinting

AIM: To screen the potential protein biomarkers in serum for the diagnosis of colorectal cancer (CRC) by the technique of proteomic fingerprint.METHODS: Proteomic fingerprint combining magnetic beads with MALDI-TOF-MS was used to profile and compare the serum proteins from 98 patients with CRC and 80 healthy blood donors. Proteomic patterns associated with CRC were identified by Biomarker Wizard Software. The model of biomarkers was constructed and evaluated using the Biomarker Patterns Software.RESULTS: A total 68 discriminating m/z peaks were identified to be related with CRC (P<0.01). The model of biomarkers based on the 4 biomarkers (2 870.7, 3 084.0, 9 180.5 and 13 748.8) was constructed by Biomarker Patterns Software. Using this model, excellent separation between the CRC and the controls was generated. The sensitivity of the model was 92.85% (91/98) and the specificity was 91.25% (73/80). Blind test data indicated a sensitivity of 86.95% (40/46) and a specificity of 85.00% (34/40).CONCLUSION: The biomarkers for CRC can be discovered in serum by MALDI-TOF-MS combining the use of magnetic beads. The pattern of combined markers provides a powerful and reliable method for diagnosis of CRC with high sensitivity and specificity.     

Analysis of proteomic spectra in serum from patients with colorectal cancer by SELDI-TOF mass spectrometry

AIM: To study the changes of proteomic spectra in serum from patients with colorectal cancer in order to detect the specific protein markers. METHODS: Proteomic spectra of sixty-four serum samples from patients with colorectal cancer (preoperation and postoperation) and forty from normal individuals were generated by IMAC-Cu proteinchip array and SELDI-TOF mass spectroscopy (surface enhanced laser desorption/ionization-time of flight). The discriminatory profiling between cancer and normal samples was analyzed by Biomarker Wizard software and Biomarker Pattern softwar. RESULTS: Nineteen differentially expressed proteins in serum were screened by analysis of protemic spectra of preoperative patients and normal individuals. Five proteins (5972.67 D, 5927.21 D, 6113.48 D, 5908.55 D and 4292.51 D) were obtained for making up marker patterns that was able to class the patients-team and normal-team. Corresponding correct ratio were 97.5% (56/64) and 80% (32/40). The proteins that overexprssed in preoperation were obviously down-regulated when postoperation. CONCLUSION: The preliminary results suggest that classification system will provide a highly accurate and innovative approach for the early diagnosis of colorectal cancer and judgment of prognosis. SELDI-TOF mass spectrometry is a useful tool for the detection and identification of new protein markers in serum.     

Expression of Akt and ERK1/2 in human osteoarthritic chondrocytes

AIM: To investigate the expression of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) in normal and osteoarthritic chondrocytes. METHODS: The samples of knee cartilage were obtained from the normal donors (n=5) and the patients (n=18) undergoing total knee arthroplasty with the diagnosis of osteoarthritis (OA). The expression of p-Akt and p- ERK1/2 in the normal and osteoarthritic cartilage tissues was detected by the method of immunohistochemistry. The chondrocytes were isolated and identified by toluidine blue staining and immunohistochemical method. The expression levels of Akt, p-Akt, ERK1/2,p-ERK1/2,phosphorylated 70-kD ribosomal protein S6 kinase(p-p70S6K) and proliferating cell nuclear antigen(PCNA) were tested in normal and osteoarthritic chondrocytes by Western blotting. Real-time fluorescence quantitative PCR was used to measured the expression levels of aggrecan and type II collagen gene in normal and osteoarthritic chondrocytes. RESULTS: The expression of p-Akt in normal cartilage was higher than that in OA cartilage. The expression of p- ERK1/2 in OA cartilage was higher than that in normal cartilage. Compared with the normal chondrocytes, the expression of p-Akt and p-p70S6K, and the mRNA levels of aggrecan and type II collagen were increased (P<0.05), and the expression of p-ERK1/2 and PCNA was decreased in OA chondrocytes (P<0.05). CONCLUSION: Akt might regulate aggrecan and type II collagen synthesis via p-p70S6K, and ERK1/2 might regulate OA chondrocyte proliferation through PCNA. Both Akt and ERK1/2 play important roles in the pathogenesis of OA.     

Differential expression profiling of proteome for systemic lupus erythematosus with quantitative proteomic technique

AIM: To identify and quantify the total proteins in peripheral blood mononuclear cells (PBMC) of systemic lupus erythematosus (SLE) patients with quantitative proteomic technique, and to establish a differential expression profile of proteome for SLE. METHODS: Four-plex isobaric tags for relative and absolute quantification coupled with multiple chromatographic fractionation and tandem mass spectrometry were used to analyze the total proteins in PBMC from healthy controls and the patients of stable SLE, active SLE and rheumatoid arthritis. The proteins were identified by database searching with peptide mass fingerprinting. The differential expression of the proteins was compared. RESULTS: More than 400 proteins were identified. Compared with healthy controls, 44 proteins were discovered to be significantly expressed by more than 2 folds in stable SLE and active SLE, among which 9 proteins were up-regulated and 35 proteins were down-regulated. Compared with rheumatoid arthritis group, 52 proteins displayed 2 or more folds of changes in stable SLE and active SLE, including 19 up-regulated proteins and 33 down-regulated ones. The up-and down-regulated proteins between active SLE and stable SLE were 17 and 13, respectively. CONCLUSION: Quantitative proteomic technique is efficiently applicable for protein identification and relative quantitation in human peripheral blood mononuclear cells. Determination of the differentially expressed proteomic profile of SLE is helpful for better understanding the pathogenesis of SLE and developing new strategies for diagnosis and treatment of SLE.     

Effects of bortezomib on Treg cells in multiple myeloma and its relationship with tumor burden and chemoresponse

AIM: To investigate the changes of regulatory T (Treg) cells in peripheral blood from multiple myeloma (MM) patients with bortezomib-based treatment, and to analyze the effects of bortezomib on Treg cells in MM and the relationship with tumor burden and chemoresponse. METHODS: Treg cells from 30 newly-diagnosed MM patients were detected by flow cytometry before and after bortezomib-based treatment. Meanwhile, 12 cases with conventional treatment were enrolled as controls. The correlation of Treg cell count with the levels of M protein and serum β₂-microglobulin (β₂-MG) was then statistically analyzed. RESULTS: The patients in early disease stage had lower Treg cell count. Decreased Treg cell count was found in 31 cases with chemoresponse (P<0.01), which did not exist in the 11 cases without chemoresponse. The patients achieved >50% serum β₂-MG reduction showed more obvious changes of Treg cell count than those achieved ≤50% serum β₂-MG reduction (P<0.05). The patients with bortezomib-based treatment showed more apparent decline in Treg cell count than the patients with conventional treatment, which were related to chemoresponse and serum β₂-MG reduction (P<0.05). CONCLUSION: Treg cell count is a reliable index of tumor budern. Both bortezomib-related treatment and conventional treatment can reduce Treg cell count, but the reduction is more obvious in bortezomib-related treatment. Such reduction is related to chemoresponse and tumor burden, suggesting the mechanism of bortezomib curing MM may be by the way of Treg suppression.     

Identification of serum differential proteins in colorectal adenoma and early malignantly transformed adenoma by proteomics

AIM: To investigate the associated proteins and sensitive biomarkers for early diagnosis of colorectal adenocarcinoma by comparing the results of differential proteomic analysis between colorectal adenoma and early malignantly transformed adenoma. METHODS: Two-dimensional gel electrophoresis was used to define patterns of protein expressions of colorectal adenoma and early malignantly transformed adenoma. Proteins expressed differentially among groups were detected, cut out and analyzed by MALDI-TOF/TOF mass spectrometry. RESULTS: Two-dimensional protein maps of colorectal adenoma and early malignantly transformed adenoma were analyzed with gel-analysis software, an average of 1 672 spots in adenoma, 1 732 in early malignantly transformed adenoma were observed. 28 spots of a 1.5-fold change were found, including 15 proteins down-regulated and 13 up-regulated in early malignantly transformed adenoma, in which 23 proteins were identified by mass spectrometry, the rate of identification was 82.14%. 13 differential proteins were attained, 8 were up-regulated and 5 were down-regulated, which was classified to 6 categories, including protease inhibitor, complement, immunoglobulin, keratoproteins, signal transduction protein and function-unknown proteins. CONCLUSION: The changes of serum proteins in early malignantly transformed adenoma from adenoma can be identified by proteomic technology. Proteins detected in the study may provide new biomarkers correlated with biological behavior of colorectal adenocarcinoma.     

菜薹BrNAP克隆及其在采后叶片衰老中的功能分析

在菜薹(Brassica rapa var. parachinensis)中克隆Br NAP1并分析其功能。Br NAP1编码区全长813 bp,编码270个氨基酸,具有NAP转录因子特有的保守结构域,属于NAP亚家族成员。Br NAP1表达量与叶片衰老程度呈正相关且受ABA诱导表达上调。亚细胞定位试验表明Br NAP1定位于细胞核。互补试验显示Br NAP1能使拟南芥atnap滞绿表型回复至野生型,过表达则能引起采后叶片早衰。双荧光素酶试验表明Br NAP1能够激活Br SAG113表达。这些说明Br NAP1是菜薹采后叶片衰老的正调控基因。     

Changes in serum TGF β1 in type 2 diabetic patients

AIM: To explore the changes in serum TGF β₁ in type 2 diabetes mellitus. METHODS: Forty-five cases type 2 diabetes mellitus patients were divided into three groups according to urine albumim excretion rate(UAER): normoalbuminuria(NA)group and microalbuminuria(MA) group and macroalbuminuria group (Overt DN). Serum TGF β₁, fasting blood glucose(FBG), HbA_1c,BUN,Cr,Ccr,lipidemia were detected in all cases. RESULTS: Serum TGF β₁ in NA, MA and ODN groups was higher than that in control. Serum TGF β₁ was positive correlation with Cr(r=0.390,P＜0.05), LDL(r=0.503,P＜0.01), HbA_1c (r=0.676,P＜0.01), and UARE(r=0.777,P＜0.01). CONCLUSION: Type 2 diabetes mellitus have a higher serum TGF β₁ than controls, serum TGF β₁ was positive correlation with HbA_1c and injury of renal function.     

Proteomic analysis of human endometrium during the implantation window

AIM: To establish a method for determining the differential expression of proteins in human endometrium during the implantation window and to analyze the correlation between altered expression of the proteins and endometrial receptivity. METHODS: A comparative proteomic strategy in a combination of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was adopted to search the proteomic alternations in the endometria of pre-receptive pre-receptive [day 2 after luteinizing hormone surge (LH+2 d)] state versus the endometria of receptive (LH+7 d) state. Validation of annexin IV was performed by Western blotting. RESULTS: Approximate 2 555±98 polypeptide spots were revealed by densitometry analysis of the 2D protein maps in LH+2 d and LH+7 d endometrial tissues resolved in the linear range of pH 3~10 on the 2D gel, in which 31 proteins were found to be significantly changed, including 17 proteins up-regulated and 14 proteins down-regulated in LH+7 d samples. These 31 identified proteins were classified into 6 functional categories of the correlation with implantation process: cell migration or assimilation, enzymic activity, signal transduction and gene regulation, immunoregulation, vascularization, and blood clotting or fibrinolysis system. The same expression trend of annexin IV was confirmed by Western blotting. CONCLUSION: Human endometrium has a differential proteomic repertoire during the window of implantation. The 6 functional categories of differentially expressed proteins in the receptive endometrium indicate that they play an important role in transforming of the endometrium during the receptive state.     

TMT-tagged quantitative proteomics analysis of differentially expressed proteins and enrichment of signaling pathways in nasopharyngeal carcinoma cells with or without SATB1 over-expression

AIM: To analyze the effects of special AT-rich sequence binding protein 1 (SATB1) expression on the protein expression profiles in human nasopharyngeal carcinoma (NPC) cells, and to enrich the differential signaling pathways through bioinformatics analysis. METHODS: SATB1 over-expressing lentivirus and negative control lentivirus were used to infect the CNE1 cells, and then the cell lines were obtained by puromycin stressed method. The total proteins of the 2 cells were extracted, and the differentially expressed proteins were screened by TMT-labeled protein quantification technique and tandem mass spectrometry. The mRNA levels of the differential protein-coding genes were verified by RT-qPCR. GO analysis was used to annotate and enrich the differentially expressed proteins, and the KEGG database was used to enrich and analyze the signaling pathways of differential proteins. RESULTS: SATB1 over-expressing CNE1 cells were established through infected with associated lentivirus. Compared with the control group, 278 differentially expressed proteins were identified in SATB1 over-expressing CNE1 cells, in which 115 were up-regulated and 163 were down-regulated. 10 representative differential protein-coding genes were verified by RT-qPCR, which showed the consistence with the proteomic results. GO analysis indicated differentially expressed proteins were mainly involved in cellular processes, single-organism processes, biological regulation, metabolic processes, protein binding and catalysis. Cell components of differentially expressed proteins mainly existed in cell part, cells and organelles. KEGG analysis showed that differentially expressed proteins were involved in signaling pathways closely related to tumors, includeing MAPK, PI3K-Akt, AMPK, JAK-STAT, p53, PPAR, Hippo and HIF-1 signaling pathways. CONCLUSION: Over-expression of SATB1 significantly alters the protein expression profiles in the NPC cells and affects multiple signaling pathways closely related to tumors. Proteomics also provides a possible macro approach to the screening of molecular mechanisms, therapeutic and prognostic targets for NPC.     

Profiling serum low molecular weight proteins in patients with chronic severe hepatitis

AIM:To profile the serum low molecular weight proteins in patients with chronic severe hepatitis with proteinchip technique. METHODS:28 cases of chronic severe hepatitis and 22 healthy controls were enrolled in the study. Serum samples were analyzed with the surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF MS) to obtain a quantitative proteomic fingerprints with molecular masses ranging from 1 kD to 10 kD. The discriminating peaks of two groups were identified. Principal component analysis was then used as the classification method for modeling the discrimination between chronic severe hepatitis patients and healthy controls. Serum was subjected to centrifugal ultrafiltration, and the unfiltered serum and the ultrafiltrates were compared using SELDI-TOF MS. RESULTS:Compared the proteomic fingerprints of severe hepatitis serum to control serum, 39 discriminating peaks were identified (P<1×10-6). Application of principal component analysis to SELDI-TOF MS data distinguished control samples from severe hepatitis samples clearly. Compared with the unfiltered serum, the SELDI-TOF MS spectrum of the filtered serum showed very few peaks with molecular masses ranging from 1 kD to 10 kD. CONCLUSION:Differentially expressed low molecular weight proteins are observed in the serum of chronic severe hepatitis patients. On the basis of the relevant literature, the biological significance of the present study is discussed.     

Screening and identification of specific serum biomarkers of Parkinson disease

AIM：To screen the possible serum biomarkers of Parkinson’s disease. METHODS：The surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to screen the serum samples from 44 cases of Parkinson’s disease and 60 control subjects. The differentially expressed protein peaks were selected and isolated with high-performance liquid chromatography (HPLC), and processed with enzyme before analysis by liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS/MS). The data mining was performed by Xcalibur program component BioWorks 3.2. RESULTS：Three differentially expressed protein peaks were selected as potential serum biomarkers from the patients of Parkinson’s disease: the protein at 8 937 m/z peak showed significant increase (27.47±16.58 in Parkinson’s disease group, and only 5.01±3.47 in control group), and the proteins at 6 636 and 8 697 m/z peaks showed significant decreases (5.43±2.66 and 20.22±9.57, respectively, in Parkinson’s disease group, and 18.85±7.56 and 51.13±26.22, respectively, in control group). The proteins at 6 636, 8 697 and 8 937 m/z peaks were identified as apolipoprotein C-I, apolipoprotein C-III and complement 3a,respectively. Combined use of these 3 biomarkers effectively distinguished the subjects between Parkinsons disease group and control group. The detection rate of the patients with Parkinsons disease was 90.0% (27/30), and the detection rate of the healthy sibkects was 92.5% (37/40). CONCLUSION：The apolipoprotein C-I, apolipoprotein C-III and complement component 3a identified as potential markers of Parkinson’s disease have diagnostic value in clinical application.     

Expression of Th cytokines in peripheral blood before and after splenectomy in immune thrombocytopenia patients

AIM: To investigate the changes of Th cytokines before and after splenectomy in immune thrombocytopenia (ITP) patients. METHODS: The QuantiGene Plex method was used to measure the mRNA expression of Th1, Th2 (IL-4, IL-5, IL-6 and IL-10), Th3 (transforming growth factor β₁,TGF-β₁), and Th17 (IL-17) cytokines in peripheral blood of ITP patients before and after laparoscopic splenectomy and those in peripheral blood of healthy controls. RESULTS: The mRNA level of IL-2 was significantly decreased in ITP patients before operation compared with the healthy controls, whereas IL-17 was obviously over-expressed. No significant difference of the other cytokines between preoperative group and the normal controls was found. After splenectomy, the expression levels of both IL-2 and TGF-β₁ were significantly higher than those in preoperative group and the normal controls. IL-2 was also significantly increased after operation, but was still lower than that in the normal controls. No significant difference of other cytokines between postoperative group and healthy controls was observed. In addition, The Th1 cytokines (IL-2 and IFN-γ) were found to be positively correlated (r=0.647, P<0.01) in preoperative patients, while no correlation was found between the other cytokines. There was a positive correlation between IL-2 and IFN-γ (r=0.787, P<0.01) in postoperative patients. IL-17 also had positive correlations with IL-2 (r=0.554,P<0.01) and IFN-γ (r=0.461,P<0.05) in ITP patients after operation, respectively. CONCLUSION: There is an imbalance of Th cytokines in ITP patients. The mechanism of splenectomy for treating ITP may be associated with the balance regulation of Th cytokines.     

Ischemia reperfusion-induced proteomic changes in rat skeletal muscle

AIM: To investigate ischemia reperfusion (I/R)-induced proteomic changes in rat skeletal muscle. METHODS: Healthy male Wistar rats were randomly divided into two groups as follows (n=6): sham group and I/R group. I/R of right hind limb was induced by 4 h ischemia followed by 24 h reperfusion. The 2-DE was applied to separate the proteins extracted from skeletal muscle tissue at the end of experiment, followed by Coomassie Brillant blue R-250 staining. Computer image analysis was used to determine the differential expression of proteins between the two groups, and 7 protein spots expressed differentially were picked out and subjected to in-gel digest and MALDI-TOP for identification. RESULTS: 354±13 proteins were detected and the match rate was (78.7±1.4)%. 10 proteins displayed significant changes after I/R, of which, 6 proteins increased and 3 proteins decreased in expression. Moreover, 2 spots in I/R group were observed, only 1 spots of which in control. 5 proteins were identified after mass spectrometry. Mitochondrial aldehyde dehydrogenase (ALDH) precursor, heat shock 27 kD protein (HSP27), an unnamed protein product (increased in I/R group), α-actin (decreased in I/R group), and nuclear transport factor 2 (NTF-2) W7a mutant were found in I/R group. CONCLUSION: I/R injury induced differential proteomic changes in rat skeletal muscle. ALDH, α-actin and HSP27 expression, and NTF-2 mutation are involved in I/R injury.     

Downregulation of beta tropomyosin protein expression in colon adenocarcinoma

AIM: To investigate the cancer associated proteins and sensitive biomakers for early diagnosis in colon adenocarcinoma by using proteomic technique.METHODS: Two-dimensional gel electrophoresis was used to define patterns of protein expression in adenocarcinoma tissue from 8 patients with matched normal colonic mucosa. Proteins expressed differently of a 2-fold change were cut and analyzed by MALDI-TOF/TOF mass spectrometry. RESULTS: Two-dimensional protein maps of adenocarcinoma and normal colonic mucosa were gained successfully. Gel-analysis software identified an average of 3 289 spots in adenocarcinoma while 3 066 in normal mucosa and statistical filtering yielded 31 spots of a 2-fold change, 18 of which were identified by using mass spectrometry, including cytokeratin 8, cytokeratin 10, S100A6, beta tropomyosin (TMβ), protein disulfide isomerase, etc. Functional analysis revealed that these proteins were associated with adenocarcinoma cellular oncogenesia, proliferation, differentiation, metastasis and so on. The results of Western blotting validated that the expression level of TM β in colon adenocarcinoma was much lower than that in matched normal colonic mucosa.CONCLUSION: Proteomic analysis can identify the proteins with variance of colon adenocarcinoma versus normal colonic mucosa. Downregulation of TM β might serve as a new biomarker of colon adenocarcinoma.     

Antagonistic effect of thalidomide on TGF-β1-induced change of CTGF gene promoter-binding proteins in HELF cells

AIM:To investigate the antagonistic effect of thalidomide (THD) on the activation of connective tissue growth factor (CTGF) gene promoter induced by transforming growth factor-β₁ (TGF-β₁) in human embryonic lung fibroblasts (HELF). METHODS:Luciferase reporter vector driven by CTGF gene promoter was used to detect the effects of TGF-β₁ and THD on the activity of CTGF gene promoter, and DNA pull-down assay with CTGF gene promoter as a probe was used to analyze the changes of CTGF promoter-binding proteins under different conditions. RESULTS:TGF-β₁ increased the activity of reporter driven by CTGF gene promoter (P<0.01), while THD significantly inhibited TGF-β₁-induced increase in the reporter activity dose-dependently (P<0.01). At the same time, THD had inhibitory effect on the TGF-β₁-induced change of CTGF gene promoter-binding proteins (P<0.05). CONCLUSION:Regulation of CTGF gene promoter-binding proteins takes part in TGF-β₁-induced activation of CTGF gene promoter, while THD has antagonistic effect on this process.