Expression Study and Clinical Correlations of MFC and CCAT2 in Breast Cancer Patients

Background: Colon cancer-associated transcript 2 [CCAT2] is a newly recognized IncRNA transcribed from the 8q24 genomic region. It functions as an oncogene in various types of cancers including breast cancer, in which it affects Wnt/p-catenin pathway. Previous studies have shown a putative interaction between this IncRNA and MYC proto-oncogene

Methods: In the current study, we evaluated the expression of CCAT2 in breast cancer tissues with regards to the expression of its target MYC. In addition, we assessed the relationship between CCAT2 and MYC expression levels in tumor tissues and the clinical prognostic characteristics of breast cancer patients

Results: MYC expression levels were significantly up-regulated in tumor tissues compared with adjacent non-cancerous tissues [ANCTs], while such analysis showed no statistically significant difference between these two tissue types in CCAT2 expression. Starkly increased CCAT2 gene expression levels were found in 12/48 [25%] of cancer tissue samples compared with their corresponding ANCTs. Furthermore, significant inverse correlations were found between CCAT2 expression and stage, as well as lymph node involvement. Besides, a significant inverse correlation was found between the relative MYC expression in tumor tissues compared with their corresponding ANCTs and disease stage

Conclusions: These results highlight the significance of MYC and CCAT2 expressions in the early stages of breast cancer development and suggest a potentially significant role for CCAT2 in a subset of breast cancer patients, which could be applied as a potential therapeutic target in these patients

Development of a probe consist of three cosmids to enumerate the chromosome 13 on uncultured lymphocytes or amniocytes using interphase FISH

To produce a reliable probe suitable for aneuploidy detection of chromosome13 on uncultured lymphocytes and amniocytes by fluorescence in situ hybridization [FISH], we used a contig of three overlapping cosmids mapped to 13q12.3. The cosmid DNA carrying the expected sequences of human chromosome 13 was isolated from host cells and labelled with biotin-11-dUTP. The hybridization and detection conditions with FITC-Avidin were optimised using a series of cultured and uncultured lymphocytes and amniocytes. Intensive signals were detected when a combination of three overlapping cosmids was used to enumerate the chromosome 13 on interphase nuclei. An average of 87 and 85.5 percent of interphase cells prepared from lymphocytes and amniocytes showed accurate number of specific signals for chromosome 13. The results obtained in present study indicate that the probe was capable of detecting the copy number of chromosome 13 on interphase cells prepared from peripheral blood or amniotic fluid cells providing that the uncultured amniotic fluid cells are free of cytoplasmic residues, RNA and protein debris

[Determination of gamma-globin induction levels by different concentrations of hydroxyurea in K 562 cell lines for beta thalassemia treatment

Hydroxyurea [HU] is currently used for beta thalassemia treatment through induction of fetal gamma-globin, In this study, effects of different concentrations of hydroxyurea on induction of gamma-globin gene in K562 cells, and inhibitory effect of siRNA against candidate gene which may be involved in HU mediated gamma-globin induction were assessed. In this eperimantal study, K562 cells were treated by different concentrations of HU [0, 50, 100 and 200 microM]. siRNA against candidate gene in HU mediated gamma-globin gene induction was transfected to K562 cells using lipofectamine 2000. The level of gammal-globin gene induction and inhibition were determined by quantitavie real-time PCR. There were 1.75-, 2.45- and 2.55- fold inductions in gamma-globin gene using 50, 100 and 200 microM HU, respectively. There has been 79.5% down regulation on candidate gene in siRNA transfected K562 cells. This study showed that gamma-globin induction in 100 microM HU is similar to 200 micro M HU treated K562 cells. There was also efficient inhibitory effect on candidate gene which may be involved in HU mediated gamma-globin induction

[Assessing the levels of cAMP response element binding protein 1 [CREB1] after siRNA mediated knockdown in K562 cells]

CREB1 is an important downstream protein for many signaling pathways. By designing efficient siRNAs against CREB1, it may be possible to assess the role of molecules involved in signaling pathways in different cell types. In this research the efficiency of CREB1 knockdown by two different siRNAs in K562 cells has been studied. siRNAs have been designed according to the criteria suggested by Reynolds et al. K562 cells were transfected by siRNA using Lipofectamine 2000. The efficiency of CREB knockdown has been assessed by quantitative relative Real-time PCR. Our results have shown that only one of the siRNAs has a high level of inhibitory effect on CREB1 gene expression. The expression of CREB1 by this siRNA was knocked-down by 87% in K562 cells. In this research, although two siRNAs were designed according to the Reynolds et al. criteria, only one showed an inhibitory effect. Reasons other than the aforementioned criteria may be involved in effectiveness of siRNAs