Nebulette expression is associated with lymph node metastasis in patients with colorectal cancer

Background:Colorectal cancer [CRC] is one of the most common cancers among men and women worldwide. Cancer metastasis is the main cause of death in patients with cancer. NEBL [nebulette, Gene ID: 10529] protein interacts with thin filaments in the cell and may functionally destabilize focal adhesion composition. There are some studies on NEBL gene expression alteration in cancer. In the presented study we aimed to analyze NEBL gene expression in patients with colorectal cancer to explore possible association of this gene with clinicopathological features in CRC

Methods:Sixty-seven fresh samples of colorectal tumors and adjacent normal tissues were collected from Iranian patients with CRC. Real time polymerase chain reaction was performed to measure the level of NEBL gene expression and its association with clinico-pathological features

Results:A significant overexpression with 3 fold increse was seen in NEBL mRNA level in tumoral tissues compared with the adjacent normal tissues. In addition there was a significant association between NEBL gene expression with lymph node metastasis in patients with CRC

Conclusion:The overexpression of NEBL has the capacity to be considred as a prognostic biomarker in patients with CRC

Employing real time PCR for the diagnosis of Huntington disease

Huntington disease [HD] is a dominantly inherited, neurodegenerative disease characterized by choreiform movement disturbances and dementia. The onset age of this disease is varied but usually is between the ages 40-50. Huntington's disease is caused by a triplet-repeat expansion in the IT15 gene [also known as huntingtin or HD] which is located on chromosome 4p3.1. Since many clinical picture of HD are indistinguishable from other distinct genetic disorders molecular test such as PCR is the only way to confirm the disease. The aim of this study was to introduce a new and fast technique for the diagnosis of Huntington disease. Blood specimens were collected from individuals suspected for Huntington disease and also people with no symptoms and family history of this disease. DNAs were extracted according to standard protocol. Using conventional PCR, patient positive for Huntington disease were diagnosed. Then employing real time PCR on the basis of difference between melting temperature [Tm] a new and fast diagnostic method was introduced. Among 29 patients suspected to be HD only 8 HD patients were confirmed using PCR and real time PCR. The numbers of CAG repeat were between 42-50 and melting temperatures were between 89-92. The concept of using melting temperature in real time PCR protocol presented in here could be employed for the rapid diagnosis of the diseases caused by the increased in triple repeat sequences. It is fast, robust and has the potential use for the prenatal diagnosis

Investigation of the genes MDR1/MRP1 and their relationship with clinical and para-clinical characteristics of colorectal cancer

Resistance to wide range of structurally and functionally different chemotherapy drugs acts as a major obstacle in the cancer therapy. There are different mechanisms that cause multi drug resistance [MDR] but the most important one is ATP Binding Cassette [ABC] transporters super-family that increases the ability of tumor cells to transport drugs out of the cells by using ATP molecule, over expression of these proteins such as MDR1 and MRP1 is responsible for MDR phenotype in cancerous cells. We analyzed the expression level of MDR1/MRP1genes by using Real time RT-PCR in 60 colorectal cancer patients also we investigate their relationship with clinical, Para-clinical characterizes of colorectal cancer patients and their responses to treatment. The expression of the MDR1 gene showed a significant increase in cancerous regions compared to adjacent normal tissue. In addition the expression of MDR1 gene showed association with histological grade of tumors but expression level of MRP1 was not significantly important. Our study once more emphasizes the effects of MDR1 over expression which impact on response to drugs in Iranian colorectal cancer patients

Transient expression assay of [A]gamma-588 [A/G] mutations in the k562 cell line

In the previous study, we have shown that the presence of A allele at position -588 in [A]gamma -globin gene was highly frequent and closely associated with fetal hemoglobin elevation among beta-thalassemia intermedia patients. Therefore, we decided to investigate whether this allele [A allele at -588] could result in an increase in [A]gamma-globin gene expression to ameliorate the severity of the disease in thalassemia patients. Three constructs containing ji locus control region, [A]gamma -globin and beta-globin genes were designed and employed in the transient expression assay. The difference among constructs was in the promoter region of, [A]gamma -globin gene [A and G alleles at -588]. A construct with T to C base substitution at -175 of, [A]gamma -globin, created by site-directed mutagenesis, was selected as positive control. The K562 cell line was transfected with the above constructs. Subsequently, the expression of, [A]gamma -globin gene was determined by quantitative real-time reverse transcription-PCR. There was not a significant increase in the expression of, [A]gamma -globin gene in the construct containing A allele comparing the one with G allele at -588. -588 [A>G] mutation does not play a major role in regulation of, [A]gamma -globin gene, suggesting that other factors may be involved

Polymorphisms of the ovine leptin gene and its association with growth and carcass traits in three Iranian sheep breeds

Leptin [LEP], the expression product of the obese gene produced primarily in the adipose tissue, is related to feed intake, growth and lipid metabolism. The aim of this study was to study the possible association between polymorphism of the LEP gene with growth and carcass traits among three Iranian sheep breeds of the Shal, Zandi and Zel varieties. A total of 180 purebred animals of the Shal, Zandi and Zel, breeds were chosen for this study. Three flocks, each comprising of 60 ewes of three breeds, were derived from Aboureyhan sheep populations. The Shal [n=18], Zandi [n=24] and Zel [n=17] lambs were screened for polymorphism of the LEP gene. Following genomic DNA extraction from whole blood samples, polymerase chain reaction [PCR] was carried out in order to amplify a 260 bp fragment of the target gene. Polymorphisms were detected using the single strand conformational polymorphism [SSCP] technique. Two genotypes of AA and AG with frequencies of 0.53 and 0.47 in the Shal 0.70 and 0.30 in the Zandi and 0.65, 0.35 in the Zel breed were observed, respectively. The frequencies of alleles A and G were 0.74, 0.26 in the Shal breed, 0.85, 0.15 in the Zandi breed and 0.82, 0.18 in the Zel breed, respectively. Chi-Square test [chi2] confirmed the Hardy-Weinberg equilibrium for the LEP loci. Average heterozygosity [30%] of the LEP locus for the three breeds was slightly low. Comparison of the sequence of the target gene available in the GeneBank with the results of the present study showed two single nucleotide polymorphisms [SNP] A?G and T?C transitions at 113 and 165 bp positions, respectively. In the Shal breed, the A113G SNP associated with an increase in cold carcass weight [p< 0.01], fat-tail percent [p< 0.05] and total body fat weight [p< 0.05]. In the Zel breed, the A113G SNP was associated with an increase in fat-tail percent [p< 0.05] and reduction in slaughter weight [p< 0.05], cold carcass weight [p< 0.01] and lean meat weight [p< 0.01]. Therefore, a significant association between SNP within the LEP gene and certain carcass traits in the Shal and Zel breeds is proposed. In the Zandi breed the A113G SNP was not associated with carcass traits but showed a reduction in weaning weight [P< 0.05]

RNAi induced inhibition of MRP1 expression and reversal of drug resistance in human promyelocytic HL60 cell line

Multidrug resistance [MDR] is a complex phenomenon in which many different genes regulating drug transport, cellular repair, detoxification and drug metabolism are involved. Nevertheless, in most drug resistant cell lines and cancer patients up-regulation of ABC-transporter genes such as MDR associated Protein [MRP1] gene could be at the basis of the drug resistance phenotype. We aimed to decrease MRP1 expression at the mRNA level to modulate drug resistance phenotype in the methotrexate-resistant HL60 cell line. We designed a small interfering RNA [siRNA] molecule against MRP1 and applied it to HL60 cell line in a 0 to 72 hours time range. siRNA could specifically inhibit gene expression by 80% of the initial mRNA level with in 36 to 48 hours. The siRNA-treated cells demonstrated 100-fold reduction in methotrexate [MTX] resistance compared to untreated cells. The data indicate that this approach may be applicable to the study of MRP1 expression and development of future strategies to reverse the MRP1 dependent drug-resistance phenotype in tumors back to a drug-sensitive one