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Objective To investigate the correlationship between DNMT3a, DNMT3b protein expressions and the state of promoter methylation of ERα gene and ERα protein expression in the development of sporadic breast cancer. Methods A total of 180 patients with sporadic breast cancer and 30 patients with breast fibroadenoma were included in this study. The expressions of DNMT3a and DNMT3b protein were detected by immunohistochemical method. The state of promoter methylation of ERα gene was detected by methylation specific PCR in 97 patients with sporadic breast cancer. Results There were no significant differences in positive expression rates of DNMT3a and DNMT3b protein between breast fibroadenoma and breast cancer. There were higher expression levels of DNMT3a and DNMT3b in breast cancer patient of Ⅲ～Ⅳstages than those of Ⅰ～Ⅱstages. The expression of DNMT3a was significantly higher in patients with lymph node metastasis than that of patients without lymph node metastasis (P＜0.05). Of 97 cases of breast cancer patients, ERα gene promoter methylation occurred in 39 cases (40.2%). The positive expression of DNMT3a protein was positively correlated with the ERα gene methylation (rS=0.250). The DNMT3a protein expression showed a significant influence to the overall survival (OS) in patients of breast cancer (P=0.035), no significant influence to the disease-free survival (DFS) (P=0.064). DNMT3b protein expression showed no significant influence to OS and DFS of patients with breast cancer (P=0.914 and 0.961). Conclusion The positive expressions of DNMT3a and DNMT3b are correlated with the invasion, metastasis and poor prognosis of sporadic breast cancer. DNMT3a was positively correlated with the state of ERα gene promoter methylation. The inhibition of DNMT3a and DNMT3b may have advantages in the prevention and treatment of sporadic breast cancer.
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Objective To observe if IL-2 gene can express in hepatic carcinoma cells by double targeting of hepatitis B virus envelope (HBVE) and alpha fetoprotein (AFP) promoter.Methods HepG2 cells,L02 cells,and HepG2.2.15 cells were cultured in vitro.HBVE was obtained by PEG8000 concentration assay,and the acquired HBVE was used to pack recombintional gene.Human AFP promoter-IL-2 recombinational gene was obtained by PCR.Then HepG2 cells and L02 cells were transfected by transient transfection and the expression of IL-2 was detected by RT-PCR and Western blot.Results IL-2 was detected in HepG2 cells by RT-PCR and Western blot but not in L02 cells.Conclusion By using HBVE as a gene transporter,human AFP promoter-IL-2 recombinational gene can express in hepatic carcinoma cells,thereby it can increase the safety of exogenous gene transfection of hepatic carcinoma cells.
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Objective To evaluate the prevalence of the DJ-1 mutation in early-onset Parkinson's disease (EOPD) patients,and analyzed the association between the certain polymorphic marker g.168_185del in intron1 and Parkinson' s disease (PD).Methods We screened all 7 exons and exon-intron boundary regions of DJ-1 by PCR and direct nucleotide sequencing in 90 Chinese patients with EOPD.We also compared the allele and genotype frequencies of the g.168_185del polymorphism between EOPD patients and controls.Results We found no causative DJ-1 mutations in our cohort of Chinese EOPD patients.But we did identified 4 known polymorphic variants,including the g.168_185del in intron 1,g.5027G ＞ A (rs17523802),g.5065T ＞ C (rs226249),and g.5094C ＞ T (rs11121064) within exon 1.Del/Ins frequencies of the g.168_185 del polymorphism were 11.1％ (10/90)and 13.3％ (14/105) in EOPD group and normal group,respectively.Ins/Ins frequencies were 88.9％ (80/90) and 86.7％ (91/105),thex2 and P value of genotype frequency were 0.222 and 0.669 between EOPD patients and controls,respectively.The insert frequencies were 94.4％ (170/180)and 93.3％ (196/210) in EOPD patients and controls,the deletion frequencies were 5.6％ (10/180) and 6.7％ (14/210),thex2 and P value of allele frequency were 0.207 and 0.679 between EOPD patients and normal,respectively.Furthermore,the P value of genotype and allele frequencies were 0.736 and 0.744 between familial EOPD patients and controls,respectively;P values of genotype and allele frequencies were 0.847 and 0.852 between sporadic EOPD patients and control group,respectively.There was no statistical difference between groups.Conclusion Mutations in DJ-1 are uncommon in Chinese EOPD patients,and no association is observed between the DJ-1 intron 1 g.168_185del polymorphism and risk of PD.
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Objective To investigate the effect of methylation of the APC gene on expression and the correlation with clinical data in pancreatic cancer.Methods Sixty postoperative tissue samples with pancreatic cancer were collected in the First Affiliated Hospital of Zhengzhou University from August 2010 to January 2011,20 benign pancreatic disease tissues were collected as control groups.APC promoter methylation and gene expression levels were detected by Methylation Specific PCR (MSP),Real Time PCR (RT-PCR) and Western blot in 60 pancreatic carcinoma,42 metastasis and 20 benign pancreatic disease tissues,then analyze the relation between methylation of the APC gene and the clinical data.Results APC promoter methlation was observed 48.53％,46.67％ and 1.16％ in pancreatic carcinoma,metastasis and benign pancreatic disease tissue,respectively.Methylation of APC in pancreatic carcinoma and metastasis increased significantly compared with control tissues (x2 =12.903,14.402; P ＜ 0.05).There were no statistically significant differences of APC expression in these tissues (P ＞ 0.05).There was a significant correlation between methylation of APC and clinicopathological stage (x2 =6.801,P ＜ 0.05),but no correlation with gender,age,tumor size,histological grade and metastasis (x2 =0.727,1.311,0.372,0.148,0.017 ; P ＞ 0.05).Conclusion The methylation of APC gene is closely related with pancreatic carcinoma inogenesis and the clinicopathological stage,but do not effect the expression of APC in tissues.
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Objective To investigate polymorphism in the promoter region of insulin-like growth factor-1(IGF-1) gene.Methods Five hundred and sixty-one neonates admitted to Peking University Third Hospital from June 1st,2010 to June 30th,2012 were recruited into the study.Gender,gestational age,birth weight and birth length were collected.Heel blood samples were collected on 3-5 days after birth.DNA was extracted to analyze the polymorphism in the promoter region of IGF-1 gene.Chi-square test,independent-sample t-test,analysis of variance and HardyWeinberg equilibrium were performed.Results Among the 561 neonates,413 were full term,and 148 were preterm; 92 were large for gestational age (LGA),433 were appropriate for gestional age (AGA),and 36 were small for gestional age (SGA).Seven different alleles and 23 genotypes in the promoter region of IGF-1 gene were identified in the population.The seven alleles were 188,190,192,194,196,198 and 200 bp respectively.The three most common genotypes were 190-192 bp,192-196 bp and 192-192 bp,whose frequencies were 23.2％ (130/561),15.0％ (84/561) and 12.8％(72/561).There were no significant differences of cytosine-adenosine (CA)19/CA19,CA19/CAno19and CAno19/CAno19 genotypes between full term and preterm infants [11.4％ (47/413) vs 16.9％(25/148),55.9％ (231/413) vs 50.7％ (75/148) and 32.7％ (135/413) vs 32.4％ (48/148)respectively,x2=2.96,1.21 and 0.00,all P＞0.05].There was no difference in the gestional age among infants with CA19/CA19,CA19/CAno19 and CAno19/CAno19 genotypes [(37.1±2.9),(37.6±3.1) and (37.4±3.1) weeks respectively,F=0.54,P=0.58].The frequency of CA19/CA19 in SGA neonates was higher than that in LGA and AGA neonates [25.0％ (9/36) vs 7.6％(7/92) and 12.9％ (56/433),x2 =7.01,P=0.03],but there were no differences in the frequency of CA19/CAno19 and CAno19/CAno19 among LGA,AGA and SGA neonates (CA19/CAno19:x2 =1.13,P=0.57; CAno19/CAno19:x2 =0.58,P=0.75).Conclusions Polymorphism exists in the promoter region of IGF-1 gene.The gestational age is not associated with the frequency of CA19 allele.
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Objective To construct human surfactant protein B (SP-B) gene promoter luciferase reporter plasmids and detect their transcriptional activities in H441 cells.Methods (1)The fragment of SP-B promoter (-218/+ 435 bp) was acquired from human genome DNA by polymerase chain reaction (PCR) amplification and then was inserted into pGM-T vector by the T4 DNA ligase.The vector was transfected into TOP10 E.coli.The positive clone was identified by DNA sequencing.The identified target SP-B promoter sequence was cloned into pGL3-basic vector to construct the recombinant vector pGL3-basic-SP-B-promoter and was identified by enzyme digestion and sequencing; (2)The pGL3-basic-SP-B-promoter vector was converted into pGL4.17-SP-B-promoter vector through enzyme digestion.The identified recombinant vectors and control plasmid pRL-TK were transfected into H441 cells by lipofectamine 2000,and luciferase assays was performed using the dual-luciferase reporter assay system.Results The sequences of SP-B promoter in the recombinant luciferase reporter plasmids were consistent with the one published on Genebank.The firefly/renilla luciferase activity ratio of pGL3-basic/pGL4.17-SP-B-promoter vector (2.8 ± 1.1,66.5±3.8) was significantly higher than pGL3-Basic,pGL4.17 control vector (0.2 ±0.1,4.3 ±0.4) with statistical significance (t =4.182,27.419,P =0.000),respectively.The SP-B promoter activity of pGL4.17-SP-B-promoter vector was significantly higher than pGL3-basic-SP-B-promoter vector (t =27.712,P =0.000).Conclusions The pGL3-basic/pGL4.17-SP-B-promoter vectors are successfully constructed with SP-B promoter activity in H441 cells and pGL4.17-SP-B-promoter vector is the better choice for further study with higher luciferase activity.
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ObjectiveTo investigate the relationship between the polymorphisms of the promoter of a disintegrin and metalloproteinase 10(ADAM10) gene and sporadic Alzheimer's disease (SAD).Methods The promoter of ADAM10 gene in 10 controls and 10 SAD patients was sequenced.Three variations were found,then these variations in 298 SAD patients (SAD group) and 315 healthy controls (control group)were genotyped by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).ResultsThree polymorphisms were found in the promoter of ADAM10 gene: -279G/A (rs653765),- 630G/T( rs514049 ) and - 921GAGA/- ( rs33926666 ).For - 921GAGA/-,there were significant differences in genotype ( GAGA/GAGA:138 (46.3％ ),GAGA/-:155(52.0％),-/-:5(1.7％))and allele frequencies (GAGA:431 (73.6％),-:165 (27.7％) ) between SAD and control (genotype:x2 =34.130,P =0.000; allele:x2 =25.972,P =0.000). For - 279G/A,there were significant differences in genotype and allele frequencies between SAD and control in the subjects without ApoEε4 allele (genotype:x2 =8.734,P=0.013; allele:x2 =5.129,P=0.024). -279G and -921GAGA were relatively protective allele types for SAD,and they were not in linkage disequilibrium.ConclusionThe polymorphisms - 279G/A and - 921GAGA/- of ADAM10 are associated with SAD.Allele G or genotype G/G of -279G/A and the GAGA/GAGA genotype or the GAGA allele of -921GAGA/- might have a protective effect on SAD.
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Objective To investigate the differences of kinesin family member 4 (KIF4A) expression between hepatic carcinoma and adjacent tissue in patients with chronic hepatitis B virus (HBV) infection,and to understand the effect of HBV on the expression of KIF4A.Methods Reverse transcriptase-polymerase chain reaction and Western blot were used to measure the expression of KIF4A in hepatic carcinoma and adjacent tissues. HepG2 cells were co-transfected with KIF4A promoter containing the luciferase gene and HBV infectious clone pHBV1.3,and luciferase activity was measured.Expression of KIF4A in HepG2 cells was measured after tranfected with different doses of pHBV1.3.The student's t-test was used for statistic analysis.Results Expression of KIF4A was much higher in hepatic carcinoma than that in adjacent tissues.HBV enhanced KIF4 A gene promoter activity and the luciferase activities were increased as the concentration of pHBV1.3 increased ( 0,0.2,0.4,0.6 and0.8 μg/mL),which were (126.8± 13.4),(219.8±16.7),(387.6±21.5),(586.5 ± 228.9 ) and (657.6 ± 35.5 ) RUL/μg protein,respectively,while the luciferase activities were (123.6± 13.8),(131.8± 14.6),(129.7-13.5),(135.3± 13.4) and (127.1± 12.7) RUL/μg protein,respectively with different doses of control plasmids transfected,and statistical analysis showed significant difference between them (t=4.875,P=0.006).And HBV upregulated KIF4A mRNA and protein expressions in HepG2 cells in a concentration-dependent manner.Conclusion Expression of KIF4A is enhanced in hepatic carcinoma and HBV can upregulate KIF4A expression.
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ObjectiveTo analyze the status of DACH1 gene promoter methylation and explore its association with the expression of DACH1 gene promoter methylation and clinical significance of endometrium carcinoma(EC).Methods From February 2004 to August 2008,a total of 80 EC tissue samples with comprehensive surgical pathology staging were collected and used for this study.Twenty normal endometrium tissues in 2008 were abtained from the fractional curettage because of dysfunctional uterine bleeding as control.All samples were confirmed pathologically.Methylation specific PCR (MSP) was performed to detect the promoter methylation of DACH1 gene,and analyze its influence on the expression of DACH1 and the relationship between DACH1 promoter methylation and clinicopathological factors in EC.DACH1 protein expression was detected by western blot.Chi-square test and Pearson test were used for statistical analysis.ResultsThe rate of promoter methylation of DACH1 gene in the EC tissues was significantly higher than that in the normal endometrium issues (30％ vs.5％,P ＜ 0.05).There was an association between the expression of DACH1 and DACH1 gene promoter methylation ( r =- 0.30,P ＜ 0.01 ).There was statistical difference between the methylation of DACH1 and the pathological grade ( P ＜ 0.05 ) or histological type ( P ＜0.05).But DACH1 gene methylation was not related with the age,stage,myometrial invasion depth and lymphnode metastasis (P ＞ 0.05 ).Conclusions DACH1gene promoter methylaion could lead to a decrease or absence in the DACH1 expression in EC.The promoter methylation of DACH1 gene may induce the inhibition of DACH1 expression,which might be one of the mechanisms of DACH1 gene inactivation in human EC.
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Objective To study the effect of follicle stimulating hormone(FSH)on the secretion of anti-mullerian hormone(AMH)in granulosa cells in patients with polycystic ovarian syndrome(PCOS).Methods From Aug.2008 to Dec.2009,33 patients with PCOS in Reproductive Medicine Research Center,Sixth Affiliated Hospital of Sun Yat-sen University were collected from 8-10 mm antral follicles and classified into the following three groups: FSH-unstimulated granulosa cells(unstimulated group,n =12),exogenous FSH-stimulated granulosa cells(exo-stimulated group,n =12)and internal FSH-stimulated granulosa cells(int-stimulated group,n =21).ELISA and real-time PCR were relatively used to measure protein and mRNA level of AMH.Luciferase reporting vector was constructed to detect the promoter activity of AMH.Results The levels of AMH secretion in PCOS granulosa cells were(11.4 ± 4.0)μg/L in unstimulated group,(7.9 ± 1.1)μg/L in exo-stimulated group and(5.6 ± 1.7)μg/L in int-stimulated group.Both the external and internal stimulation of FSH may suppress AMH secretion significantly(P ＜0.05).The mRNA level of AMH in PCOS granulosa cells were 2.5 ± 1.2 in unstimulated group,which were higher than 1.5 ± 0.5 in exo-stimulated group and 1.1 ± 0.7 in int-stimulated group significantly(P ＜0.05).The luciferase activity of AMH in PCOS granulosa cells were 11.5 ± 2.3 in unstimulated group,8.7 ± 2.4 in exo-stimulated group and 6.8 ± 2.4 in int-stimulated group.The luciferase activity of AMH in unstimulated group was significantly higher than those in exo-stimulated and int-stimulated groups(P ＜0.05).Conclusion FSH may inhibit the excessive secretion of AMH and stimulate follicle growth in PCOS granulosa cells by suppressing activity and expression of promoter.
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Objective To inactivate Death-associated protein kinase 1 gene (DAPK1) by transfecting complementary methylated oligonucleotides and studies its effect on the proliferation of myelogenous leukemia cell line K562. Methods Methylated oligonucleotides complementary to DAPK1 gene promoter were transfected into K562 cells by Iipo2000. Methylation specific PCR (MSP) and Reverse transcription PCR (RT-PCR) were applied to detect DAPK1 gene promoter methylation status and its mRNA expressions respectively. MTT was used to detect the proliferation of K562 cells pre- and post- oligonucleotides transfection. Results DAPK1 gene promoter in non-treated and control groups were unmethylated with detectable mRNA expressions, but it became methylated with inhibited mRNA expressions after methylated oligonucleotide transfection. Proliferation in methylated oligonucleotide treatment group was significantly higher than that in non-treated and control groups. Conclusion Complementary methylated oligonucleotides could inactivate DAPK1 gene and inhibit its expression in K562 cells, which could promote its proliferation.
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Objective To explore the relationship between CA repeats polymorphism in the promoter region of IGF-1 gene and MS in the Han nationality.Methods 1047 subjects were recruited from general population of Dongcheng District in Beijing.MS was diagnosed based on the criteria for MS in 2005 by IDF.Genomic DNA was extracted by standard methods.PCR,Genescan,Genotyper and direct sequencing were conducted to screen CA repeats polymorphism in the promoter region of the human IGF-1 gene.Levels of plasma glucose,lipids,serum insulin and IGF-1 were determined.BMI and ISI were calculated.Results The prevalence of MS in (CA) 19 homozygote was lower than that in (CA) 19 heterozygote (9.1% vs 18.3%,x2 = 8.55,P ＜ 0.01) and without (CA) 19 (9.1% vs 24.0%,x2 = 18.05,P ＜ 0.01).The level of serum IGF-1 had differences among the three groups [ (114.0 ± 52.6) μg/L vs (136.6 ± 80.5) μg/L vs (129.2±49.1) μg/L,F =3.16,P ＜0.05],(CA)19 homozygote had lower serum IGF-1 than (CA)19heterozygote and without (CA) 19.BMI,WC,TG,FIns,2hIns and ISI had difference among the three groups (P ＜0.05).Conclusions (CA)19 repeats polymorphism in the promoter region of IGF-1 gene was significantly associated with MS in Han nationality.
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Objective To explore the relation between IL-10 promoter region gene polymorphism and irritable bowel syndrome (IBS).Methods By polymerase chain reaction combined with restrition fragment length polymophism (PCR-RFLP),gene type of IL-10 promoter -1082 and -819 sites in 313 IBS patients and 281 controls was analyzed.Results The distribution of IL-10-1082 and-819 allele frequencies in IBS group,control group and total was in accordance with Hardy-Weinberg equilibrium law.The frequency of IL-10-819 T allele in diarrhea subtype (79.8％) and mixed IBS subgroup (77.1％) was significantly higher than that in control group (65.7％).There were no significant differences in IL-10-1082 A/G allele frequency between each subtypes and control group (P＞0.05),however there was statistically difference between diarrhea subtype and mixed IBS subgroup (P＜0.05).The frequency of-819 T/T genotype in IBS group (51.1 ％ )was significantly higher than that of control group (40.2％),the frequency of C/T genotype was significantly lower than that of control group,and the difference was statistically significant (all P＜0.05).The IL-10-819 T/T allele frequency of all IBS subtypes was significantly higher than that of control group; however C/T allele genotype frequency of all IBS subtypes was significantly lower than that of control group,and the difference was statistically significant (all P ＜ 0.05). There was no significant difference of C/C allele genotype between subtypes (P＜0.05).There was no significant difference of -1082 allele genotype between IBS group and control group (P＞0.05).The frequency of -1082 A/A genotype in diarrhea subtype of IBS patients (93.3％) was significantly higher than that of mixed IBS subtype (82.4％),while the frequency of A/G genotype was lower than that of mixed IBS subtype,and the difference was statistically significant (all P ＜ 0.05 ); there was no significant difference between other IBS subtypes and control group (P＞0.05).Conclusion IL-10-819 promoter T/Tgenotype may be related to IBS pathogenesis.
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Objective To investigate the relationship between single nucleotide polymorphism (SNP) of Fas ligand (FasL) and fulminant hepatitis B in Han Chinese. Methods HBV infected subjects were enrolled in this case-control study, including 233 cases of inactive HBsAg carrier, 68 patients with fulminant hepatitis B,100 cases of spontaneous hepatitis B clearance, 102 patients with hepatitis B virus (HBV) related cirrhosis and 112 patients with HBV related primary hepatocellular carcinoma. The blood samples and clinical data were collected. FasL-844T/C polymorphisms of enrolled subjects were examined by TaqMan real time fluorescent genotyping polymerase chain reaction (RT-PCR). A adjusted odds ratios (OR)and 95% confidence intervals (CI)were calculated using the Logistic regression model. Results After adjusting the factors of gender and age, binary Logistic regression analyses indicated that the genotype frequencies of FasL-844 CC,CT,TT in inactive HBsAg carriers were 50. 64% ,39. 91% and 9. 44% respectively, and those in cases of fulminant hepatitis B were 79. 41%, 17. 65% and 2. 94%, respectively. The analysis also revealed that FasL-844CC genotype in inactive HBsAg carriers was high risk factor of developing fulminant hepatitis B (OR =4. 729,95%CI:0. 510 - 21. 282,P = 0. 043), while there were no statistic significances in other cases (P>0. 05). Conclusion The inactive HBsAg carriers harboring FasL-844CC may have greater susceptibility to fulminant hepatitis B, which need arouse high attention.
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Objective To investigate AXIN2 mRNA expression level in hepatocellular carcinoma (HCC) , and to analyze the effect of AXIN2 gene methylation status on its mRNA expression and HCC genesis and development. Methods Fifty-three surgical excised HCC specimens and paired adjacent non-cancerous specimens, seven normal liver specimens and five HCC cell lines were collected. The expression of AXIN2 at mRNA level and the methylation status of AXIN2 gene promoter were determined by quantitative PCR. Results The expression of AXIN2 mRNA was lower in HCC tissues (0.1629 + 0.0679) than that in adjacent non-cancerous tissues (0. 4155 + 0. 2330), and there was significant difference (Z= -2. 567, P = 0. 010). The methylation level of AXIN2 gene in HCC and adjacent non-cancerous tissues (39. 77% ±3. 89%, and 36. 92% ±2. 81%) was significantly higher than that in normal liver tissues (7. 38% ±2. 40% , t=-3. 663 ,P = 0. 009;t= -4. 591 ,P = 0. 007).AXIN2 gene was hypermethylated in all five HCC cell lines. There was a negative correlation between AXIN2 mRNA expression level and the degree of methylation ( r = -0. 458, P = 0. 032). The methylation level was higher in TNM Ⅲ patients of HCC than that in TNM Ⅰ and Ⅱ patients (P =0.008). Conclusion The down-regulation of AXIN2 gene mRNA expression is correlated with its hypermethylation status. The low expression of AXIN2 mRNA and the abnormal methylation of promoter may be one of the important mechanism of HCC genesis and development.
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Objective To investigate the association between hepatitis B virus (HBV)genotype, the mutations in HBV basic core gene promoter(BCP), pre C/C gene region and treatment response to interferon (IFN)α-1b. Methods Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients were treated with IFNα-Ib for 6 months and were followed up for 6 months after the end of treatment. Restriction Fragment Length Polymorphism (RFLP) was used for determining HBV genotype. HBV DNA was amplified by polymerase chain reaction (PCR) and analyzed for BCP and pre C/C gene region by sequencing. Measurement data were compared using t test and analysis of variance. Enumeration data were compared using chi-square test, Fisher exact probability test.Logistic regression analysis was utilized for multi-factor analysis. Results There were 39 patients who completed the treatment and follow up in this study. At the end of treatment, 16(41.0%) patients showed response to the IFNα-lb treatment. At the end of follow-up, four out of 16 patients who achieved on treatment response relapsed. Among 3a patients, 29 (74.4 %) were infected with genotype B and 10 (25. 6%) with genotype C. The treatment response rates were not significant different between the groups with different genotypes. The double mutation pattern (T1762/A1764) was found in eight (20. 5%) patients. The response rates to IFNα-lb treatment were not significant different between the group with and without double mutation pattern. A1896 mutation was detected in eight patients at baseline. Three of them became HBeAg negative at the end of treatment and returned to HBeAg positive during follow-up. The non-lyphocyte epitope mutations, L60V and I97L, were found in 15 patients (38. 5%) and 14 patients (35.9%), respectively. At the end of follow-up, the patients with 60V had a significantly lower HBeAg seroconversion rate and HBV DNA undetectable rate compared to the patients with 60L (Fisher exact probability test; P = 0.0126 and 0.0069,respectively). The HBV DNA undetectable rates in the patients with 97I were significantly lower than those in patients with 97L both at the end of treatment and the end of follow-up (Fisher exact probability test; P= 0.0484 and 0. 0024, respectively). Logistic regression analysis results showed that there was no association between the above viral mutations and the treatment response to IFNαlb. Conclusions There is no association between HBV genotype, BCP double mutation pattern and IFN-α treatment response. The non-lyphocyte epitope mutations, L60V and I97L, may have impact on IFN-α treatment response.
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Objective To investigate the relationship between HBV genotypes and YMDD motif mutations or BCP mutations in Xiangtan of Hunan Province. Methods HBV genotypes, YMDD motif mutations and BCP mutations were analyzed in 952 HBV infected patients. Results HBV genotyping showed that 698 HBV type B patients and 115 HBV type C patients accounted for 73.32% and 12.08% respectively of all the participants. The rest 139( 14.60% )were genotype B and C mixed infection( B + C ). The analysis of YMDD motif mutations showed that 844 YMDD wild-type which accounted for 88.66% of all the subjects and the remainder were YMDD mutation types, of which 54( 5.67% ) carried YVDD, 53( 5.57% ) YIDD,and 1 YVDD and YIDD mixed infection. Basic Core Promoter mutations showed that 1762A/1764G ( wild type )accounted for 70.59% and 1762T/1764A( mutant ) accounted for 19.75%. The rest 92 patients were 1762T/1764A and 1762A/1764G mixed infection. This study showed no significant difference in the rate of YMDD mutation( 10.04% vs 10.43% ,χ2 =0.017,P＞0.05 ) ,but a significant difference in the types of YMDD mutation(χ2 = 4.836, P ＜ 0.05 )between HBV types B and C. The YVDD mutation was more commonly seen in genotype C( 9.57% ) than in genotype B( 5.88% ). The BCP mutation rate showed a significant difference( 27.36% vs 46.09%, χ2 = 16.478, P ＜ 0.01 ). Genotype C was more frequent than genotype B. The BCP mutation rate showed no significant difference between YMDD Wild-type and YMDD mutation types( 28.67% vs 35.51%, χ2 = 2.139, P ＞ 0.05 ), but most of BCP mutations happened in YVDD mutant type( 61.11% ). Conclusions ( 1 ) The predominant HBV genotypes in Xiangtan were genotype B and genotype C, the major genotype was type B, which display the characteristics of epidemiology in Southern China. ( 2 ) Determination of HBV genotypes before lamivudine therapy was probably not an important pretreatment investigation to predict antiviral responses. ( 3 ) Detection of HBV genotypes, YMDD motif mutations and BCP mutations will contribute to the correct evaluation of prognosis and timely proper management of HBV patients.
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Objective To establish quantitative method for detection of methylation level of DLC-1 promoter with HRM technology to analyze its association with pathological parameters in prostate cancer.Methods 89 prostate cancer tissue samples and 10 matched normal tissue samples were enrolled into this study.Prostate cancer cells were obtained by LCM.DNA was extracted and modified for methylation determination.CpGenome Universal Methylated DNA was chosen as 100% methylation sample.Then the calibrators representative of 100%,80%,50%,30%,10% and 0% methylation levels were prepared with dilution in a DNA sample of peripheral blood from healthy subjects(100% non-methylation).The DLC-1 methylation levels in prostate cancer tissue samples were detected with HRM.The associations of methylated level with age of patients,PSA value,TNM stage were investigated respectively.ResultsThe melting curves representing 100%,80%,50%,30%,10% and 0% methylation levels were aligned from right to left.The methylation levels of 10 adjacent normal samples and 35 prostate cancer samples were overlapped with 0% methylated calibrator.The methylation levels of 5 cancer samples ranged between 0% and 30%.The methylation levels of 29 cancer samples ranged between 31% and 80%.The methylation levels of 20 cancer samples ranged between 81% and 100%.HRM could be used to reliably detect the as low as 10% methylation for each assay,whereas methylation specific PCR(MSP) could be used to detect 30% methylation level.No significant association between methylation level and patients' age(X~2=3.29,P=0.19),PSA level(X~2=2.04,P=0.36) was found.However,DLC-1 methylation was higher in the prostate cancer tissues with advanced TNM stage(X~2=9.04,P=.01).Conclusions The quantitative method for DLC-1 methylation with HRM is successfully established.It is convenient with good reproducibility and high sensitivity.DLC-1 methylation could be used as the molecular marker for estimation of malignancy in prostate cancer.
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Objective To detect the FHIT gene promoter methylation and protein expression in mycosis fungoides(MF).Methods Tissue specimens were collected from 48 patients with MF and 18 normal human controls.FHIT protein expression was determined by immunohistochemistry,and methylation status of FHIT gene by methylation-specific PCR.Results Abnormal methylation of FHIT gene was found in 26(54.2%)out of the 48 specimens.Thirty(63.5%)specimens of MF were negative for FHIT protein,which was observed in all the control specimens.The promoter methylation of FHIT was closely correlated with the protein expression of FHIT,but unrelated to the sex of,tumor staging or lymph node metastasis in patients with MF.Conclusion The FHIT gene promoter methylation may contribute to the inactivation and abnormal expression of FHIT protein in MF.
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Objective To investigate the association between promoter polymorphisms of interferon-alpha receptor-1 (IFNAR1) gene and the treatment response to interferon-alpha (IFN-α) in patients with chronic hepatitis B (CHB). Methods Sixty-one CHB patients who consented to receive IFN-a therapy were enrolled in this study. The subjects were treated with recombinant IFN-α2b 500 MU intramuscular injection qod for 48 weeks. The treatment responses were monitored.Meanwhile, the promoters of IFNAR1 gene in these patients were sequenced. Measurement data were analyzed by t test and enumeration data were analyzed by Chi square test. Results Twenty-two treated patients achieved complete response. Eight patients achieved partial response and 31 had no response. Polymorphisms were identified in the promoter of IFNAR1 gene, which included C/T substitution at locus -408, C/T substitution at locus-3 and GT microsatellite repeat sequence at locus-77 [-77(GT)n]. The three polymorphisms were in linkage and composed some haplotypes,such as - 408C/-77(GT)5/-3C. The response rate to IFN-α in CHB patients with genotypes -408C/-77(GT)5/-3C, -408C/-77(GT)5/-3C, and-408C/-77(GT)5/-3C, non -408C/ - 77(GT)5/-3C in IFNAR1 gene promoter was higher than that in patients with genotype non -408C/-77(GT)5/-3C, non-408C/- 77(GT)5/- 3C (61. 0% vs 25. 0%, x2=6. 961, P =0.008). Conclusions CHB patients with genotype-408C/ - 77(GT)5/ - 3C, -408C/-77(GT)5/-3C and -408C/ -77(GT)5/ -3C, non -408C/ -77(GT)5/-3C in the promoter of the IFNAR1 gene are prone to have better response to IFN-a treatment. Polymorphisms in the promoter of IFN-αgene are associated with the treatment response to IFN-α in CHB patients.