ABSTRACT
<p><b>OBJECTIVE</b>To construct eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 wild-type or its variants (W39R, R264C, W39R-R264C) and to observe its expression in MCF-7 and Bcap-37 cells.</p><p><b>METHODS</b>The aromatase WT cDNA sequence was obtained by RT-PCR amplification and cloned into the eukaryotic expression vector pcDNA3.1(+). pcDNA3.1(+)-CYP19-GFP plasmid was then used as the template for site-directed mutation to create variant constructs (W39R, R264C, W39R-R264C). pcDNA3.1(+)-CYP19-GFP was transfected and expressed in MCF-7 and Bcap-37 cells.</p><p><b>RESULT</b>The construction of pcDNA3.1(+)-CYP19-GFP plasmid was confirmed by enzyme digestion and DNA sequencing. pcDNA3.1(+)-CYP19(W39R)-GFP, pcDNA3.1(+)-CYP19(R264C)-GFP, pcDNA3.1(+)- CYP19(W39R-R264C)-GFP plasmids were confirmed by DNA sequencing. The MCF-7 and Bcap-37 cells transfected with the pcDNA3.1(+)-CYP19-GFP plasmid expressed reporter gene of GFP.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmids have been constructed and expressed in MCF-7 and Bcap-37 cells successfully, which lays the foundation for the research of biological activities of CYP19 variant allozymes.</p>
Subject(s)
Humans , Aromatase , Genetics , Cell Line, Tumor , Genetic Vectors , Green Fluorescent Proteins , Genetics , Mutagenesis, Site-Directed , Plasmids , Genetics , Recombinant Fusion Proteins , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To explore the activation of the signal transduction pathways related with the carcinogenesis of sporadic colon cancers.</p><p><b>METHODS</b>A gene microarray monitoring activation of 8 signal transduction pathways (PathwayFinder GEArray) was used to screen the differentially expressed genes between colorectal cancer and normal colon tissue. The differentially expressed genes were further analyzed by RT-PCR, using RNA extracted from cancer tissue and matched normal colon mucosa of 35 patients with colorectal cancer.</p><p><b>RESULTS</b>The expression of hsf1, hsf27 and inos was increased in colon cancer compared with normal colon mucosa using PathwayFinder GEArray. The RT-PCR results showed that the expression of hsf1 was detected in 86% of patients(30/35)and the expression of inos detected in 63% patients(22/35).</p><p><b>CONCLUSION</b>Hsf1 induces heat shock stress signaling pathway, which might play a role in the carcinogenesis of sporadic colorectal cancer.</p>