-
[show abstract]
[hide abstract]
ABSTRACT: To explore the expression status of Vigilin (high density lipoprotein binding protein) in various cell lines and human hepatocellular carcinoma (HCC) tissues.
The expression of Vigilin was measured semiquantitatively with western blot in hepatic cancer, cervical cancer and normal cell lines. The samples of 59 hepatocellular carcinoma tissues, 59 adjacent liver tissues and 33 distant non-tumor liver tissues were collected, Vigilin expression in the above samples was detected by immunohistochemistry.
Vigilin expressed in all cell lines, but no expression was found in peripheral blood lymphocytes. The expression levels of Vigilin in tumor cell lines were higher than those in normal cell lines (P < 0.05). Most of the hepatic cells expressed Vigilin, but the expression levels were different (tumor tissues: 0.2226 +/- 0.054, adjacent tissues: 0.2060 +/- 0.056, distant tissues: 0.1820 +/- 0.038, P < 0.001). Highly expression of Vigilin was observed in 54% of tumor tissues, 35% adjacement tissues, and 6% of distant non-tumor tissues, respectively.
Vigilin may have relationship with HCC progression and proliferation.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 03/2011; 42(2):170-3.
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the function of CTCF and understand the pathogenesis of tumors better, we produced rabbit polyclonal antibody of human transcription factor CTCF protein and detected its expression in several kinds of human cancer cells and tissues. GST fusion protein of human CTCF-N domain was purified by GSTrap-FF affinity chromatography and was successfully expressed under induction of IPTG in E. coli BL21. Western blotting analysis demonstrated that the polyclonal antibody can recognize the endogenous CTCF from HepG2, MCF-7 and HeLa cells specifically. The produced antibodies can be used for gene expression regulation and tissue distribution study at protein level.
Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi 04/2010; 27(2):379-83.
-
[show abstract]
[hide abstract]
ABSTRACT: To evaluate the relationship between the low-density lipoprotein cholesterol (LDL-C)/high-density lipoprotein cholesterol (HDL-C) ratio and HDL subclass distribution and to further examine and discuss the potential impact of LDL-C and HDL-C together with TG on HDL subclass metabolism.
Small-sized prebeta1-HDL, HDL3b and HDL3a increased significantly while large-sized HDL2a and HDL2b decreased significantly as the LDL-C/HDL-C ratio increased. The subjects in low HDL-C level (< 1.03 mmol/L) who had an elevation of the LDL-C/HDL-C ratio and a reduction of HDL2b/prebeta1-HDL regardless of an undesirable or high LDL-C level. At desirable LDL-C levels (< 3.34 mmol/L), the HDL2b/prebeta1-HDL ratio was 5.4 for the subjects with a high HDL-C concentration (> or = 1.55 mmol/L); however, at high LDL-C levels (> or = 3.36 mmol/L), the ratio of LDL-C/HDL-C was 2.8 in subjects, and an extremely low HDL2b/prebeta1-HDL value although with high HDL-C concentration.
With increase of the LDL-C/HDL-C ratio, there was a general shift toward smaller-sized HDL particles, which implied that the maturation process of HDL was blocked. High HDL-C concentrations can regulate the HDL subclass distribution at desirable and borderline LDL-C levels but cannot counteract the influence of high LDL-C levels on HDL subclass distribution.
Lipids in Health and Disease 01/2010; 9:69. · 2.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the demethylation effect of CDP on P16 and E-CADHERIN genes.
Breast cancer cell lines T47D and MDA-MB-435 were treated with CDP and DNA methyltransferase inhibitor 5-azacytidine (5-aza-C). The methylation of P16 and E-CADHERIN gene promoters were measured by methylation-specific PCR (MSP). The RNA transcription was determined by reverse transcription-PCR(RT-PCR).
1) The methylation-specific fragments of P16 gene promoter existed in T47D cells after 25, 50 and 75 micromol/L of CDP treatment for 6 days. An absolute demethylation on P16 gene occurred after treatment with 100 micromol/L of CDP. The unmethylation-specific fragments appeared in T47D cells after being treated with 25, 50, 75 and 100 micromol/L of CDP for 6 days. The RNA expression of P16 was detected after treatment with 75 and 100 micromol/L of CDP. 2) After being treated with 50 micromol/L of CDP, the methylation-specific fragments of CpG island in P16 gene promoter still existed in T47D cells. The unmethylation-specific fragments in T47D cells started to appear after 24 hours of treatment and lasted until 144 hour of treatment. The RNA expression was detected after 144 hours of treatment. 3) The demethylation on E-CADHERIN gene and genomic DNA or RNA transcription were not detected in MDA-MB-435 cells.
CDP has concentration- and time-dependent demethylation effect on P16 gene in T47D cells, but not on E-CADHERIN gene in MDA-MB-435 cells, which indicates that CDP has substantial diversity in molecular activities.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 09/2009; 40(5):798-802.
-
[show abstract]
[hide abstract]
ABSTRACT: To explore possible relationship among expression of human high density lipoprotein binding protein(VIGILIN), H19 and the insulin-like growth factor 2 (IGF2) mRNA in HepG2 cell cycle and investigate the role of VIGILIN in controlling imprinting genes of H19 and IGF2 mRNA expression.
We investigated time course cell cycle distribution of HepG2 cells by FACS, analyzed VIGILIN, H19 and IGF2 mRNA expression at the indicated times using RT-PCR, RNAi and real-time PCR.
Cell-cycle of HepG2 cells was approximately 20 h. 0 h-9 h and 20 h-28 h, 9 h-20 h and 28 h-39 h were S-phase and G2/M-G1-phase, respectively. Firstly, cells were synchronized by serum-starvation for 24 h. As expected, VIGILIN transcription was up-regulated with expression peaks at 20 h and 60 h after serum stimulating by the addition of 10% fetal calf serum. In parallel, H19 mRNA had a high expression level at 6 h and 43 h, and IGF2 mRNA was also increasing with cell-cycle. The expression profiles of human VIGILIN, H19, and IGF2 mRNA were ascending with cell-cycle. In addition, the knock-down of VIGILIN expression by transfecting HepG2 cells with shRNA expression plasmid pSIREN-VIG inhibited the expression of human VIGILIN, which led to the expression of H19 mRNA decrease by 12.08%, and IGF2 mRNA increase by 30.13%.
The expression of VIGILIN and H19 mRNA was the cell-cycle dependent and had something to do with each other. The results clearly shed light on the roles of VIGILIN in controlling expression of the imprinted H19 and IGF2 genes.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 09/2009; 40(5):770-4.
-
[show abstract]
[hide abstract]
ABSTRACT: To express human VIGILIN N terminus gene in E. coli, prepare the polyclonal antibody and study the subcell localization of human VIGILIN.
The N-terminal sequence of human VIGILIN was amplified by PCR and cloned into the polyclonal site of pGEX-4T-2 expression vector. The fusion protein was expressed under induction of IPTG in E. coli BL21. The extracted GST fusion protein was purified by GSTrap-FF affinity chromatography. The polyclonal antibody against human VIGILIN N was prepared by immunizing New Zealand white rabbits using the purified fusion protein as immunogen and analyzed the titer and specificity of the antiserum by ELISA and Western blot. Through immunofluorescence staining, the distribution of VIGILIN in cell was observed.
Expression of the GST fusion protein was induced with 1 mmol/L IPTG at 28 C for 3 hours. The antibody titer was 1:16000. Western blot analysis demonstrated that the polyclonal antibody can recognize VIGILIN specifically. VIGILIN present in both the cytoplasm and the nucleus. Its distribution in the nucleus concentrated on the inner layer of nuclear membrane and the region close prominent area of DAPI staining.
The human VIGILIN fusion protein was successfully expressed. The polyclonal antibody against human VIGILIN was generated and was further applied to the study of distribution of VIGILIN in cells. This study will provide a substantial base for further clarification of the quality and function of VIGILIN.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 04/2009; 40(2):185-9.
-
[show abstract]
[hide abstract]
ABSTRACT: To clone ctc f cDNA, N, Zn and C fragments separately into expresstion vector, purify and identify the expressed proteins.
Using the recombinant plasmid pGEM7Zf (-)-ctc f as template for PCR, pGEX-4T-2-ctc f, pGEX-4T-2-ctc f-N, pGEX-4T-2- ctc f-Zn and pGEX-4T-2- ctc f-C recombinants were constructed successfully. After transformed into E. coli BL21 cells, the recombinants were confirmed by enzyme digestion and sequence analysis. After optimizing the IPTG inducing condition, the purified GST fusion proteins with affinity chromatography were conformed by Far-Western blotting.
The recombinant plasmids pGEX-4T-2-ctc f, pGEX-4T-2- ctc f-N, pGEX-4T-2-ctc f-Zn and pGEX-4T-2-ctc f-C were confirmed by restriction enzyme assay and sequencing. All GST fusion proteins, CTCF, CTCF-N, CTCF-Zn and CTCF-C were successfully expressed at the optimal parameters and purified with affinity chromatography, and specifically recognized by anti-GST antibody.
Ctc f, ctc f-n, ctc f-Zn and ctc f-c gene recombinants were constructed successfully and their corresponding fusion proteins were successfully purified with affinity chromatography and identified.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 02/2009; 40(1):1-5.
-
[show abstract]
[hide abstract]
ABSTRACT: To prolong serum half-life of human Erythropoietin for better efficacy, a new form of recombinant human erythropoietin (rhEpo-L-Fc) was generated by fusion of a full length human erythropoietin gene and the Fc fragment of human IgG1 with flexible linker sequence. The fusion gene rhEPO-L-Fc was constructed by PCR, then inserted into expression vector pOptiVEC-TOPO, and expressed in Chinese Hamster Ovary cells deficient in the DHFR enzyme(CHO-dhfr-). The chimeric protein was purified by Protein A affinity chromatography, showed expected molecular weight and demonstrated a similar bioactivity compared to that of the native recombinant human erythropoietin (rhEPO) in an EPO-dependent cell-based assay. In vivo pharmacokinetic studies showed that the rhEPO-L-Fc had an elimination half-life of 27 h. In vivo efficacy studies showed that a single dose administration of rhEPO-L-Fc in rats increased the reticulocyte number in the peripheral blood significantly. These results demonstrated that the new engineered rhEPO-L-Fc may become alternative therapeutic approach to extend the half-time of rhEPO to treat anemia.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2008; 24(11):1874-9.
-
[show abstract]
[hide abstract]
ABSTRACT: The full-length of human vigilin encodes 1289 amino acids, containing 14 KH domains. We adopted a five step PCR technique to clone the full-length vigilin coding regions to investigate the functions of full-length vigilin, N-terminal of vigilin, C-terminal of vigilin and different KH.
Vigilin was dissected into five fragments according to the restrictive endounclease enzymes site of vigilin. The total RNA was extracted from the livers of healthy adults died from accident. The cDNA was amplified by RT-PCR. Then the amplified PCR product was digested and inserted into pUC118 vector and subcloned into pUC118 vector.
Eight gene fragments and full-length of human vigilin were obtained. The gene sequence of vigilin was identical with its cDNA sequence reported (NM:005336) in the GenBank.
The full-length vigilin coding region has been cloned successfully.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 12/2008; 39(6):877-81.
-
[show abstract]
[hide abstract]
ABSTRACT: To construct a shRNA eucharyotic expression plasmid against human VIGILIN and explore possible relation between human VIGILIN and HepG2 cell cycle.
We constructed the shRNA eucharyotic expression plasmid targeted human VIGILIN, and transfected HepG2 cells with shRNA expression plasmid pSIREN-VIG, then determined the expression of VIGILIN mRNA and protein in HepG2 cells by RT-PCR and Western-blot, analysed alteration of cell cycle using FACS.
The plasmid pSIREN-VIG can effectively and specifically inhibit the expression of human VIGILIN. After transfection 48 hours, the expression of VIGILIN was significantly decreased. Due to knockdown of human VIGILIN, cell cycle is impaired and cells are arrested in G2/M phase. The proportion of G2/M phase of all groups were listed as: C group (untreated wild HepG2 cells) 2.4%, M group (HepG2 cells treated with transfection reagent) 4.9%, G group (HepG2 cells transfected with pSIREN-GFP) 6.5% and V group (HepG2 cells transfected with pSIREN-VIG) 9.4%.
We have successfully constructed a shRNA expression plasmid which could effectively and specifically inhibit the expression of human VIGILIN.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 08/2008; 39(4):527-30, 539.
-
[show abstract]
[hide abstract]
ABSTRACT: In this study, 39 human hepatocellular carcinoma (HCC) tissues and 7 normal adult liver tissues were screened for heterozygous polymorphisms in IGF2, H19, and the differentially methylated region of H19 (H19DMR) using PCR–RFLP and PCR sequencing. The imprinting of IGF2 and H19 was examined by RT-PCR–RFLP, while the methylation profile of H19DMR was detected by bisulfite sequencing from every informative sample. Of the informative HCC samples 47.06% (8 of 17) demonstrated a gain of imprinting of IGF2, and 21.74% (5 of 23) of the informative HCC samples demonstrated a loss of imprinting of H19. Interestingly, we found three methylation profiles for H19DMR in the informative HCC samples: hyper-, medium-, and hypomethylated profiles. Furthermore, the hypomethylated and hypermethylated profiles were immediately associated with aberrant imprinting of IGF2 and H19.
Genomics 06/2008; · 3.02 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To analyze the relationship between plasma apoB100 concentrations, apoB100/apoA-I ratio and the alteration of HDL subclasses distribution.
The apoA-I contents of plasma HDL subclasses were quantitated by 2-dimensional gel electrophoresis coupled with immunodetection in 506 subjects.
The subjects in the highest tertile of apoB100 groups had significantly higher small-sized prebeta(1)-HDL, however lower large-sized HDL(2a), HDL(2b) than the subjects in the lowest tertile of apoB100 group. Furthermore, a significant down in the HDL(2b)/prebeta(1)-HDL values (from 6.8 to 1.6) with a rise in apoB100/apoA-I ratio (from 0.4 to 1.4) were observed. Compared to subjects with apoB100/apoA-I ratio<0.9, the subjects with apoB100/apoA-I ratio > or =0.9 had significantly higher small-sized prebeta(1)-HDL whereas lower HDL(3a), HDL(3b) and large-sized HDL(2a), HDL(2b.) Pearson correlation revealed that concentrations of apoB100 were positively correlated with prebeta(1)-HDL but negatively correlated with HDL(2a) and HDL(2b), and in multivariate analysis, all HDL subclasses were independently associated with the apoB100/apoA-I ratio.
The apoB100 concentrations, especially apoB100/apoA-I ratio could reflect sensitively the alteration of HDL subclasses distribution. And HDL subclasses distribution characteristics of hyperlipidemic subjects appeared in the subjects with apoB100/apoA-I ratio > or =0.9, which indicated the efficiency of RCT was weakened and the maturation of HDL was blocked.
Clinica Chimica Acta 02/2008; 388(1-2):148-55. · 2.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: This study was to establish BALB/c murine model featured with the human-mouse chimeras from umbilical cord blood transplantation.
Thirty BALB/c nude mice were exposed to 350 cGy radiation under the sublethal condition. The nuclear cells from fresh umbilical cord blood were injected into the mice of experimental group via tail vein, in which the mice were further allocated to A, B and C sub-group with given 1.0 x 10(7), 2.0 x 10(7) and 3.0 x 10(7) nuclear cells per mouse respectively, and simultaneously the equal volume normal sodium (NS) was injected into the mice of control group. White blood cells from peripheral blood in experimental group and the control group were detected at week 1, 2, 3, 4 and 8 after transplantation. Human CD34+ and CD45+ from peripheral blood in experimental group were detected by using flow cytometry analysis on week 4, 6 and 8 after transplantation in order to be the human-mouse chimeras known.
No difference in the number of white blood cells showed between experimental groups and control group before blood cell transplantation and after transplantation conducted on for 8 weeks (P > 0.05), but the number of white blood cells from experimental groups and control group wasn't totally same on week 1, 2, 3 and 4 after cell transplantation conducted (P < 0.05). As compared with pre-transplantation, the number of white blood cells for post-transplantation at different time in experimental groups and control group was decreasing, and the lowest on 1 week, then going up and to the level of pre-transplantation on 4 week in experimental groups and on 8 week in control group. CD34+ and CD45+ cells in peripheral blood for nude mice appeared on 4 week, but the total number was little. The number of CD34+, CD45+ cells in experimental group A was less than those in experimental group B and C at all the stage (P < 0.05), but there was no difference between experimental group B and C(P > 0.05). There was obvious correlation between CD34+ and CD45+ on 4 week (r = 0. 903, P < 0.05), but this correlation didn't appear on 6 and 8 week (P > 0.05), which might be related to the amount of sample.
The BALB/c murine model can be successfully established with the human-mouse chimeras from umbilical cord blood transplantation.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 01/2008; 39(1):44-7.
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the reactivation of hypermethylated GSTP1 (glutathione S-transferase P1) promoter activity by a component of natural drug, CDP.
The recombinant plasmid of pGL3-GSTP1 pro(m) containing hypermethylated GSTP1 gene promoter was constructed and then used to transiently transfect the human breast cancer cell line MCF-7 cell. After treatment with CDP and 5-aza-C, The luciferase activity in cell lysates were assayed.
Low promoter activities were found in hypermethylated GSTP1 promoter. The promoter activities were reactivated and in a CDP dose-dependent mode.
CDP has the ability to reactivate the hypermethylated GSTP1 gene promoter activity.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 10/2007; 38(5):761-5.
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the allelic expression of IGF2/H19 in human hepatocellular carcinoma and to analyze the relationship between the imprint status of IGF2 and that of H19.
Heterozygotes of IGF2 and of H19 were identified by restriction fragment length polymorphism (RFLP). Allelic expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and RFLP.
Forty seven point one percent (47.1%, 8 of 17) of HCC samples were demonstrated to have the gain of imprinting (GOI) of in IGF2 gene, and 45.5% (5 of 11) of HCC samples were found to have the loss of imprinting (LOI) for H19 gene. No relationship was observed between the imprinting status of IGF2 and that of H19.
IGF2 GOI and H19 LOI are common in HCC, and the imprinting of IGF2 could be independent from that of H19 in adult liver.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 02/2007; 38(1):49-52.
-
[show abstract]
[hide abstract]
ABSTRACT: To construct a combination method of methylation sensitive restriction enzyme and semi-nested touch down PCR assay for studying the promoter region methylation status of P16 gene in human hepatocellular carcinoma.
According to the sequence of CpG rich promoter region of P16 gene, three primers were designed and synthesized for semi-nested touch down PCR assay to examine the promoter region methylation status of P16 gene. 340 bp segment of this region was cloned into vector pMD18-T; the plasmid was transformed into E. coli JM109 to harvest an extended quantity, then the plasmid was treated by CpG methylase M. Sss I, the methylated plasmid was named P16Pm+. This P16Pm+ is validated by digestion of Hpa II and is employed in studying the specificity and sensitivity of this constructed method. After construction, the method was used to examine the promoter region methylation status in P16 gene of 40 DNA samples from human HCCs and three DNA samples from normal human liver tissue.
It was confirmed that the specificity and sensitivity of this method are solid and reliable (100 fg). It was found that 12/40 (30%) of hepatocellular carcinoma showed promoter region methylation in P16 gene whereas none (0/3) of the normal tissues was methylated in the promoter region in P16 gene.
Promoter region methylation in P16 gene may take part in human hepatocellular carcinogenesis. The constructed method is simple, cost-effective and is of high specificity and sensitivity, thus suggesting its potential application to detecting promoter methylation in population-based studies.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 02/2007; 38(1):53-6.
-
[show abstract]
[hide abstract]
ABSTRACT: To purify an active extracellular region of the TNF related apoptosis inducing ligand (TRAIL) protein.
According to the high-usage codons in Escherichia coli and the multiple cloning site of expression vector pTWIN1 of a self-splicing prokaryotic expression system, the extracellular region of TRAIL gene was designed and synthesized, which was cloned into pMD18-T vector. After pMD18-T/TRAIL and pTWIN1 were digested by Nru I and EcoR I, the target fragment purified was linked to the expression vector pTWIN1, which was transferred into the competent cell JM109, and positive recombination was screened. After positive recombination vector pTWIN1/TRAIL (identified with restrictive endonuclease digesting and sequencing) was transferred into the ER2566, the expression was induced by different IPTG concentration at different temperature and culture time. The expression products were analyzed by 150 g/L SDS-PAGE.
The extracellular region of TRAIL gene was obtained by PCR, and was constructed successfully in a self-splicing prokaryotic expression vector pTWIN1/TRAIL. By 0.3 mmol/L IPTG at 15 degrees C for 14 to 16 hours, the soluble target protein was expressed efficiently.
High-expression level of the extracellular region of TRAIL fusion protein was attained by use of E. coli ER2566, and the soluble target protein without any additional amino acid was successfully purified by simple treatment.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 10/2006; 37(5):670-4, 678.
-
[show abstract]
[hide abstract]
ABSTRACT: To express recombinant Ancylostoma caninum anticoagulant peptide-c2 (AcAPc2), a whole cDNA fragment encoding AcAPc2 was achieved by ligation- PCR and inserted into prokaryotic expression vector pTWIN1 for constructing the specific self-splicing prokaryotic expression vector, pTWIN1-AcAPc2; positive recombinants were transformed into E. coli ER2566 for expression research. The recombinant protein, AcAPc2-intein2-CBD, was soluble and expressed in E. coli ER2566 (about 30.1% fusion protein in total protein). AcAPc2-intein2-CBD was characterized to be 41 KD by SDS-PAGE and identified by Western-blot. The recombinant fusion protein was purified to a efficiently high degree by chitin affinity chromatography. After the process of specific self-splicing induced by beta-Mercaptoethanol, the target protein, AcAPc2, was obtained, characterized to be 21 KD by SDS-PAGE and migrated as a dimmer. Molecular weight of AcAPc2 conformed to native dimmer. Bio-information analysis indicated relationship between secondary construction of AcAPc2 and biologic function. These findings greatly facilitate the purification of AcAPc2 and are very important for the additional studies on its anti-coagulation mechanism and its clinical application as anti-coagulation medicine.
Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi 07/2006; 23(3):630-4.
-
[show abstract]
[hide abstract]
ABSTRACT: Glutathione S-transferases, enzymes that prevent cells from damage mediated by oxidant and electrophilic carcinogens, may be early critical determinants of carcinogenesis. To explore the aberrant promoter CpG island methylation of the GSTP1 gene as a biomarker for screening hepatocellular carcinoma (HCC) high risk individuals and for the early detection of HCC, we analyzed its methylation in the tumor and non-tumor tissues and serum samples from 26 patients with HCC, as well as serum from 8 liver cirrhosis patients by methylation-specific PCR (MSP).
Twenty-three of 26 (88.5%) tumor tissues and 18 of 26 (69%) corresponding non-tumor tissues displayed GSTP1 promoter CpG island hypermethylation. Similarly, GSTP1 promoter hypermethylation was detected for the first time in 16 of 32 (50%) of circulating tumor DNA in the peripheral serum from HCC patients and 4 of 8 (50%) cirrhosis tissues and 3 of 8 (37.5%) corresponding serum DNA from cirrhosis patients. The aberrant methylation of the GSTP1 gene in the serum of patients is in agreement with tumor methylation status (P = 0.004). None of the 12 normal PBMC samples were methylation positive.
These data indicate that the epigenetic aberrance of promoter CpG island hypermethylation of the GSTP1 gene may contribute to the hepatopathogenesis of HCC and is a potential valuable biomarker for noninvasive disease monitoring and HCC early diagnosis.
Clinical Biochemistry 05/2006; 39(4):344-8. · 2.08 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: nm23-H1 gene is a well-known tumor metastasis suppression gene. Our previous study has found that transfection of wild type nm23-H1 gene can significantly downregulate the ERK1/2 activity of human high-metastatic large cell lung cancer cell line L9981. The aim of this study is to investigate the influence of nm23-H1 and exogenous ERK1/2 pathway inhibitor U0126 on the extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.
The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell lines, L9981 (parent cell line with nm23-H1 gene hetero-deletion), L9981-nm23-H1 (transfected with nm23-H1 gene ) and L9981-PLXSN (transfected with vector) were detected by Western blot and immunoprecipitation technique after treating with U0126 (40μmol/L for 20 minutes). The in vitro proliferative and invasive abilities among the above three lung cancer cell lines were determined by MTT and improved Boyden chamber methods.
The phosphorylated ERK1/2 expression level and relative activity in L9981-nm23-H1 lung cancer cell line were remarkably lower than those in L9981 and L9981-PLXSN lung cancer cell lines after being treated with U0126 (P < 0.01), but there was no significant difference between L9981 and L9981-PLXSN lung cancer cell lines. No significant difference of total ERK1/2 expression level was observed among the three lung cancer cell lines (P > 0.05) after being treated with U0126. The in vitro proliferation and invasion of L9981-nm-23H1 lung cancer cell line were remarkably lower than those of L9981 and L9981-PLXSN lung cancer cell lines (P < 0.01 ), but no significant difference was found between L9981 and L9981-PLXSN lung cancer cell lines (P > 0.05 ); U0126 could significantly down-regulate the in vitro proliferation and invasion of L9981 lung cancer cell line (P < 0.01).
Blocking the activity of ERK1/2 in L9981 lung cancer cell line and transfecting the nm23-H1 gene into the L9981 lung cancer cell line may produce similar cell biological behavior changes, namely the significant reduction of in vitro proliferation and invasion of L9981 lung cancer cell line. These results indicate that the molecular mechanism which nm23-H1 gene reverses invasion and proliferation of the human high-metastatic large cell lung cancer cell line may be related to its effects of down-regulating the activity of the key kinase ERK1/2 of Ras-to-MAPK signal transduction pathway.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 01/2006; 9(4):307-11.
