ABSTRACT
BACKGROUND: Studying whole blood DNA methylation as a risk marker has valuable applications in either diagnosis or staging of breast cancer. We investigated whole blood DNA methylation status of VIM, CXCR4, DOK7, and SPDEF genes in breast cancer patients in comparison to healthy control subjects. MATERIALS AND METHODS: 60 patients with breast cancer and 40 healthy controls were examined. Genomic DNA isolated from peripheral blood and restriction enzyme polymerase chain reaction (REP) method was applied for analysis. Real-time PCR was used to confirm methylation status of the aforementioned genes and therefore to find out the methylation differences between normal and breast cancer subjects. RESULTS: Level of DOK7 promoter hypomethylation in normal and breast cancer samples was significant (P-value = 0.001). The study, also, showed that hypomethylation of VIM and CXCR4 genes are significant in patients compared with normal cases (P-value < 0.05). Furthermore, SPDEF promoter hypomethylation was not significantly differed between normal and breast cancer samples (P-value = 0.2). CONCLUSIONS: Hypermethylation of DOK7 gene in DNA from patients affected with breast cancer offers a biomarker for diagnosis of the breast cancer. This study indicates that methylation status of VIM and CXCR4 genes changes in breast cancer; so, they can be used as molecular biomarkers in breast cancer prognosis.
ABSTRACT
Objective: MicroRNAs [miRNAs] are a class of non-coding RNAs [ncRNAs] that tran-scriptionally or post-transcriptionally regulate gene expression through degradation of their mRNA targets and/or translational suppression. However, there are a few reports on miRNA-mediated expression regulation of long ncRNAs [lncRNAs]. We have previ-ously reported a significant upregulation of the lncRNA SOX2OT and its intronic coding gene, SOX2, in esophageal squamous cell carcinoma [ESCC] tissue samples. In this study, we aimed to evaluate the effect of induced overexpression of miR-211 on SOX2OT and SOX2 expression in vitro
Materials and Methods: In this experimental study, we performed both bioinformatic and experimental analyses to examine whether these transcripts are regulated by miRNAs. From the list of potential candidate miRNAs, miR-211 was found to have complementary sequences to SOX2OT and SOX2 transcripts. To validate our finding experimentally, we transfected the NT-2 pluripotent cell line [an embryonal carcinoma stem cell] with an expression vector overexpressing miR-211. The expression changes of miR-211, SOX2OT, and SOX2 were then quantified by a real-time polymerase chain reaction [RT-PCR] approach
Results: Compared with mock-transfected cells, overexpression of miR-211 caused a significant down-regulation of both genes [P<0.05]. Furthermore, flow-cytometry analysis revealed a significant elevation in sub-G1 cell population following ectopic expression of miR-211 in NT-2 cells
Conclusion: We report here, for the first time, the down-regulation of SOX2OT and SOX2 genes by an miRNA. Considering the vital role of SOX2OT and SOX2 genes in pluripotency and tumorigenesis, our data suggest an important and inhibitory role for miR-211 in the aforementioned processes
ABSTRACT
Fetal DNA in maternal plasma and serum has been shown to be a useful material for prenatal fetal sex determination during early gestational ages. Non-invasive prenatal diagnosis is now possible at 8[th] week of pregnancy, by maternal blood sample testing. The purpose of this study was to evaluate two DNA extraction methods from mother plasma and its routine clinical application in bovine fetus gender determination with non-invasive method. Maternal blood samples were taken from 40 pregnant cows during the 8[th]-38[th] weeks of gestation. DNA was extracted from 350 micro l of maternal plasma with two salting-out and phenol-chloroform methods. The absorption in A[260] and purity [A[260]/A[280]] of extracted DNA were detected by ultraviolet spectrophotometer. Three micro l of the extracted DNA with phenol-chloroform method was used as a template. The PCR reaction was carried out to amplify the fragments of X and Y chromosomes of amelogenin, TSPY and BC1.2 genes. The difference between the mean absorption of DNA extracted by phenol-chloroform method and salting-out method was not significant in A260 [p>0.05, p=0.3549], but the difference between mean purity [A260/A280] of DNA extracted by phenol-chloroform method and salting-out method was significant [p<0.001]. X chromosome fragment was detected in all 40 samples and Y chromosome fragments were detected in 25 plasma samples which were delivered a male calf. The sensitivity and specificity of test was 100% with no false negative and false positive results. The results showed that phenol-chloroform method is a simple and sensitive method for isolation of fetal DNA in maternal plasma
ABSTRACT
In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction [PCR] analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses. In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 micro l maternal plasma. Two primer pairs for bovine amelogenin gene [bAML] and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome. The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results. The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses