[Expression levels of key components of the hedgehog pathway and an investigation of the correlation of this pathway with expression levels of claudin-1 in invasive breast carcinoma]

Objective: Breast cancer is considered a heterogeneous disease, characterized by different biological and phenotypic features which make its diagnosis and treatment challenging. We have sought to investigate the expression levels of key components of the Hedgehog signaling pathway, correlation between the signal transducer Smo, and clinicopathologic features [lymph node metastasis and metastasis stage] in invasive breast carcinoma. Also, we examined the inverse correlation between expression levels of Smo and Claudin-1 [an important gene involved in cell tight junctions]

Methods: In this case-control study, we assessed 36 pairs of tumor and adjacent normal tissue specimens obtained from patients with invasive ductal breast carcinoma. The expression levels of key components of Hedgehog signaling [Smo, Gli1 and Ptch], Claudin-1, E-cadherin, and MMP2 were measured by qRT-PCR. The correlations between Smo expression with some clinicopathologic parameters were also analyzed

Results: We found up-regulation of Hedgehog signaling in invasive breast carcinoma samples compared to normal adjacent tissues. Upregulation of the signal transducer Smo correlated with tumor stages and lymph node metastasis of the breast tumors. Interestingly, this correlation was affected by the expression of Her2. A significant correlation existed between expression levels of the signal transducer Smo and Claudin-1, E-cadherin as an epithelial cell marker, and MMP2 as a metastasis-related gene in advanced metastatic tumor samples

Conclusion: Taken together, our study revealed a new layer of molecular complexity which should be considered in the management of patients with invasive breast carcinoma. The results suggested a key role for Hedgehog signaling in invasive breast carcinoma. In terms of the inverse correlation between expression levels of Claudin-1 and Hedgehog signaling, Claudin-1 could serve as a candidate gene in diagnostic studies. Thus, its clinical significance should be further clarified

Production of recombinant lentiviruses expressing mir-16 by transient transfection of 293t cells

The aim of the present study was the production of recombinant lentviruses that express miR-16. After transduction, altered expression levels of miRNA and its target protein were analyzed. A DNA fragment that contained the miR-16 precursor was cloned in a lentiviral plasmid. Lentiviral vector particles were produced by transient calcium phosphate co-transfection of 293T cells with the combined lenti-miR, structural and packaging plasmids. Viral supernatants were harvested and concentrated by ultracentrifuge. Virus titration was determined by fluorescent microscopy and flow cytometry. Altered expression levels of miR-16 were evaluated by real-time PCR; its protein target was evaluated by Western blot. The identity of DNA was established by colony-PCR, enzymatic digestion of positive clones, and DNA sequencing. After co-transfection of 293T cells with the combined lenti-miR, structural and packaging plasmids, viral particles were concentrated and the virus titer determined. Maximum expression of the GFP reporter gene was obtained in more than 80% of the cells transduced with lentivirus at MOI=1. Real-time PCR assay showed that miR-16 expression levels significantly increased in transduced cells compared with the control group. As shown by Western blot analysis, miR-16 overexpression downregulated Bcl-2 expression at the protein level. This lentivirus expression system could be considered as a tool for efficient delivery of produced miRNAs to cells

Chronic myeloid leukemia as a stem cell-derived malignancy

Chronic myeloid leukemia [CML] is a myeloproliferative disease of the hematopoietic stem cells, characterized by the presence of the Philadelphia [Ph] chromosome. Although imatinib inhibits the BCR-ABL kinase activity, clinical experiences confirm that imatinib may not target CML stem cells in vivo. The identification of signaling pathways responsible for the self-renewal properties of leukemic stem cells in CML will help in the discovery of novel therapeutic targets. Here we review signaling pathways including Wnt/beta-catenin, Hedgehog, Alox5, and Foxo which play crucial roles in the maintenance of stem cell functions in CML. It is thought that inhibition of key genes that are part of self-renewal associated signaling pathways may provide an effective way to reduce aberrant stem cell renewal in CML

[Detection of unknown deletions in alpha globin genes in alpha thalassemia carriers using real-time PCR]

Alpha-thalassemia is one of the most prevalent hemoglobin disorders in the world and it is a common hereditary condition caused by deletion of one or more alpha-globin genes. Common alpha-thalassemia deletions like 3.7 kb, 4.2 kb, 20.5 kb and Med can be detected by Multiplex PCR. There are, however, some unknown deletions that can not be detected by the mentioned method or even by direct DNA sequencing. In the present study, Real-time PCR was used to determine the presence or absence of unknown deletions. Real-time PCR was performed using intercalating dye SYBR Green I and alpha1, alpha2 and CLCN7 genes were amplified. Data analysis was conducted using comparative threshold method [delta delta CT] for determination of Gene dosage of alpha1-globin and alpha2-globin genes. The results showed the ratio of 0.90 +/- 0.16 for normal individuals and the ratio of 0.32 +/- 0.15 for carrier samples with deletions. In addition, Melting curve analysis confirmed the specific amplification of target genes. The Real-time PCR assay is simple, rapid, and reliable. It can be applied for direct determination of unknown deletions in Alpha-thalassemia carriers