Proteomic analysis between pathological scars and normal skin

AIM: To investigate and screen the sensitive proteins in the formation mechanism of pathological scars by comparing the results of differential proteomic analysis between pathological scars and normal skin.METHODS: Two-dimensional gel electrophoresis was used to detect the protein expression profiles in 8 keloid patients, 8 hypertrophic scar patients and 3 matched normal skin patients.The proteins that showed differential expression of over 4-fold change were cut and analyzed by MALDI-TOF/TOF mass spectrometry.RESULTS: A two-dimensional protein profiling comparison between pathological scars and normal skin was successfully established.On average, 2 978 spots in keloid, 2 975 spots in hypertrophic scar and 3 053 spots in normal skin were identified using gel analysis software.Compared with normal skin, there were totally 36 differentially-expressed proteins in keloid and hypertrophic scar identified from the spots of over 4-fold change, including 16 proteins in both keloid and hypertrophic scar (8 up-regulated and 8 down-regulated), 11 only in keloid (9 up-regulated and 2 down-regulated) and 9 only in hypertrophic scar (4 up-regulated and 5 down-regulated).CONCLUSION: Proteomic analysis can identify the proteins with variance of pathological scars versus normal skin, thus providing probable new clues to reveal the formation mechanism of pathological scars.     

Expression of A20 in patients with systemic lupus erythematosus

AIM: To investigate the expression and regulation of A20 in healthy individuals and the patients with systemic lupus erythematosus (SLE). METHODS: The expression levels of A20, NF-κB, MALT1, and MALT1V1 in peripheral blood mononuclear cells (PBMC) of the patients with SLE (including 2 cases with scleroderma, 1 case with rheumatoid arthritis, and 1 case with lymphoma) were analyzed by real-time PCR. RESULTS: A significantly lower A20 expression level was found in the PBMC from SLE group compared with the healthy controls, while the expression levels of MALT1 and NF-κB were also decreased. In addition, no significant correlation between A20 and NF-κB expression levels in healthy group was observed, but a positive correlation was found in SLE group (P<0.05). A significant positive correlation between MALT1 and NF-κB expression levels in healthy group (P<0.05) was observed, and no significant correlation was found in SLE group. The expression level of MALT1V1 in SLE group was significantly lower than that in healthy control group, and there was a positive correlation between A20 and MALT1V1 in healthy volunteers (P<0.01), but that did not exist in SLE group. CONCLUSION: The characteristics of the expression pattern of MALT1-A20-NF-κB in the SLE patients were presented. Lower level of A20 expression was found in the SLE patients, in particular with other autoimmune disease or lymphomas, indicating the lower immune tolerance in SLE. The positive correlation of A20 and NF-κB may relate to positive regulation of MALT1.     

Differential expression profile of long non-coding RNA in hepatic tissue between drug-induced liver injury and immune liver injury

AIM: To analyze the expression profile of long non-coding RNA(lncRNA) in the liver tissues of drug-induced liver injury(DILI) and immune liver injury(ILI). METHODS: The technique of lncRNA microarray was used to inspect the lncRNA expression profile in the mouse liver tissues that the liver injury was induced by acetaminophen or concanavalin A. The raw data of lncRNA were pretreated for normalization. RESULTS: Compared with normal hepatic tissue, the lncRNA which had more than 1.5-fold variation and significant difference(P<0.05) by statistical analysis were regarded as lncRNA with differential expression. A total of 68 lncRNA with differential expression were found in the hepatic tissues of DILI, with 21 increased more than 1.5 folds and 47 reduced more than 1.5 folds. A total of 60 lncRNA with differential expression in the liver tissues of ILI were observed, with 17 increased more than 1.5 folds and 43 reduced more than 1.5 folds. In all lncRNA, 8 was simultaneously up-regulated in 2 liver injury models, accounting for 38% and 47% respectively, while 28 was simultaneously down-regulated in 2 liver injury models, accounting for 59% and 65% respectively. CONCLUSION: lncRNA expression profiles of DILI and ILI change significantly in comparison with normal hepatic tissue, and there are also differences between 2 hepatic damage models. The simultaneous changes of lncRNA may participate in the same or similar pathophysiological process, while the differences may be involved in relatively particular mechanisms.     

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.     

Analysis of expressive proteome in human hepatocarcinoma cell HepG2 transfected with miR-26a mimics

AIM: To determine whether microRNA-26a(miR-26a) is involved in development of liver cancer by analysis of proteomic expression profile of human hepatocarcinoma cell HepG2 transfected with miR-26a mimics.METHODS: HepG2 cells were cultured by a routine method and transfected with miR-26a mimics for 48 h for cell cycle analysis. The expressive proteome profiles of HepG2 cells with or without miR-26a mimics treatment were established by the methods of two-dimensional electrophoresis separation following lysis of the cells and extraction of the proteins. The proteomic expression profiles were analyzed by comparative proteomics technique to discover the important protein spots with differential expression. The identification of the proteins was conducted by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS). RESULTS: miR-26a brought down the proliferation of HepG2 cells. Total 11 protein spots with alteration of expressive amounts more than 2 times were successfully identified in the proteomic expression profile of HepG2 cells treated with miR-26a mimics, including annexin A1, peroxiredoxin 4, proliferating cell nuclear antigen, apolipoprotein A1, cytochrome C oxidase subunit 5A, cyclin E2, ribose-phosphate pyrophosphokinase 3, cyclin-dependent kinase 1 and phosphatidylethanolamine-binding protein 1. Among these, the expression of 3 protein spots was up-regulated and 8 of them was down-regulated.CONCLUSION: miR-26a contributes to the anti-cancer effect by expressive regulation of the proteins mentioned above, or directly or indirectly controls the proliferation, differentiation and death of hepatocarcinoma cells.     

Gene expression pattern of periphery blood mononuclear cells in patients with active lupus nephritis

AIM: To find new gene function associate with active lupus nephritis (LN) through study on the difference in gene expression of peripheral blood mononuclear cells between LN patients and healthy controls by gene chip. METHODS: The CSC-GE-80 chip containing 8 000 spots of cDNAs were used to investigate the difference of the expression. Both the total RNA from peripheral blood mononuclear cells of active LN patients and healthy donors were reversely transcribed to cDNA with the incorporation of fluorescent( cy3 and cy5) labeled dCTP to prepare the hybridization probes. After hybridization, the gene chip was scanned for the fluorescent intensity. The differentially expressed genes were screened. We repeated that in three groups of LN patients and healthy controls, respectively, and only the genes that have differential expression in all three chips were considered associated with LN. RESULTS: 75 genes were identified to be differently expressed in all three groups of LN patients as compared with healthy controls, including 42 up-regulated genes and 33 down-regulated ones. CONCLUSION: The present study represents a global view of gene expression of LN and provides important clues for further study of LN related genes. And it also suggests defensin α₁, S100A8, S100A9 may be involved in the pathogenesis of LN.     

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.     

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.     

Comparative proteomics analysis of human osteosarcomas and benign tumors of bone

AIM: To analyze the proteomic components of the tissue from human osteosarcoma and benign tumor of bone, and to search the diagnostic markers of osteosarcoma. METHODS: Five samples of osteosarcoma and 5 additive samples from benign bone tumor were analyzed by a series of methods including immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2DE), deep purple staining. The ImageMaster 2DE analysis-software, as well as peptide mass fingerprint based on matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database searching were used for data processing. RESULTS: The average spots in osteosarcoma and the benign tumor of bone were 1 386±101 and 1 270±202, respectively. 22 peptide mass fingerprint (PMF) maps were obtained by MOLDI-TOF-MS and identified by searching the SWISS-PROT database. Compared with those in benign tumor of bone, 15 proteins were significantly up-regulated, especially zinc finger protein 133 (Znf133), lamin B and tailless complex polypeptide 1 (TCP-1). 7 down-regulated proteins were observed, with the absence of protein kinase C inhibitor protein 1 (KCIP-1) in osteosarcoma. CONCLUSION: The results suggest that an obviously differential proteomic expression exists between the osteosarcoma and benign tumor of bone. The overexpression of Znf133, lamin B, TCP1 and low-expression of KCIP-1 may play an important role in the development of osteosarcoma, and may serve as diagnostic markers/therapeutic targets of osteosarcoma.     

Therapeutic effect of sodium arsenite on rheumatoid arthritis in rats

AIM:To study the molecular mechanism of rheumatoid arthritis (RA) and the effects of sodium arsenite on AP-1 and MIP-1. METHODS:32 Wistar female rats were randomly divided into 4 groups: normal control (C), arthritis (A), low concentration of sodium arsenite (LSA) and high concentration of sodium arsenite group (HSA).The LSA group and the HSA group were treated with sodium arsenite (0.5 mg·kg^-1·d^-1 and 1.0 mg·kg^-1·d^-1) through abdominal cavity injection for 20 days. The normal control group and the model group were treated with saline (0.2 mL/d). The AP-1 and MIP-1α expression of synovium in four groups were determined by immunohistochemistry. Light microscope was used to observe the synovium with HE staining. RESULTS:Compared with C group, the expression of AP-1 and MIP-1α in the synovium up-regulated in A group (P<0.01) and were inhibited by sodium arsenite treatment (P<0.05), especially in HSA group. CONCLUSION:The activated AP-1 and MIP-1α play an important role in the development of rheumatoid arthritis. Sodium arsenite down-regulated the expression of AP-1 and MIP-1α and may have some therapeutic effects in RA.     

Effect of miR-23b-3p on viability and apoptosis of rheumatoid arthritis synovial fibroblasts by targeting XIAP

AIM:To investigate the effect of microRNA-23b-3p (miR-23b-3p) on the viability and apoptosis of rheumatoid arthritis synovial fibroblasts by targeting X-linked inhibitor of apoptosis protein (XIAP). METHODS:The expression of miR-23b-3p and XIAP was detected by RT-qPCR. The TargetScan was used to predict the targeting regulatory relation between miR-23b-3p and XIAP, and then the regulatory relation was confirmed by dual-luciferase reporter assay. After the miR-23b-3p mimic and inhibitor were transfected into the cells, the expression of miR-23b-3p and XIAP was detect by RT-qPCR. The effect of miR-23b-3p on the viability and apoptosis was measured by CCK-8 assay and flow cytometry. The protein expression levels of Ki67 and Bcl-2 were determined by Western blot. RESULTS:The expression level of miR-23b-3p was down-regulated significantly (P<0.05), and XIAP was up-regulated significantly in rheumatoid arthritis synovial fibroblasts (P<0.05). The miR-23b-3p mimic significantly inhibited XIAP expression and the cell viability, promoted the apoptosis, and down-regulated the expression of Ki67 and Bcl-2 (P<0.05). The effects of miR-23b-3p inhibitor were the opposite. CONCLUSION:miR-23b-3p inhibits the viability and promotes apoptosis of rheumatoid arthritis synovial fibroblasts by targeting XIAP.     

橡胶树死皮病黄色体差异表达蛋白的初步分析

橡胶树"死皮病"给橡胶种植业带来严重的危害.为了更好地了解和阐明死皮病发生、发展的分子机制,研究应用双向凝胶电泳技术(two-dimensional gel electrophshiya/oresis,2-DE)比较橡胶树死皮株与健康株胶乳黄色体蛋白质组表达的差异.采用TCA/丙酮沉淀法提取橡胶树死皮株与健康株黄色体蛋白质并采用固相pH梯度(immobjlized pH graalient,IPG)双向凝胶电泳分离两种材料蛋白质,凝胶经银染显色后,用PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质.成功获得橡胶树死皮株与健康株胶乳黄色体的双向凝胶电泳图谱.鉴定出13个蛋白差异点,其中10个上调表达,3个下调表达.并应用质谱技术鉴定了其中部分表达差异的蛋白质点,对渗调蛋白进行功能分析,认为渗调蛋白在死皮株中表现下调的情形可能与死皮病的发生有一定的关系.     

Gene and protein alterations in the hippocampus of rats with temporal lobe epilepsy

AIM:To examine the expression profiles of both genes and proteins in hippocampus of rats with temporal lobe epilepsy (TLE) for revealing the molecular mechanisms of TLE and looking for the candidate targets and new therapeutic approaches in clinical practice.METHODS:Rat temporal lobe epilepsy was induced by administration of lithium chloride and pilocarpine (LiCl-PILO).The expression spectra of genes and proteins were constructed through the techniques of cDNA microarray,two-dimensional (2D) electrophoresis and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Subsequently,the differentially expressed genes and proteins were identified and analyzed.RESULTS:There were 192 genes of differential expression observed in hippocampal tissues of LiCl-PILO-induced temporal lobe epilepsy,and 159 genes have been registered in Genbank database,in which 84 genes were up-regulated while 75 genes were down-regulated.78 protein spots of differential display were screened out,in which 31 proteins were detected to be down-regulated and 47 were up-regulated.Finally,5 proteins were identified.CONCLUSION:These genes and proteins found in our study may play pivotal roles in the pathogenic mechanisms of epilepsy and may promise new therapeutic targets for refractory epilepsy in the future.     

Abnormal activity of ROCK contributes to increased adhesion and migration of peripheral blood T cells from patients with systemic lupus erythematosus

AIM: To investigate the role of Rho kinase (ROCK) in the regulation of adhesion and migration of the T cells from systemic lupus erythematosus (SLE) patients. METHODS: The T cells were isolated by RosettSep T cell purification kit. ROCK activity was assessed by Western blotting. T cell migration was examined by Transwell chambers. RESULTS: Compared with the T cells from healthy controls and rheumatoid arthritis patients, the activity of ROCK in ex vivo T cells from SLE patients was significantly increased. In vitro, the adhesion and migration of the T cells from SLE patients were also increased. Furthermore, the adhesion and migration of the T cells from SLE patients were inhibited by a specific ROCK inhibitor Y27632. CONCLUSION: The results indicate that ex vivo T cells from SLE patients exhibit increased activity of ROCK. Alteration of ROCK activity may contribute to the abnormal adhesion and migration of T cells from SLE patients.     

Analysis of serum biomarkers in human knee osteoarthritis

AIM: To analyze the proteomic components of the sera from knee osteoarthritis patients and normal people, and to search proteins that might serve as serum biomarkers for osteoarthritis diagnosis, treatment or pathogenesis. METHODS: Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was applied to analyze the sera obtained from the patients with knee osteoarthritis (n=4) and normal controls (n=4). The differentially expressed proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Western blotting analysis was applied to confirm the results. RESULTS: Comparative proteomic data of serum from the patients with osteoarthritis was successfully obtained. Eight differentially expressed protein spots were observed. Five up-regulated and 3 down-regulated proteins were identified. Western blot analysis confirmed that α₂-macroglobulin was increased. CONCLUSION: There are significant differences between serum proteins obtained from the patients with knee osteoarthritis and normal controls. α₂-Macroglobulin might be utilized as potential biomarkers for the diagnosis and treatment of osteoarthritis.     

Proteomic study on mice hepatic cell membrane proteins after exposed to hypoxia

AIM: To identify the differential expression of mice hepatic cell membrane proteins after hypoxic exposure.METHODS: The hepatic cells of C57BL/6 mice were cultured for 8 h under hypoxic or normoxic conditions and the membrane proteins were extracted. The differentially expressed membrane proteins were separated by two-dimensional electrophoresis. The technique of matrix-assisted laser desorption/ionization mass time-of-flight spectrometry(MALDI-TOF-MS) was used to analyze the proteins.RESULTS: Compared with the proteins extracted from the cells under normoxic condition with a threshold of 1.5-fold, 28 differentially expressed proteins were identified in the proteins extracted from the cells under hypoxic condition according to the database NCBInr 20071130, in which 9 were down-regulated and 7 were up-regulated, including prohibitin, cytochrome b-c1 complex subunit 1 and p22HBP.CONCLUSION: The results suggest that prohibitin, cytochrome b-c1 complex subunit 1 and p22HBP might play important roles in sensing of cellular oxygen and the related signal transduction pathways.     

Relationship between IL-1 gene polymorphism and severity of rheumatoid arthritis and secretion of IL-1

AIM: To determine the influence of interleukin-1α(IL-1α) and β(IL-1β) gene polymorphisms on rheumatoid arthritis(RA) disease severity and secretion of IL-1β. METHODS: The study included 136 RA patients and 102 healthy controls. PCR-RFLP was used to detect site mutation at IL-1 gene. Meanwhile the IL-1β was also measured in the supernatant of the cultured and stimulated peripheral blood mononuclear cells(PBMC). RESULTS: No difference in the allele frequencies or genotypes of the IL-1α gene polymorphisms was found between the controls and RA patients.IL-1β allele 2 was overrepresented in patients with erosive RA but not in nonerosive patients. The patients with IL-1β allele 2 had a higher swollen joint index, higher tender joint index and erythrocyte sedimentation rate than those without IL-1β allele 2.The IL-1β in supernatant of stimulated PBMC from patients with IL-1β allele 2 had a higher level than that from those without allele 2. CONCLUSION: IL-1 gene polymorphisms may influence the occurrence of RA. Detection of IL-1β allele 2 have a potential prognostic value in RA.     

Rosiglitazone inhibits osteoclastogenesis in rheumatoid arthritis by down-regulating RANKL expression and suppressing ERK phosphorylation

AIM: To investigate the effects of rosiglitazone on fibroblast-like synoviocyte (FLS)-induced osteoclastogenesis in rheumatoid arthritis (RA) and the related mechanism. METHODS: RA-FLS were cocultured with peripheral blood monocytes from healthy volunteers in the presence of macrophage colony-stimulating factor (M-CSF) and rosiglitazone. Osteoclasts were assayed by tartrate-resistant acid phosphatase (TRAP) staining. Resorption lacunae area was identified by toluidine blue staining and quantified by image analysis software. The mRNA expression of RANKL and OPG was evaluated by real-time PCR, and the protein levels of RANKL, OPG, p-ERK, p-p38 and p-JNK were measured by Western blot. RESULTS: Compared with control group (without rosiglitazone treatment), rosiglitazone at concentration of 15 μmol/L significantly decreased the number of osteoclasts (P<0.01) and resorption lacunae area (P<0.05). The expression of RANKL at mRNA and protein levels was significantly down-regulated by rosiglitazone at concentration of 15 μmol/L, while the mRNA and protein expression of OPG was up-regulated (P<0.01). Rosiglitazone (15 μmol/L) significantly decreased the protein level of p-ERK (P<0.05), but not the protein level of p-p38 or p-JNK (P>0.05). CONCLUSION: Rosiglitazone inhibits RA-FLS-induced osteoclast formation and its resorption activity by down-regulating RANKL expression and ERK phosphorylation, suggesting that rosiglitazone may inhibit RA osteoclastogenesis and bone resorption.