Advances in cardiovascular-related biomarkers to predict diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is a common chronic complication of diabetes mellitus. One of the most common types is distal symmetric poly-neuropathy, which begins as bilateral symmetry pain and hyperesthesia and gradually progresses into hypoesthesia with nerve fibre disorder and is frequently accompanied by depression and anxiety. Notably, more than half of patients with DPN can be asymptomatic, which tends to delay early detection. Furthermore, the study of adverse outcomes showed that DPN is a prominent risk factor for foot ulceration, gangrene and nontraumatic amputation, which decreases quality of life. Thus, it is essential to develop convenient diagnostic biomarkers with high sensitivity for screening and early intervention. It has been reported that there may be common pathways for microvascular and macrovascular complications of diabetes. The pathogenesis of both disorders involves vascular endothelial dys-function. Emerging evidence indicates that traditional and novel cardiovascular-related biomarkers have the potential to characterize patients by subclinical disease status and improve risk prediction. Additionally, beyond traditional cardiovascular-related biomarkers, novel cardiovascular-related biomarkers have been linked to diabetes and its complications. In this review, we evaluate the association between major traditional and nontraditional car-diovascular-related biomarkers of DPN, such as cardiac troponin T, B-type natriuretic peptide, C-reactive protein, myeloperoxidase, and homocysteine, and assess the evidence for early risk factor-based management strategies to reduce the incidence and slow the progression of DPN.

Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells.

Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells.

Renal tissue thawed for 30 minutes is still suitable for gene expression analysis.

Some biosamples obtained from biobanks may go through thawing before processing. We aim to evaluate the effects of thawing at room temperature for different time periods on gene expression analysis. A time course study with four time points was conducted to investigate the expression profiling on 10 thawed normal mice renal tissue samples through Affymetrix GeneChip mouse gene 2.0 st array. Microarray results were validated by quantitative real time polymerase chain reactions (qPCR) on 6 candidate reference genes and 11 target genes. Additionally, we used geNorm plus and NormFinder to identify the most stably expressed reference genes over time. The results showed RNA degraded more after longer incubation at room temperature. However, microarray results showed only 240 genes (0.91%) altered significantly in response to thawing at room temperature. The signal of majority altered probe sets decreased with thawing time, and the crossing point (Cp) values of all candidate reference genes correlated positively with the thawing time (p<0.05). The combination of B2M, ACTB and PPIA was identified as the best choice for qPCR normalization. We found most target genes were stable by using this normalization method. However, serious gene quantification errors were resulted from improper reference genes. In conclusion, thirty minutes of thawing at room temperature has a limited impact on microarray and qPCR analysis, gene expression variations due to RNA degradation in early period after thawing can be largely reduced by proper normalization.

NANOG promotes liver cancer cell invasion by inducing epithelial-mesenchymal transition through NODAL/SMAD3 signaling pathway.

NANOG is a major transcription factor essential to the stem cell self-renewal and is associated with tumor malignancy, but the NANOG signaling in cancer metastasis is still elusive. In this study, we determined the expression of NANOG in hepatocellular carcinoma (HCC) and investigated its underlying mechanism in the metastasis of HCC. The expression levels of NANOG were examined in tumor tissues by immunohistochemistry. Functional effect of NANOG was investigated both in vivo and in vitro. Our data shows that high level of NANOG expression correlates with metastasis and low survival rate in HCC. HCC cells overexpressing NANOG are characterized by active epithelial-mesenchymal transition (EMT), and exhibit increased ability of invasion, soft agar colonization, sphere formation and drug resistance, whereas SB-431542, an antagonist of activin receptor-like kinase (ALK) receptors, attenuates EMT and invasion of HCC cells. NANOG activates NODAL and CRIPTO-1 to promote SMAD3 phosphorylation and SNAIL expression. The transcriptional activity of NODAL gene is dependent on two NANOG binding motifs in its promoter region. This study shows a significant correlation between the NANOG expression and the expression of NODAL, P-SMAD3 or SNAIL, and the combination of NANOG and P-SMAD3 is a potential predictor of poor prognosis of HCC. Additionally, cells in the tumor edge area displays higher NANOG expression than cells in the tumor center. These results present novel mechanistic insight into an important role of NANOG in HCC metastasis, and suggest a potential application of NANOG in HCC prognosis and treatment.

[Applicability of the multiplex quantitative antibody array system for early diagnosis of hepatocellular carcinoma].

OBJECTIVE: To develop an early and accurate detection method for hepatocellular carcinoma (HCC) based on detection of tumor-associated serum markers using a multiplex quantitative antibody array. METHODS: The double-antibody sandwich principle was used to establish an antibody array composed of eight cancer-related serum markers, including alpha-fetoprotein (AFP), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-b1), and vascular endothelial growth factor (VEGF). Serum samples from 160 cases of clinically diagnosed HCC and from 58 cases of liver cirrhosis (LC; controls) were obtained to test the array. Sixty percent of the samples were randomly selected for use as the training set (HCC, n = 96; LC, n = 36), and the remaining 40% was used as the test set (HCC, n = 64; LC, n = 22). The SPSS statistical software was used to perform logistic regression analysis and to create a diagnostic model. RESULTS: When used with the training set, the model had sensitivity of 93.3%, specificity of 83.3%, and accuracy of 90.9%. When used with the test set, the model had sensitivity of 89.0%, specificity of 77.3%, and accuracy of 86.0%. The traditional serum AFP value (cut-off value of 20 ng/mL) showed 70.0% diagnostic sensitivity, 59.0% specificity, and 64.0% accuracy. CONCLUSION: The newly developed multiplex quantitative antibody detection system has high sensitivity and specificity. The diagnostic model with AFP and seven other cancer-related factors was superior to the traditional AFP only approach for early diagnosis of liver cancer, indicating its potential clinical value.

iTRAQ-2DLC-ESI-MS/MS based identification of a new set of immunohistochemical biomarkers for classification of dysplastic nodules and small hepatocellular carcinoma.

The study aims to develop novel clinical immunohistochemical biomarkers for distinguishing small hepatocellular carcinoma (sHCC) from dysplastic nodules (DN). iTRAQ-2DLC-ESI-MS/MS technique was used to screen immunohistochemical biomarkers between precancerous lesions (liver cirrhosis and DN) and sHCC. A total of 1951 proteins were quantified, including 52 proteins upregulated in sHCC and 95 proteins downregulated in sHCC by at least 1.25- or 0.8-fold at p < 0.05. The selected biomarker candidates were further verified using Western blotting and immunohistochemistry. Furthermore, receiver operation characteristics (ROC) curves and logistic regression model were carried out to evaluate the diagnostic values of the biomarkers. Finally, aminoacylase-1 (ACY1) and sequestosome-1 (SQSTM1) were chosen as novel candidate biomarkers for distinction of sHCC from DN. A constructed logistic regression model included ACY1, SQSTM1, and CD34. The sensitivity and specificity of this model for distinguishing sHCC from DN was 96.1% and 96.7%. In conclusion, ACY1 and SQSTM1 were identified as novel immunohistochemical biomarkers distinguishing sHCC from DN. In conclusion, expression levels of CD34, ACY1, and SQSTM1 can be used to establish an accurate diagnostic model for distinction of sHCC from DN.

[Identification of metastasis-related osteopontin expression and glycosylation in hepatocellular carcinoma].

OBJECTIVE: To test expression level and glycosylation level of OPN in HCC cell lines with different metastatic potential and HCC tissues, and investigate the correlation between the glycosylation change and the liver cancer transporting as well as its significance. METHODS: The level of OPN expression in liver cancer tissue(6 cases of non-metastasis and 7 cases of metastasis)as well as HCC cell lines with different metastatic potential (L02, Hep3B, MHCC97L, MHCC97H, HCCLM3, HCCLM6)was identified by immunohistochemistry and Western Blot, and then OPN was purified from HCC tissues by immunoprecipitation, followed by glycosylation detection of OPN from non-metastatic and metastatic HCC tissues by multiple lectin blot. Data were analyzed by t-test and variance analysis. RESULTS: Different levels of OPN expression were observed in HCC cell lines with different metastatic potential (F = 5.04, P = 0.008). Additionally, OPN expression level in HCC tissues with metastasis was higher than that in non-metastasis group (t = 2.447, P < 0.05). Relative optical density value was 0.69 ± 0.21 and 0.45 ± 0.14 respectively. OPN in liver cancer tissue was successfully purified using immunoprecipitation. Followed lectin blotting result showed that OPN protein in metastasis group showed lower affinity to MAL, PHAE, DSA, ConA as compared with that in non-metastasis group (P < 0.05). CONCLUSIONS: The expression of OPN was positively correlated with the enhanced metastasis potential of HCC. OPN from metastasis HCC tissues presented lower level of some specific glycan structures such as a2, 3- sialic acid, bisecting GlcNAc, biantennary, muti-antennary and high mannose type N-glycan structure. This study not only indicates the role of OPN in HCC metastasis for the first time, but also provide experimental support for the mechanism of the function of OPN in the transportation of liver cancer cells as well as offer potential target for clinical treatment.

[Effects of AKR1B10 gene silence on the growth and gene expression of HCC cell line MHCC97H].

OBJECTIVE: To explore the biological function and possible underlying mechanism of aldo-keto reductase family 1 member B10 (AKR1B10) gene during hepatocarcinogenesis. METHODS: A pair of chemically synthesized small interfering RNA (siRNA) targeting on AKR1B10 was transfected into liver cancer cell line MHCC97H by LipofectamineTM 2000. After confirming the interfering effects of AKR1B10-siRNAs through Quant SYBR Green polymerase chain reaction (Real-time PCR), Western blot and enzymatic activity assay, the capabilities of proliferation and apoptosis of the transfected cells were observed by CCK-8 assay and flow cytometry analysis, and the expressions of a group of tumor-related gene such as c-myc, c-fos, N-ras were observed through Real-time PCR. RESULTS: The expressions of AKR1B10 and the enzymatic activity were down-regulated significantly in AKR1B10-siRNA-transfected cells. Compared with mock and blank control groups, cell growth in AKR1B10-siRNA-transfected group was inhibited by 26.6%+/-3.1% at 72h after transfection. The ratio of apoptotic cells was 37.3%+/-1.0% in AKR1B10-siRNA-transfected group, which was significantly higher than that in mock and blank control groups (P < 0.01). Real-time PCR showed that the expressions of oncogene c-myc, c-fos and N-ras, and the proliferation-associated gene ki-67 were down-regulated in AKR1B10-siRNA-transfected cells, while the expressions of apoptosis-promoting gene caspas-3 and bax were up-regulated. CONCLUSIONS: AKR1B10 might promote proliferation, inhibit apoptosis and then induce malignant transformation of hepatocytes by regulating the expression level of some tumor-related genes.

Array-based profiling of the differential methylation status of CpG islands in hepatocellular carcinoma cell lines.

Alterations in the DNA methylation status particularly in CpG islands are involved in the initiation and progression of many types of human cancer. A number of DNA methylation alterations have been reported in hepatocellular carcinoma (HCC). However, a systematic analysis is required to elucidate the relationship between differential DNA methylation status and the characteristics and progression of HCC. In the present study, a global analysis of DNA methylation using a human CpG-island 12K array was performed on a number of HCC cell lines of different origin and metastatic potential. Based on a standard methylation alteration ratio of ≥2 or ≤0.5, 58 CpG island sites and 66 tumor-related genes upstream, downstream or within were identified. This study showed a series of CpG island methylation alterations in the HCC cell lines. The expression of various oncogenes, tumor suppressor genes and other key genes were up- or downregulated, respectively, resulting in CpG island hypomethylation or hypermethylation accordingly. To conclude, a foundation has been provided for screening CpG island methylation profiles as HCC biological markers.

[Comparison of the serum proteomes of pathological stages during hepatocarcinogenesis].

OBJECTIVE: To compare the 2-DE profiles for serum proteins of different pathological stages during hepatocarcinogenesis. METHODS: Sera from hepatocellular carcinoma patients, cirrhosis patients, chronic hepatitis patients and healthy controls were collected. After sonication, albumin and immunoglobulin (IgG) depletion, and desalination, sera were subjected to 2-DE, the differential protein spots were identified by MALDI-TOF-MS. Western blot was used to validate these differentially expressed proteins. RESULTS: 2-DE sera protein profiles were obtained from the patient suffering from HCC, liver cirrhosis, chronic hepatitis, healthy controls in each group. From optimized 2-DE gel images of the above groups, 96 protein spots with more than 2-fold difference in intensity between the two groups were selected by image master 6.0 software, differential proteins including haptoglobin, SAA1 and SP40 were identified by MALDI-TOF-MS/MS. 7 different spots within more than 30 protein spots belonged to the same haptoglobin family. The differential expression of haptoglobin was confirmed by western blot. CONCLUSIONS: Four protein expression patterns have been identified during the pathological stages of hepatocarcinogenesis. Haptoglobin is significantly increased from liver cirrhosis to HCC. It implies that haptoglobin might be a potential biomarker in the early diagnosis of liver cancer.

[Differentially expressed proteins in the precancerous stage of rat hepatocarcinogenesis induced by diethylnitrosamine].

OBJECTIVE: To screen the differentially expressed proteins especially at the precancerous stage of diethylnitrosamine (DEN) induced hepatocarcinogenesis by comparative proteome research. METHODS: Rats were divided into normal and DEN groups and sacrificed periodically. The liver samples were stained with gamma-glutamyl transpeptidase (GGT) and HE to distinguish the preneoplastic lesion (pre-HCC) from the normal and HCC tissues. The two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF-MS/MS) were then applied to analyze the differentially expressed protein between pre-HCC and normal tissues, pre-HCC and HCC, as well as HCC and normal tissues. A few of the candidate proteins such as laminin receptor 1 (67LR) and agmatinase were validated by Western blot and RT-PCR. RESULTS: Totally, there were 82 proteins that differentially expressed two fold or more in one kind of tissues sample than the other, 47 of which occurred in the pre-HCC tissues. Eight proteins including 67LR were consistently up-regulated from normal tissue to pre-HCC and then to HCC tissues, while 22 proteins including agmatinase showed progressively down-regulated in these tissues samples. CONCLUSION: The protein expression profiles are different during the process of hepatocarcinogenesis. Further study on the differentially expressed protein, especially these upregulated in the precancerous stage such as 67LR and agmatinase, might contribute to prevention and early diagnosis of human HCC.

[Protein profiles of multinodular hepatocellular carcinoma with multicentric occurrence or with intrahepatic metastasis].

OBJECTIVE: To analyze the protein expression profiles of multinodular hepatocellular carcinoma (HCC) with multicentric occurrence (MO) or with intrahepatic metastasis (IM). METHODS: 5 IM and 6 MO patients were divided into groups of IM1, IM2, MO1 and MO2 according to the size of node of HCC. Two dimensional gel electrophoresis (2-DE) and mass spectrum were used to analyze the protein expression profiles. Western blot was used to confirm the results obtained by mass spectrum. RESULTS: 2-DE of IM1, IM2, MO1 and MO2 indicated that 30 protein dots were differentially expressed in these tumors. By mass spectrum, 25 proteins were identified. Gene ontology classification indicated that these proteins are associated to cell movement, signal transduction, oxidoreduction, lipid metabolism, and amino acid metabolism. CONCLUSION: The protein expression profiles of IM is different from that of MO, 2-DE and mass spectrum can be used to identify the molecular markers of IM and MO of HCC.

[Analysis of the expression of Slit/Robo genes and the methylation status of their promoters in the hepatocellular carcinoma cell lines].

OBJECTIVE: To analyze the expression of genes in the Slit/Robo signaling pathway, and the methylation status of their promoters in hepatocellular carcinoma (HCC) cell lines. METHODS: Genomic DNA and total RNA were isolated from 9 HCC cell lines of different metastatic ability (Hep3B, HepG2, PLC/PRF/5, SMMC-7721, BEL-7402, MHCC97-H, MHCC97-L, LM3, LM6) and a control cell line L-02. The expression profiles of Slit1, Slit2, Slit3, Robo1, and Robo3 were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The methylation status of the promoters was detected by methylation specific polymerase chain reaction (MSP). RESULTS: The promoters of Slit1, Slit2 and Slit3 genes were almost methylated in all the HCC cell lines. The Slit1 and Slit3 RNAs were not detected in most of the cell lines. Furthermore, the mRNA Slit2 was decreased gradually as the metastatic potential of the cell lines increased. As the candidate ligand of the Slit2 gene, Robo1 was frequently methylated in HCC cell lines whereas its mRNA was detected in all of these cells except SMMC-7721, BEL-7402 and L-02. Robo3 was unmethylated in HCC cell lines while its mRNA was not detected in these HCC cell lines. CONCLUSION: The hypermethylation status of Slit/Robo signaling pathway related genes is a universal event in the HCC. The hypermethylation status of Slit1, Slit2, Slit3 genes associated with the loss of expression or reduced expression. Those data suggest that Slit/Robo pathway may play a significant role in the progress or metastasis of HCC.

Application of SELDI-TOF-MS to identify serum biomarkers for renal cell carcinoma.

PURPOSE: Early diagnosis is critical for improving the outcome of patients with renal cell carcinoma (RCC). In this study, we applied a proteomic approach to identify serum biomarkers associated with different stages of renal tumor development. MATERIALS AND METHODS: The protein expression profiles in patient serum samples were analyzed using surface enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF-MS). The subjects included 65 patients with renal cell carcinomas, 34 with benign renal tumors, and 69 normal controls. A diagnostic decision tree was developed and validated based on the differentially expressed proteins between the serum of patients with small (

[Analysis of gene expression profile during malignant transformation of rat liver oval-like cells].

OBJECTIVE: To investigate the changes of gene expression profile during malignant transformation of rat liver oval-like cells and to analyze the significances of these changes. METHODS: MNNG initiated WB-F344 cells were exposed to H2O2 once a week (repeated 21 times) to induce their malignant transformation. The characteristics of the transformed cells were confirmed by morphology, genetics and soft agar assay. Then, gene expression profiles of the transformed cells at different time points were evaluated using rat Cancer PathwayFinder Oligo Microarray. RESULTS: The transformed cells possessed heteroploid karyotypes with anchoring-independent growth characteristics. Transmission electron microscopy showed that the transformed cells had more cellular organelles, gap junctions and microvilli than the controls. The 21 differential expression genes were mainly involved in regulating cell proliferation, apoptosis, adhesion and motility. The expression of PTEN was gradually up-regulated and cdkn1a was gradually down-regulated in three groups of cells. Myc, fos, and casp-8 were first up-regulated in WB-5 cells and then down-regulated in WB-21 cells. CONCLUSION: The disorder of proliferation and apoptosis may play an important role in malignant transformation of WB cells. Cdkn1a, PTEN, myc and fos may be crucial factors involved in the process.

[Proteomic analysis in combination with CT diagnosis to distinguish renal cell carcinoma from renal benign masses].

OBJECTIVE: To identify the proteomic differences between renal cell carcinoma (RCC) and renal benign masses and to evaluate the diagnostic value of parallel and serial test combining with CT and surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS). METHODS: Serum samples were collected from 96 patients with renal tumors, 62 RCC cases and 34 renal benign mass cases, all of which had been evaluated by CT before surgery. The sera were analyzed using IMAC-Cu2+ ProteinChip system by SELDI-TOF-MS. The decision tree was generated by Biomark Pattern based on the sera of 42 RCC cases and 22 renal benign mass cases and was blind-tested by the rest sera samples. The parallel method and serial method combining CT and SELDI were also used to distinguish RCC and renal benign masses. RESULTS: The sensitivity and specificity of the decision tree were 85.7% (36/42) and 90.9% (20/22) respectively. The sensitivity and specificity of the double-blind test were 75.0% (15/20) and 83.3% (10/12) respectively. CT showed higher sensitivity but lower specificity in detecting RCC. While combining CT with SELDI-TOF-MS, the sensitivity and specificity could be improved. CONCLUSION: Three peaks with the molecular weights of 4657.56, 2955.95, and 3278.00 were detected which are potentially useful for differentiating RCC and renal benign masses. Using serial method combining CT and the decision tree based on these three proteins improves the sensitivity and positive predictive values of diagnosing RCC to 100%.

Screening serum hepatocellular carcinoma-associated proteins by SELDI-based protein spectrum analysis.

AIM: To find out potential serum hepatocellular carcinoma (HCC)-associated proteins with low molecular weight and low abundance by SELDI-based serum protein spectra analysis, that will have much application in the diagnosis or differentiated diagnosis of HCC, as well as giving a better understanding of the mechanism of hepato-carcinogenesis. METHODS: Total serum samples were collected with informed consent from 81 HCC patients with HBV(+)/cirrhosis(+), 36 cirrhosis patients and 43 chronic hepatitis B patients. Serum protein fingerprint profiles were first generated by selected WCX2 protein chip capture integrating with SELDI-TOF-MS, then normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard. Comparative analysis of the intensity of corresponding protein fingerprint peaks in normalized protein spectra, some protein peaks with significant difference between HCC and cirrhosis or chronic hepatitis B were found. RESULTS: One hundred and twenty-eight serum protein peaks between 2000 and 30000 Da were identified under the condition of signal-to-noise > 5 and minimum threshold for cluster > 20%. Eighty-seven of these proteins were showed significant differences in intensity between HCC and cirrhosis (P < 0.05). Of the above differential proteins, 45 proteins had changes greater than two-fold, including 15 upregulated proteins and 30 downregulated proteins in HCC serum. Between HCC and chronic hepatitis B, 9 of 52 differential proteins (P < 0.05) had intensities of more than two-fold, including 2 upregulated proteins and 7 downregulated proteins in HCC serum. Between cirrhosis and chronic hepatitis B, 28 of 79 significant differential proteins (P < 0.05) changes greater than two-fold in intensity, including 17 upregulated proteins and 11 downregulated proteins in cirrhosis serum. For the analysis of these leading differential proteins in subtraction difference mode among three diseases, the five common downregulated proteins in HCC serum (M/Z 2870, 3941, 2688, 3165, 5483) and two common upregulated proteins (M/Z 3588, 2017) in HCC and cirrhosis serum were screened. CONCLUSION: Because the interference of unspecific secreted proteins from hepatitis B and cirrhosis could be eliminated partly in HCC serum under subtraction difference analysis, these seven common differential proteins have the obvious advantage of specificity for evaluating the pathological state of HCC and might become novel candidate biomarkers in the diagnosis of HCC.

[Screening for differential methylation status by CpG island microarray in the hepatocellular carcinoma cell lines].

OBJECTIVE: To profile the methylation alterations of CpG islands in hetpatocellular carcinoma cell lines. METHODS: A global analysis of DNA methylation using the Human CpG-island 12K Array (HCGI12K) from Canada University Health Network was performed on nine human hepatocellular carcinoma (HCC) cell lines (Hep3B, HepG2, PLC/RPF/5/RPF/5, SMMC-7721, BEL-7402, MHCC97-H, MHCC97-L, HCCLM3, HCCLM6) and a control cell line Chang's liver. Metastatic potential related alterations were also screened in MHCC97 series cell lines (MHCC97-H, MHCC97-L, HCCLM3, HCCLM6), using MHCC97-L, a cell line with low metastatic potential, as control. To screen the key genes which are hypermethylation or hypomethylation in the HCC cell lines compared with the normal liver cell line by normalization processing and cluster analysis of microarray data. Two randomly selected genes was analyzed by methylation specific PCR to verify the chip results. RESULTS: By a standard of methylation alteration ratio > or = 2 or < or = 0.5, fifty-eight CpG island cloning sites and sixty-six upstream or downstream tumor-related genes were identified. The genes were oncogenes, tumour suppressor genes and their ligand genes, apoptosis-related genes, cell proliferation and differentiation genes, cell cycle-related gene and cell signaling pathway key genes such as Wnt, ras, and FGF pathway-related genes. The methylation specific PCR results were consistent with those obtained by chips. CONCLUSION: The results of this study demonstrate that there are a series of CpG island methylation alterations in HCC cell lines. The expression of many oncogenes, tumor suppressor genes and other key genes may be up- or down-regulated, respectively, because of their CpG island hypomethylation or hypermethylation accordingly. It may provide a basis for screening HCC biological markers by CpG island methylation profilling.

[Screening low molecular weight protein biomarkers relevant to portal vein tumor thrombi in serum of patients with hepatocellular carcinoma].

OBJECTIVE: To screen low molecular weight protein biomarkers relevant to portal vein tumor thrombi (PVTT) in serum of hepatocellular carcinoma (HCC) patients. METHODS: Serum samples were obtained from 12 healthy volunteers, 12 HCC patients without PVTT and 12 HCC patients with PVTT. Using two-dimensional gel electrophoresis (2-DE) in which the second dimension was 16% SDS-PAGE, serum protein images of the 3 groups were analyzed by ImageMaster software. The differential protein spots were further identified by MALDI-TOF MS/MS. RESULTS: Comparing the results using 12.5% SDS-PAGE gel, there were more protein bands (between 3 x 10(3) and 20 x 10(3)) and low molecular weight (MW) protein spots (less than 20 x 10(3)) were clearly shown in the 16% SDS-PAGE gel. Fifteen differential protein spots representing 5 proteins were found in the 3 groups by inter-class comparison and they were then identified. Compared with those in the healthy group, apolipoprotein A-I, lipoprotein CIII, transthyretin and DNA topoisomerase II were all down regulated in HCC groups and haptoglobin-2 was over expressed. All 5 proteins decreased more in the PVTT group than in the non-PVTT group. CONCLUSION: The expression of low MW serum protein obviously changes in the beginning and in the progressive stage of HCC, and differentially expressed low MW proteins might be potential biomarkers in an early prognostic prediction and surveillance in the treatment for HCC and PVTT.