Protein and gene expression characteristics of heterogeneous nuclear ribonucleoprotein H1 in esophageal squamous cell carcinoma.

AIM: To investigate the expression characteristics of heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) mRNA and protein in cell lines and tissues of esophageal squamous cell carcinoma (ESCC). METHODS: Western blotting was used to assess the expression of HNRNPH1 protein in seven ESCC cell lines and 30 paired fresh tissue specimens. The subcellular localization of HNRNPH1 was determined by immunofluorescence in ESCC cells. The RNA sequencing data from 87 patients with ESCC were obtained from the cancer genome atlas (TCGA), and the expression and clinical characteristics analysis of different transcript variants of HNRNPH1 were evaluated in this dataset. In addition, immunohistochemistry was carried out to detect the expression of HNRNPH1 protein in 125 patients. RESULTS: The expression of HNRNPH1 protein varied across different ESCC cell lines. It was exclusively restricted to the nucleus of the ESCC cells. There are two transcript variants of the HNRNPH1 gene. Variant 1 was constitutively expressed, and its expression did not change during tumorigenesis. In contrast, levels of variant 2 were low in non-tumorous tissues and were dramatically increased in ESCC (P = 0.0026). The high levels of variant 2 were associated with poorer differentiated tumors (P = 0.0287). Furthermore, in paired fresh tissue specimens, HNRNPH1 protein was overexpressed in 73.3% (22/30) of neoplastic tissues. HNRNPH1 was significantly upregulated in ESCC, with strong staining in 43.2% (54/125) of tumor tissues and 22.4% (28/125) of matched non-cancerous tissues (P = 0.0005). Positive HNRNPH1 expression was significantly associated with poor tumor differentiation degree (P = 0.0337). CONCLUSION: The different alternative transcript variants of HNRNPH1 exhibited different expression changes during tumorigenesis. Its mRNA and protein were overexpressed in ESCC and associated with poorer differentiation of tumor cells. These findings highlight the potential of HNRNPH1 in the therapy and diagnosis of ESCC.

[Chinmedomics strategy to discover effective constituents and elucidate action mechanism of Nanshi capsule against kidney-yang deficiency syndrome].

The chinmedomics method was used to explore the effect of Nanshi capsule on endogenous metabolites of rats with kidney-yang deficiency syndrome, investigate the metabolites and metabolic pathways closely related to kidney-yang deficiency syndrome (KYDS)and identify the therapeutic basis of Nanshi capsule(NPC)as well as its action mechanism for KYDS. The routine biochemical indexes of serum were detected and histomorphology was observed. Based on the chinmedomics technology platform, discriminatory analysis in multivariate modes was conducted for rat blood and urine, thus to investigate the biomarkers of KYDS and the therapeutic effect of NPC against KYDS. Meanwhile, the main constituents of NPC in rat serum were also detected to analyze its correlation between the constituents in vivo and the biomarkers of KYDS, and determine the potential effective compounds for therapeutic effect. Eleven biomarkers of KYDS were identified in the rat models, involving steroid hormone biosynthesis, tryptophan metabolism and tyrosine metabolism. It was found that NPC could regulate steroid hormone biosynthesis, tryptophan metabolism and tyrosine metabolism; PCMS analysis showed that caffeic acid, 2-hydroxy-1-methoxy-anthraquinone, 1-hydroxy-2-methoxyanthraquinone, ferulic acid glucuronide conjugation, deacetylasperulosidic acid, cynaroside, betaine and umbelliferone were the main effective compounds of NPC for KYDS. In this study, cynaroside, betaine, umbelliferone and other compounds in NPC could integrally regulate the disturbance of metabolic profile in KYDS by improving the hormone synthesis, hormone synthesis pathway, hormone synthesis and release pathway in tyrosine metabolism and linoleic acid synthesis pathway in linoleic acid metabolism. These results indicated that the NPC had the characteristics of multi-pathway, multi-target and overall regulation in the treatment of KYDS. Chinmedomics approach can provide methodology support to discover innovative drug from traditional Chinese medicine.

Aberrant expression of peroxiredoxin 1 and its clinical implications in liver cancer.

AIM: To investigate the expression characteristics of peroxiredoxin 1 (PRDX1) mRNA and protein in liver cancer cell lines and tissues. METHODS: The RNA sequencing data from 374 patients with liver cancer were obtained from The Cancer Genome Atlas. The expression and clinical characteristics of PRDX1 mRNA were analyzed in this dataset. The Kaplan-Meier and Cox regression survival analysis was performed to determine the relationship between PRDX1 levels and patient survival. Subcellular fractionation and Western blotting were used to demonstrate the expression of PRDX1 protein in six liver cancer cell lines and 29 paired fresh tissue specimens. After bioinformatics prediction, a putative post-translational modification form of PRDX1 was observed using immunofluorescence under confocal microscopy and immunoprecipitation analysis in liver cancer cells. RESULTS: The mRNA of PRDX1 gene was upregulated about 1.3-fold in tumor tissue compared with the adjacent non-tumor control (P = 0.005). Its abundance was significantly higher in men than women (P < 0.001). High levels of PRDX1 mRNA were associated with a shorter overall survival time (P = 0.04) but not with recurrence-free survival. The Cox regression analysis demonstrated that patients with high PRDX1 mRNA showed about 1.9-fold increase of risk for death (P = 0.03). In liver cancer cells, PRDX1 protein was strongly expressed with multiple different bands. PRDX1 in the cytosol fraction existed near the theoretical molecular weight, whereas two higher molecular weight bands were present in the membrane/organelle and nuclear fractions. Importantly, the theoretical PRDX1 band was increased, whereas the high molecular weight form was decreased in tumor tissues. Subsequent experiments revealed that the high molecular weight bands of PRDX1 might result from the post-translational modification by small ubiquitin-like modifier-1 (SUMO1). CONCLUSION: PRDX1 was overexpressed in the tumor tissues of liver cancer and served as an independent poor prognostic factor for overall survival. PRDX1 can be modified by SUMO to play specific roles in hepatocarcinogenesis.

Monitoring disease progression and treatment efficacy with circulating tumor cells in esophageal squamous cell carcinoma: A case report.

This study investigated whether changes in circulating tumor cell (CTC) numbers reflect tumor progression and treatment efficacy in esophageal squamous cell carcinoma (ESCC). A 47-year-old male patient with ESCC is presented in this case study. The patient was evaluated for a series of serum tumor markers and subjected to radiological examinations before and after surgery and during follow-up over the course of five years. In addition, the CTCs in 7.5 mL of peripheral blood were enriched by magnetic-activated cell sorting negative selection and identified by immunofluorescence staining. Serum tumor markers remained within normal ranges and were discordant with imaging scans during the follow-up. Initially, one CTC was detected in the peripheral blood sample, and 14 were observed seven days after the operation. After 12 wk, subcutaneous metastases and bone metastases occurred, and the number of CTCs increased to 84. After 48 wk, lung metastases were noted, and the CTC level was 21. At 104 wk, the number of CTCs was 14, and disease recurrence was detected by positron emission tomography-computed tomography. The CTC counts were in accord with the imaging studies at several time points. The additional information provided by CTC enumeration could thus facilitate monitoring of disease status and treatment efficacy and provide support for treatment decisions.

Influence of Per3 genotypes on circadian rhythmicity in flight cadets after militarized management.

OBJECTIVE: The purpose of this study was to explore the effect of PERIOD3 (PER3) genotypes on circadian rhythmicity in flight cadets after militarized management. METHODS: We performed a preliminary study in 146 newly enrolled male flight cadets. Venous blood samples were collected, and genotyping of PER3 (4/5) was determined by using PCR. The morningness-eveningness questionnaire (MEQ) survey was given to flight cadets upon enrollment and after militarized management for 24 months respectively. Comparison of frequency distribution of PER3 genotypes between cases and controls (120 well-matched civilians) was performed using the X(2) test. We also compared the circadian rhythmicity upon enrollment and 24 months after enrollment in flight cadets, and analyzed the connection of changes in circadian clock with PER3 genotypes. RESULTS: The frequency distribution of PER3 genotypes in flight cadets was not significantly different from that in controls subjects. MEQ survey results showed chronotype within flight cadet group varied widely at the two time-points: the moderately morning type (50%) and the neither type (41.1%) upon enrollment; the neither type (76.7%) and the moderately morning type (21.2%) 24 months after enrollment. The circadian rhythm of individuals with the PER3 (5/5) genotype showed no significant difference before and after 24 months of militarized management, whereas notable changes were found in individuals with the PER3 (4/4) genotype (n=116, X(2) =37.26, P < 0.001). CONCLUSION: In conclusion, we provide some evidence that circadian rhythm of flight cadets with the PER3 (5) allele are less likely to be affected compared to those with the PER3 (4) allele.

Identification of biomarkers for hepatocellular carcinoma by semiquantitative immunocytochemistry.

AIM: To investigate the expression of key biomarkers in hepatoma cell lines, tumor cells from patients' blood samples, and tumor tissues. METHODS: We performed the biomarker tests in two steps. First, cells plated on coverslips were used to assess biomarkers, and fluorescence intensities were calculated using the NIH Image J software. The measured values were analyzed using the SPSS 19.0 software to make comparisons among eight cell lines. Second, eighty-four individual samples were used to assess the biomarkers' expression. Negative enrichment of the blood samples was performed, and karyocytes were isolated and dropped onto pre-treated glass slides for further analysis by immunofluorescence staining. Fluorescence intensities were compared among hepatocellular carcinoma (HCC) patients, chronic HBV-infected patients, and healthy controls following methods similar to those used for cell lines. The relationships between the expression of biomarkers and clinical pathological parameters were analyzed by Spearman rank correlation tests. In addition, we studied the distinct biomarkers' expression with three-dimensional laser confocal microscopy reconstructions, and Kaplan-Meier survival analysis was performed to understand the clinical significance of these biomarkers. RESULTS: Microscopic examination and fluorescence intensity calculations indicated that cytokeratin 8/18/19 (CK) expression was significantly higher in six of the seven HCC cell lines examined than in the control cells, and the expression levels of asialoglycoprotein receptor (ASGPR) and glypican-3 (GPC3) were higher in all seven HCC cell lines than in the control. Cells obtained from HCC patients' blood samples also displayed significantly higher expression levels of ASGPR, GPC3, and CK than cells from chronic HBV-infected patients or healthy controls; these proteins may be valuable surface biomarkers for identifying HCC circulating tumor cells isolated and enriched from the blood samples. The stem cell-like and epithelial-mesenchymal transition-related biomarkers could be detected on the karyocyte slides. ASGPR and GPC3 were expressed at high levels, and thus three-dimensional reconstructions were used to observe their expression in detail. This analysis indicated that GPC3 was localized in the cytoplasm and membrane, but that ASGPR had a polar localization. Survival analyses showed that expression of GPC3 and ASGPR is associated with a patient's overall survival (OS). CONCLUSION: ASGPR, GPC3, and CK may be valuable HCC biomarkers for CTC detection; the expression of ASGPR and GPC3 might be helpful for understanding patients' OS.

Novel method for extracting exosomes of hepatocellular carcinoma cells.

AIM: To develop a novel method for the rapid and efficient extraction of exosomes secreted by tumor cells. METHODS: Unlike the traditional extraction method, the supernatants of cell cultures were concentrated, and the exosomes were isolated promptly and effectively using a novel nanomaterial called ExoQuick. Coomassie brilliant blue staining was used for protein quantification, and the morphology of the exosomes extracted by both methods was visualized by transmission electron microscopy. Exosome marker proteins were detected by Western blot analysis. Two potential hepatoma-associated proteins, tissue transglutaminase 2 (TGM2) and annexin A2, were analyzed. RESULTS: The exosomes separated by the new extraction assay based on the nanomaterial were disc-shaped, intact vesicles with lipid bilayer membranes. They were approximately 30-100 nm in diameter, which is similar to the diameter of exosomes isolated by the traditional method. The protein concentration of exosomes extracted by the new method was approximately 780 µg/10(8) cells, and therefore, it was 19 times higher than that of exosomes extracted in the traditional manner. There were differences between the total proteins of Huh-7 cells and the exosomal proteins. Typical exosome proteins, such as the transmembrane protein CD63 and heat shock protein 70, were confirmed. Two potential hepatoma-associated proteins were also identified. TGM2 was first found to exist in the exosomes of human liver cancer cells, but annexin A2 was not secreted into exosomes. CONCLUSION: The new extraction method based on the nanomaterial is quick and efficient. The cancer-associated protein TGM2 can be secreted through an exosome-mediated non-classical secretion pathway, and it may be a valuable tumor marker.

Expression and phosphorylation of stathmin correlate with cell migration in esophageal squamous cell carcinoma.

Microtubules play extensive roles in cellular processes, including cell motility. Stathmin is an important protein which destabilizes microtubules. The essential function of stathmin is closely associated with its phosphorylation status. Stathmin is overexpressed in many human cancers and has a significant relationship with clinical characteristics such as grade, tumor size and prognosis. We demonstrated that stathmin was overexpressed in ESCC tissues using both 2-DE and immunohistochemistry analysis. In addition, overexpression of stathmin was significantly correlated with histological grade in ESCC. However, no correlation was found with age, gender and lymph node metastasis. Knockdown of stathmin with siRNA impaired cell migration in KYSE30 and KYSE410 cells. When EC0156 cells were treated with paclitaxel, stathmin was stably phosphorylated and migration was impaired. These observations suggest that stathmin may have a more important function in ESCC development and migration. The present study provides further understanding of the importance of stathmin in ESCC therapy or diagnosis.

Clinical significance of serum expression of GROß in esophageal squamous cell carcinoma.

AIM: To determine the association between serum levels of growth-related gene product ß (GROß) and clinical parameters in esophageal squamous cell carcinoma (ESCC). METHODS: Using enzyme-linked immunosorbent assay, serum GROß levels were measured in ESCC patients (n = 72) and healthy volunteers (n = 83). The association between serum levels of GROß and clinical parameters of ESCC was analyzed statistically. RESULTS: The serum GROß levels were much higher in ESCC patients than in healthy controls (median: 645 ng/L vs 269 ng/L, P < 0.05). Serum GROß levels were correlated positively with tumor size, lymph node metastasis, and tumor-node-metastasis (TNM) staging, but not with gender or the histological grade of tumors in ESCC patients. The sensitivity and specificity of the assay for serum GROß were 73.61% and 56.63%, respectively. CONCLUSION: GROß may function as an oncogene product and contribute to tumorigenesis and metastasis of ESCC.

A suppressor of multiple extracellular matrix-degrading proteases and cancer metastasis.

Cancer metastasis remains the most poorly understood process in cancer biology. It involves the degradation of extracellular matrix (ECM) proteins by a series of 'tumour-associated' proteases. Here we report the identification of a novel protease suppressor, NYD-SP8, which is located on human chromosome 19q13.2. NYD-SP8 encodes a 27 kD GPI-anchored cell surface protein, which shows structural homology to urokinase plasminogen activator receptor (uPAR). Co-immunoprecipitation experiments showed that NYD-SP8 binds to uPA/uPAR complexes and interfere with active uPA production. Overexpression of NYD-SP8 results in reducing activities of the three major classes of proteases known to be involved in ECM degradation, including uPA, matrix metalloproteinases (MMPs) and cathepsin B, leading to suppression of both in vitro and in vivo cancer cell invasion and metastasis. These data demonstrate an important role of NYD-SP8 in regulating ECM degradation, providing a novel mechanism that modulates urokinase signalling in the suppression of cancer progression.

Rapid detection of hepatitis B virus surface antigen by an agglutination assay mediated by a bispecific diabody against both human erythrocytes and hepatitis B virus surface antigen.

Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens.

[Mediastinal lymph nodes micro-metastases in patients with clinical stage I-II lung cancer].

OBJECTIVE: To investigate micro-metastasis in mediastinal lymph nodes (mLN) of patients with clinical stage I approximately II lung cancer and its clinical significance. METHODS: A total of 181 mLN from 32 lung cancer patients in clinical stage I approximately II were collected during operation and their frozen sections at two different levels were examined immunohistochemically (IHC) with an anti-epithelial cell monoclonal antibody Ber-Ep4. Routine HE staining was done for comparison. The results were processed by Chi-square tests in SPSS 10.0 soft ware. RESULTS: Fifteen of the 32 patients (46.9%) were found to have micro-metastasis in 21 of 181 mLN (11.6%) examined by immunohistochemical staining though routine histopathological examinations were negative. Of those 15 cases, micro-metastasis was detected in 9 only by IHC and in 6 both by IHC and HE stainings. The positive rate of micro-metastasis in N0, N1, and N2 stratified by routine pathology was 36.8% (7/19), 33.3% (2/6) and 85.7% (6/7), respectively (N0 vs N2, P < 0.05). When stratified according to clinical staging (cTNM), pathological staging (pTNM) and pathological staging on the basis of IHC (iTNM), the frequencies of N2 cases were 0, 18.8% and 46.9%, respectively (differences among the three groups: P < 0.01). Nine cases reported as N0(7) and N1(2) by routine histopathological examination were found to have micro-metastasis in mLN by IHC staining, therefore they were actually N2 cases. CONCLUSION: IHC staining with a monoclonal antibody specific for epithelial cells (Ber-Ep4) is more sensitive in the detection of mediastinal micro-metastais than routine HE staining. Underestimation of the extent of mLN metastasis by cTNM and/or pTNM stagings frequently exists in patients with clinically early lung cancer.

Alterations in expression, proteolysis and intracellular localizations of clusterin in esophageal squamous cell carcinoma.

AIM: To investigate biogenesis and intracellular localizations of clusterin to elucidate the potential molecular mechanisms implicated in tumorigenesis of esophageal mucosa. METHODS: Semi-quantitative RT-PCR for multi-region alteration analysis, Western blot for different transcriptional forms and immunohistochemical staining for intracellular localizations of clusterin were carried out in both tissues and cell lines of ESCC. RESULTS: The N-terminal deletions of the clusterin gene and the appearance of a 50-53 ku nuclear clusterin, an uncleaved, nonglycosylated, and disulfide-linked isoform, were the major alterations in cancer cells of esophagus. Naturally the 40 ku clusterin was located in the connective tissue of the lamina propria of epithelial mucosa and right under the basal membrane of epithelia, but it was disappeared in stromal mucosa of esophagus and the pre-matured clusterin was found positive in cancerous epithelia. CONCLUSION: The N-terminal deletion of clusterin may be essential for its alterations of biogenesis in ESCC.

Overexpression of annexin 1 in pancreatic cancer and its clinical significance.

AIM: To investigate the expression of annexin I in pancreatic cancer and its relationship with the clinicopathologic factors, and to evaluate its potential clinical significance. METHODS: Annexin I expression was analyzed by Western blot and immunohistochemical staining in pancreatic adenocarcinoma and multi-tissue microarrays (MTAs). RESULTS: Western blot analysis showed that annexin I was overexpressed in 84.6% (11/13) pancreatic ductal adenocarcinomas. Immunohistochemistry analysis of pancreatic cancer in MTAs showed that annexin I protein was 71.4%(30/42) positive which was markedly increased compared with that in the tumor matched normal pancreas tissues 18.4%(7/38) (P<0.01). In the meantime, the high expression of annexin 1 was correlated with the poor differentiation of pancreatic adenocarcinoma. CONCLUSION: Annexin 1 overexpression is a frequent biological marker and correlates with the differentiation of pancreatic cancer during tumorigenesis.

[Significance of CEA, SCC and Cyfra21-1 serum test in esophageal cancer].

OBJECTIVE: To study the clinical significance of serum CEA, SCC and Cyfra21-1 test in the diagnosis, prediction of prognosis and postoperative monitor of recurrence in esophageal cancer. METHODS: The concentration of serum CEA and Cyfra21-1 was measured by electrochemiluminescence immunoassay (ECLIA) using Elecsys 2010, CEA kit and Cyfra21-1 kit. Serum SCC was measured by microparticle enzyme immunoassay (MEIA) using IMx System and SCC kit. Serum of 206 patients with esophageal cancer (203 squamous cell carcinoma, 2 small cell carcinoma and 1 adenosquamous carcinoma) was measured preoperatively, 71 of whom also measued 8 to 12 days after resection. RESULTS: The cut-off value of CEA and Cyfra21-1 was < or = 3.25 ng/ml and < or = 2.61 ng/ml, which were determined by the data of 45 healthy Chinese measured during the same period. The positive ratios of serum CEA and Cyfra21-1 in 206 cases were 29.1% and 45.1%. The combined positive ratio of CEA and Cyfra21-1 was 57.3%. The CEA positive ratios, according to the pathological stage of 165 resectable patients, were 16.6% (stage I), 26.8% (II) and 30.8% (III). For Cyfra21-1, they were 27.8%, 37.5% and 50.5%. For CEA combined with Cyfra21-1, they were 38.9%, 50.0% and 63.7%. The mean value of CEA, SCC and Cyfra21-1 (especially SCC and Cyfra21-1) was found to be well correlated with the tumor volume, TNM stage and depth of tumor invasion. Patient with bulky tumor or advanced tumor (T4) usually had much higher mean value than those with early stage tumors. One week after radical resection, the level of the three tumor markers fell to normal level in 92.9% of 71 patients. The level of serum CEA and Cyfra21-1 varied greatly in a small part of the patients. Extremely elevated serum CEA and Cyfra21-1 usually indicated advanced lesion or tumor metastasis. CONCLUSION: Preoperative and postoperative measurement of serum CEA, SCC and Cyfra21-1 (especially Cyfra21-1) is helpful in the diagnosis, prediction of prognosis and monitor of postoperative recurrence in patients with esophageal cancer.

[Application of proteomic approach for solid tumor marker discovery].

In the post-genome era, the major mission of biology is to understand the functions of genome. To well know the whole book of genes, the proteins coded by different genes have to be studied more deeply since they are the final representatives of the specific genes. Proteomics is the key issue in the bioscience research. Using the two dimensional electrophoresis technique, the total proteins of tumor cells or tissues can be profiled first by their molecular weight and isoelectric points, after that, the specific interested proteins can be identified via mass spectrometry analysis combining with database searching. Cancer proteomics is a main part of the proteomics, by which we can understand the protein profiles during the different stages of the tumorigenesis and it brought a new hope for discovery of the tumor-specific biomarkers. Recently using proteomic tools with the laser capture microdissection (LCM) technique, more achievements have been made and many promising candidates of tumor-markers have been identified even most of them have not affirmed yet. This article will give a brief review about that.

Translocation of annexin I from cellular membrane to the nuclear membrane in human esophageal squamous cell carcinoma.

AIM: To investigate the alteration of the annexin I subcellular localization in esophageal squamous cell carcinoma (ESCC) and the correlation between the translocation and the tumorigenesis of ESCC. METHODS: The protein localization of annexin I was detected in both human ESCC tissues and cell line via the indirect immunofluorescence strategy. RESULTS: In the normal esophageal epithelia the annexin I was mainly located on the plasma membrane and formed a consecutive typical trammels net. Annexin I protein also expressed dispersively in cytoplasm and the nuclei without specific localization on the nuclear membrane. In esophageal cancer annexin I decreased very sharply with scattered disappearance on the cellular membrane, however it translocated and highly expressed on the nuclear membrane, which was never found in normal esophageal epithelia. In cultured esophageal cancer cell line annexin I protein was also focused on the nuclear membrane, which was consistent with the result from esophageal cancer tissues. CONCLUSION: This observation suggests that the translocation of annexin I protein in ESCC may correlate with the tumorigenesis of the esophageal cancer.

Loss of clusterin both in serum and tissue correlates with the tumorigenesis of esophageal squamous cell carcinoma via proteomics approaches.

AIM: To identify the differentially secreted proteins or polypeptides associated with tumorigenesis of esophageal squamous cell carcinoma (ESCC) from serum and to find potential tumor secreted biomarkers. METHODS: Proteins from human ESCC tissue and its matched adjacent normal tissue; pre-surgery and post-surgery serum; and pre-surgery and normal control serum were separated by two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. The silver-stained 2-DE were scanned with digital ImageScanner and analyzed with ImageMaster 2D Elite 3.10 software. A cluster of protein spots differentially expressed were selected and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). One of the differentially expressed proteins, clusterin, was down-regulated in cancer tissue and pre-surgery serum, but it was reversed in post-surgery serum. The results were confirmed by semi-quantitative reverse-transcription (RT)-PCR and western blot. RESULTS: Comparisons of the protein spots identified on the 2-DE maps from human matched sera showed that some proteins were differentially expressed, with most of them showing no differences in composition, shape or density. Being analyzed by MALDI-TOF-MS and database searching, clusterin was differentially expressed and down-regulated in both cancer tissue and pre-surgery serum compared with their counterparts. The results were also validated by RT-PCR and western blot. CONCLUSION: The differentially expressed clusterin may play a key role during tumorigenesis of ESCC. The 2DE-MS based proteomic approach is one of the powerful tools for discovery of secreted markers from peripheral.

[Cloning of human uracil N-glycosylase and its detection in cancer tissues by quantitative RT-PCR].

The uracil in DNA comes from either the misincorporation of dUTP in place of dTTP or deamination of cytosine. In the latter case, it can result in a GC to AT transition mutation if the uracil is not removed before DNA replication. Base excision repair (BER) is a major pathway for removing DNA lesions arising from endogenous processes as well as those induced by exposure to exogenous chemicals or irradiation. BER is initiated by DNA glycosylases that excise aberrant bases from DNA by cleavage of the N-glycosidic bond linking to the base of its deoxyribose sugar. Uracil N-glycosylase (UNG) is the enzyme responsible for the first step in the BER pathway that specifically removes uracil from DNA. The UNG gene undergoes both temporal and spatial regulation mainly at the level of transcription. Normally cancer cells undergo over-proliferation and up-regulate their UNG during tumorigenesis. In this study we examine the correlation between UNG level and carcinogenesis, and explore the possibility of using UNG as a marker for cancer diagnosis. Human UNG gene was amplified from the total RNA of the human choriocarcinoma cell line, JEG-3, by RT-PCR. After purification, the 942bp full-length UNG cDNA coding sequence was digested with EcoR I and Sal I, and cloned into the digested pET-21 to construct a recombinant vector, pUNG. The UNG protein was expressed under the control of T7 promoter in E. coli BL21 (DE3) cells induced with IPTG. After ultrasonic treatment, the cell lysate and precipitate were analyzed by SDS-PAGE and a 39kD band was detected. The plasmid was serially diluted at appropriate concentrations and employed as standards in the subsequent quantification. Total RNAs were extracted from 18 pairs of clinical samples, each pair contains a sample of esophageal squamous cell carcinoma (ESCC) tissue and its surrounding normal esophageal epithelia. The copy numbers of UNG mRNA in these RNA samples were determined by real-time quantitative RT-PCR using a Lightcycler (Roche). UNG was present in 13 cases of ESCC (13/18, n = 18) but absent in all of the normal tissues. The results indicated that there was a correlation between high level of UNG expression and the carcinogenesis of ESCC.