Polymerease Chain Reaction Research Articles

The effects of up-regulation of microRNA-148a on the expression of anti-oncogene preproenkephalin, p16 and ras association domain family 1A in pancreatic carcinoma AsPC-1 cells

Objective To investigate the effect of up-regulation of microRNA (miRNA, miR)-148a on the methylation and protein expression of anti-oncogene preproenkephalin (ppENK), p16, ras association domain family 1A (RASSF1A) and the proliferation of pancreatic cancer AsPC-1 cells. Methods MiR-148a mimics were transfected into AsPC-1 cells mediated by Lipofectamine 2000. AsPC-1 cells were devided into three groups, blank group (transfected Lipofectamine), mimincs-NC group (transfected negative mimics) and mimincs-148a group (transfected miR-148a mimics). At 72 h after transfection, the expression of miR-148a was analyzed by real-time quantitative polymerase chain reaction (Real-time PCR). The CpG island methylation status of anti-oncogene ppENK, p16, RASSF1A were measured by methylation specific polymerease chain reaction (MSP). The expression of DNA methy transferas 1 (DNMT1), ppENK, p16 and RASSF1A protein were detected with Western blotting. The cell proliferation activity was examined by methyl thiazol tetrazolium (MTT) assay. Results The expression of miR-148a in mimincs-148a group was significantly higher than those in blank group and mimincs-NC group (4.63±1.32 vs. 1.00±0.30 vs. 0.93±0.37, P=0.002), while the expression of DNMT1 protein in mimincs-148a group was significantly lower than those in blank group and mimincs-NC group (1.00±0.00 vs. 1.04±0.19 vs. 0.27±0.15, P=0.001). ppENK, p16 and RASSF1A genes were positive methylation in blank group and mimincs-NC group, while ppENK and p16 genes were negative methylation, and RASSF1A gene was partial methylation in mimincs-148a group. The expression of ppENK, p16 and RASSF1A protein in mimics-148a group were significantly higher than those in blank group and mimics-NC group (ppENK: 2.06±0.32 vs. 1.00±0.00 vs. 1.01±0.21, P=0.002; p16: 2.02±0.12 vs. 1.00±0.00 vs. 0.96±0.12, P=0.000; RASSF1A: 1.95±0.37 vs. 1.00±0.00 vs. 1.09±0.12, P=0.002). The cell proliferation activities in mimincs-148a group were significantly slower than those in in blank group and mimincs-NC group (P=0.026). Conclusion Up-regulation of miR-148a could lead to demethylation of ppENK, p16 and RASSF1A gene promoter CpG island and up-regulate its protein expression, and inhibit cell proliferation in pancreatic cancer AsPC-1 cells. The mechanism may be related to the inhibition of DNMT1 expression induced by miR-148a. Key words: Pancreatic cancer; MicroRNA-148a; DNA methy transferas 1; Anti-oncogene; Methylation; Cell proliferation

Relationship between S100A2 expression and promoter methylation of gastric carcinoma

Objective To analysis the relationship between the expression of S100A2 and promoter methylation of gastric carcinoma. Methods Expression of S100A2 was investigated by using streptavid-in-peroxidase (SP) immunohistochemical staining and promoter methylation was assayed by methylation specific polymerease chain reaction (MSP) in 100 cases of carcinoma, 20 cases normal gastric tissues. Results The expression of S100A2 in normal gastric cases was higher than its in carcinoma (χ2=18.352, P=0.001). The expression of S100A2 was related with tumor differentiation, depth of infiltration and lymph node metastasis (χ2=6.732, 4.537, 10.531, P=0.009, 0.049, 0.002). There was a inverse association between expression of S100A2 and promoter methylation (χ2=12.421, P=0.002). Conclusion The expression of S100A2 was associated with the occurrence and development of gastric carcinoma. The negative expression of S100A2 always suggested poor differentiation, high invasive and metastatic potention. It may be helpful to evaluate prognosis of this cancer. The promoter methylation might lead to the down or loss expression of S100A2 and was associated with the occurrence and development of gastric carcinoma. Key words: Gastric carcinoma; S100A2; Promoter methylation

Methylation of p16 and death-associated protein kinase promoter gene regions in the blood serum of patients with non-invasive urinary bladder cancer

Objective To investigate p16 and death-associated protein kinase (DAPK) promoter methylation in the blood serum of patients with non-invasive urinary bladder cancer. Methods p16 and DAPK promoter methylations in blood serum of patients with non-invasive urinary bladder cancer (experimental group, n=25) and healthy volunteers (control group, n=25) were detected by methylation specific polymerease chain reaction (MSP), and their clinical implications were analyzed. Results The methylation rate of p16 in experimental group was 52% (13/25), higher than 24% (6/25) in control group(χ2=4.160, P=0.041); The methylation rate of DAPK in experimental group was 60% (15/25), higher than 32% (8/25) in control group (χ2=3.945, P=0.047). Both of them had statistically significant difference. The methylation rate of p16 and DAPK was not correlated with smoking (χ2=0.187, P=0.665; χ2=0.109, P=0.741), age (χ2=0.000, P=0.991; χ2=0.038, P=0.845), gender (χ2=0.746, P=0.382; χ2=1.288, P=0.256) or the grade of tumor (P=1.000; P=1.000). Conclusion p16 and DAPK promoter methylations are closely related to non-invasive urinary bladder cancer. Key words: Bladder cancer; p16; Death-associated protein kinase; Methylation

Value of runt related transcription factor 3 gene CpG island methylation in induced sputum in condition and prognosis evaluation for lung adenocarcinoma

Objective To study the value of runt related transcription factor 3 (Runx3) gene CpG island methylation in induced sputum in condition and prognosis evaluation for lung adenocarcinoma. Methods Methylation specific polymerease chain reaction (MSP) was used to detect the Runx3 gene promoter methylation by induced sputum in patient with lung adenocarcinoma (LAC group) and non-cancer patients (CON group). The Runx3 gene CpG island methylation rate in different clinicopathological factors was compared. The R0 radical resection, disease-free survival time and 3-year survival rate between patient with Runx3 gene CpG island methylation and unmethylation were compared. Results The Runx3 gene promoter methylation rate in LAC group was significantly higher than in CON group (62.5% vs. 2.5%, P<0.05). The Runx3 gene promoter methylation rate in pleural effusion, degree of differentiation, tumor diameter, lymph node metastasis, distant metastasis and cTNM staging were significantly different (χ2=5.861, 4.417, 5.273, 4.110, 10.240, 10.559, all P<0.05). The R0 radical resection (72.0% vs. 93.3%), disease-free survival time [(1.7±0.4) years vs. (2.4±0.6) years] and 3-year survival rate (40.0% vs. 66.7%) in patient with Runx3 gene CpG island methylation were significantly lower than in patients with Runx3 gene CpG island unmethylation (χ2=4.083, t=6.261, χ2=4.320, all P<0.05). Conclusion Runx3 gene CpG island methylation in induced sputum correlates with lung adenocarcinoma’s condition and prognosis. Key words: Lung adenocarcinoma; Runt related transcription factor 3; Methylation

Studies on tumor suppressor gene apoptosis protease-activating factor1, ARF tumor suppressor , RUNT-related transcription factor 3 methylation in tissue of colorectal cancer

Objective To investigate abnormal promoter methylation status of apoptotic protease activating factor-1 (APAF1), ARF tumor suppressor (p14ARF) and RUNT-related transcription factor 3 (RUNX3) genes and analyze the value of these genes in the diagnosis of colorectal cancer. Methods Methylation specific polymerease chain reaction (MSP) was used to detect the methylation status of APAF1, p14ARF and RUNX3 genes in 70 cases of colorectal cancer tissues. Results The methylation rate of APAF1, p14ARF and RUNX3 in tumor tissue of colorectal cancer was 78.5%, 54.2% and 47.1% respectively. RUNX3 and APAF1 genes were highly expressed in pathological stage Ⅱ, and p14ARF and APAF1 in pathological stage Ⅲ. Aberrant methylation rate of APAF1, p14ARF and RUNX3 genes in colorectal cancer was more significantly different than the polyp of colon (P<0.05). Conclusion Aberrant methylation status of APAF1, p14ARF and RUNX3 is closely related with incidence of colorectal cancer. Key words: Colorectal cancer; Tumor suppressor gene; Methylation specific polymerease chain reaction

A New Female Case with 47,XXY Karyotype and SRY

Objective: Numerical abnormalities of sex chromosome, 47,XXY karyotype, have the clinical phenotype of Klinefelter syndrome. Very few 47,XXY cases with a female phenotype have been reported. The testicular feminization syndrome is a female phenotype with a male karyotype (46,XY). Most of these cases have SRY positive SRY (testis-determining factor). The genetic explanation of this phenomenon is not very clear. Methods: Clinical examination, conventional cytogenetic analysis, fluorescence insitu hybridization and molecular genetic analysis polymerease chain reaction were done on a 17 year old female patient. Results: Clinical examination revealed that the case had a female external genitalia, breast development and pubic hair. Some clinical findings of the patients are unique and not similar to the other cases reported in the literature. The present case has different clinical features including normal phenotype, breast development and normal gentelia, tubes, ovaries, but hypoplastic uterus. Cytogenetic analysis and fluorescence in situ hybridization revealed that the Y chromosome was normal; both fluorescence in situ hybridization (FISH) and the polymerase chain reaction (PCR) shown the presence of SRY gene. However, PCR analysis revealed the deleted sequences of AZF region. Conclusion: This is a new distinct female case with 47,XXY in spite of the presence of a Y chromosome and the normal SRY. Undertake published cases in the literature and present case, each case should be considered as a different identity in female phenotype with 47,XXY karyotype and SRY, but the deletion of AZF region on the long arm of chromosome Y.

Expression of microRNA-375 in esophageal squamous cell carcinoma and its methylation regulation

Objective To investigate the expression of microRNA (miRNA)-375 and its genomic promoter DNA methylation in esophageal squamous cell carcinoma (ESCC).Methods The expression of miRNA-375 in ESCC and the matched normal esophageal tissues (from the tumor edge ＞ 3 cm) was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR).DNA methylation status was examined by methylation specific polymerease chain reaction (MSP).The association between miRNA-375 expression and clinical factors were analyzed statistically.Results As compared with normal controls,miRNA-375 was significantly decreased in ESCC.The methylation levels in ESCC tissues were significantly higher than in the matched normal esophageal tissues (P ＜ 0.05).The low expression of miRNA-375 was significandy associated with ESCC TNM stage (P ＜ 0.05).Conclusion The lower expression of miRNA-375 and aberrant hypermethylation of the CpG island promoters in ESCC may play a crucial role in the carcinogenesis of ESCC. Key words: Esophageal squamous cell carcinoma; MicroRNA-375; DNA methylation

Correlation analysis of ras association domain family 1A gene methylation and environmental factors to hepatocellular carcinoma

Objective To investigate the relationship between ras association domain family 1A gene (RASSF1 A) methylation and environmental factors to the hepatocellular carcinoma (HCC).Methods The methylation of RASSF1A was determined by methylation specific polymerase chain reaction (PCR) in 80 genomic DNAs from patients with HCC.Aflatoxin B1 (AFB1)-DNA adducts and polycyclic aromatic hydrocarbons (PAHs)-DNA adducts in tumor tissues were determined by immunohistochemistry.The association between RASSFI A methylation and environmental factors taking into consideration of the clinical biological material was statistically analyzed.Results RASSF1A promoter hypermethylation was detected in 60 of the 80 HCC tissues (75.0％).Of the paired corresponding adjacent tissues from the 80 HCC patients,aberrant methylation was detected in 33 (41.3％).The positive rate for RASSF1A methylation was significantly higher in the HCC tissues than in the paired controls (P ＜O.O1 ).For AFBI-DNA adducts,the positive rate by methylation specific polymerease chain reaction (MSP) was significantly higher in positive cases (43/51,84.3％ ) than in negative cases (17/29,58.6％,P＜0.01 ).For PAHs-DNA adduct,the positive rate by MSP was also significantly higher in positive cases ( 39/45,86.7％ ) than in negative cses ( 21/35,60.0％,P ＜ O.05 ).Conclusion Statistically significant correlations were observed between RASSF1A methylation and AFB1-DNA adducts or PAHs-DNA adducts in the HCC tissues. Key words: Hepatocarcinogenesis; Aflatoxin B; Ras association domain family 1A gene; Methylation

Study the relationship between the gene regulation and microsatellite instability of the cyclooxygen-ase-2,hMLH1 in colon carcinoma

Objective To investigate the cyclooxygenase-2 (COX-2),human mut-lhomologue 1 (hMLH1) of genes promoter methylation and microsatellite instability (MSI) frequency in 42 cases of human colon carcinoma and normal tissues, and to evaluate the relationship between them and occurrence of colon carcinoma. Methods Methylation specific polymerease chain reaction ( MSP) and polymerase chain reaction (PCR) methods were employed in this study in order to investigate the promoter methylation of these genes and 5 loci MSI frequency in human colon carcinoma and normal tissues. Results Of 42 colon carcinoma cases,the total frenquency of MSI was 43.86 % ( 18/42). The MSI frenquency no significant discrepancy was found among the 5 loci (P > 0.05). The number of MSI, MSS was 18 and 24, respectively. Cases with methylation of COX-2 and hMLH1 gene promoter CpG islands in colon carcinoma were 13 and 15 ,and they were not detected in normal tissues. 8 cases with methylation of COX-2 gene promoter CpG islands only occurred in MSI-H colon carcinoma, and 6 cases both with methylation of COX-2 and hMLH1 gene promoter CpG islands was observed in MSI-H. Conclusion The methylation rate of hMLH1 gene promoter CpG islands in MSI colon carcinoma was higher than that in MSS colon carcinoma. It suggested that detecting the methylation of hMLH1 gene promoter CpG islands might be a useful method for determining the colon carcinoma type. Key words: Colon carcinoma; Cyclooxygenase-2; hMLH1; Methylation specific polymerease chain reaction

A novel mutation of the MEN1 gene in a Chinese kindred with multiple endocrine neoplasia type 1

Germline mutations in the MEN1 gene are well documented as the genetic cause of multiple endocrine neoplasia type 1 (MEN1). In this study, we performed genetic analysis by direct MEN1 gene mutation analysis on a Chinese MEN1 family. The two patients in this family were diagnosed as MEN1 by the typical clinical findings of parathyroidoma, insulinoma and pituitary adenoma. The coding sequences, including 9 coding exons and exon/intron boundaries of the MEN1 gene were amplified by polymerase chain reaction (PCR) and subjected to direct sequencing. Sequence analysis showed a same novel insertion mutation in exon 3 (c.433_434ins CTTC) in both patients, resulting in an open reading frames shift and produced a premature termination codon. None of the other family members had this insert mutation. In conclusion, we add a new mutation of MEN1 gene in Chinese patients with MEN1, and it would be useful for the diagnosis of the disease.

Host Plant Resistance to Pathogen with Reference to Induction of Systemic Acquired Resistance (SAR) in Tobacco (Nicotiana Tabacum Var. Samsun) When Infected with Tobacco Mosaic Virus (TMV)

A model study based on induction of SAR via infecting different lines of tobacco (Nicotiana tabacum var. Samsun) with Tobacco mosaic virus (TMV) was carried out at the Department of Applied Biology, University of Helsinki, Finland in December, 2006. The objective was to demonstrate the onset of defence response, spread of SAR in tobacco plants infected with TMV and detection of expression of marker gene, PR protein gene (PR1) using northern blotting and real time polymerease chain reaction (PCR). Localized necrosis was observed at 3 and 10 days post inoculation (dpi) only in TMV-inoculated NN plants in contrast to absence of similar localized symptom in the corresponding inoculated nn plants except systemic yellowing at 10 dpi. The data generated by northern blot analysis as well as real-time PCR showed existence of variation among experimental treatments with respect to PR1 gene expression. Northern blotting and real-time PCR gave similar results except a difference that was observed for sample 10 probably signifying high precision and sensitivity of real-time PCR in indicating PR1 expression compared to northern blotting. The RNA ladder was not visible in agarose gel and it was not possible to observe the actual result. This confirmed the importance of proceeding to the application of northern blotting. The result of this study indicated localized induction of necrotic lesions in infected tobacco var. Samsun (NN) as indicator for the HR programmed cell death and PRl expression as indicator for SAR in systemic leaves. All plants of nn variety are susceptible and showed only systemic mosaic symptoms in contrast to localized lesions expressed in NN plants. The result of this study indicated that the NN gene in TMV-infected tobacco var. Samsun carrying N allele recognized the corresponding Avirulence gene of the virus inducing the SAR in systemic leaves as indicated via expression of PR1 protein and hypersensitive reaction (HR) in infected leaves as visualized by localized necrosis in infected leaves. This is in agreement with the theory of gene-for-gene interaction in plant defence responses. Keywords : Gene-for-Gene; Inoculation; Pathogenesis-Related proteins East African Journal of Sciences Vol. 2 (2) 2008: pp. 184-188

Treatment of Bile Acid Malabsorption Using Ileal Stem Cell Transplantation

We hypothesized that ileal stem cell clusters transplanted into a segment of jejunum can be used to treat bile acid malabsorption. In adult Lewis rats, a 15-cm segment of jejunum was isolated with its blood circulation left intact and partially stripped of enterocytes using luminal high-velocity perfusions with 3mmol/L ethylenediamine tetra-acetic acid solutions. Continuity was restored by anastomosing the proximal and distal gut. Ileal stem cell clusters were harvested from neonatal Lewis rats and transplanted into the stripped segments to generate a "neoileum." After 4weeks, recipients underwent resection of the native ileum, and the isolated neoileum was anastomosed in its place. After an additional 4weeks, a 48-hour stool collection was performed. The engrafted segment was harvested for taurocholate uptake studies, ileal bile acid transporter (IBAT) protein by immunohistomorphometry, and IBAT mRNA quantitation by reverse transcription polymerease chain reaction. Data were analyzed by ANOVA/t-test. Rats undergoing ileectomy, jejunectomy, or sham operations served as controls. Total bile acid loss in the stool was markedly lower in rats with a neoileum compared with rats with an ileectomy (p < 0.001). Total taurocholate uptake was notably increased in the neoileum compared with the jejunum (p < 0.001). IBAT protein signal intensity was considerably higher in the neoileum compared with jejunum (p < 0.001). IBAT mRNA amounts in the neoileal group were comparable with those in normal rat ileum and were considerably higher (p = 0.003) than in the jejunum. Ileal stem cell clusters were used to establish a new zone of bile acid uptake and IBAT expression in a jejunal segment. This neoileum eliminated loss of bile acids in the stool after ileectomy. This is the first time that transplantation of intestinal stem cell clusters has been shown to correct a clinical malabsorption syndrome.

Mutational abrogation of the PTEN/MMAC1 gene in gastrointestinal polyps in patients with Cowden disease

Background & Aims: To understand the molecular etiology of Cowden disease–associated gastrointestinal polyps, we analyzed the mutational status of PTEN/MMAC1, a recently identified Cowden disease gene located at 10q23, in gastric hamartomas, colonic adenoma, and juvenile polyps of 3 patients with Cowden disease. Methods: Messenger RNA expression, gene deletion, and sequence alteration of PTEN/MMAC1 were evaluated by quantitative polymerease chain reaction (PCR), PCR–single-strand conformation polymorphism, and sequencing analysis. Results: Germline missense mutation at codon 289 (AAA to GAA, Lys to Glu) and deletion of the wild-type allele were detected in the polyps of 2 patients with Cowden disease in the same family. Germline allelic deletion and transcriptional silencing of the remaining allele, probably caused by abnormal methylation, were also observed in a gastric hamartoma of 1 patient. Conclusions: The germline mutation and alteration of the remaining allele observed in this study strongly support that PTEN/MMAC1 functions as a tumor suppressor in Cowden disease. This study is the first to show that the mutational abrogation of PTEN/MMAC1 plays a causal role in the genesis of gastrointestinal polyps in Cowden disease, providing molecular genetic evidence that colonic adenoma, juvenile polyp, and gastric hamartoma could be included in the manifestations of Cowden disease. GASTROENTEROLOGY 1998;115:1084-1089

Rapid diagnosis of influenza infection by PCR method--detection of influenza virus HA gene in throat swab

We studied the detection of the HA gene of human influenza viruses in throat swabs obtained from the outbreaks of influenza in school children utilizing the polymerase chain reaction (PCR) method. Sensitivity and specificity of the PCR method was compared to conventional virus isolation using MDCK cells. Three pairs of primers for PCR in detecting the HA genes of AH1, AH3, and B influenza viruses showed both subtype and type specificity. The dilution experiments showed that influenza viruses, as few as 1.1-3.5 plaque-forming units per 50 microliters, were sufficient for the detection of HA genes by PCR method and the detection rate by PCR method was 2-3 fold higher than that by conventional method. Our results showed that the PCR method was a fast, sensitive and reliable method for the diagnosis of influenza infections.