Didriksenflindt2783

Copyright© 2020 Avicenna Research Institute.Background Zinc-finger Enhancer Binding protein (ZEB1) acts as a transcription factor to promote cancer progression through regulating Epithelial to Mesenchymal Transition (EMT). It is well-known that ZEB1 mRNA expression is directly induced by both Estrogen Receptor (ER) and Progesterone Receptor (PR). Moreover, Androgen Receptor (AR) and PR could bind to the same regulatory element. Since it has been shown that AR overexpresses in Gastric Cancer (GC) as a male-predominant tumor, the goal of this study was to evaluate whether AR could regulate ZEB1 expression in GC. Methods The expression profile of ZEB1 in 60 fresh GC and adjacent non-tumor tissues and 50 normal gastric specimens was assessed by qRT-PCR, and the association of ZEB1 expression with clinicopathological features was investigated. Furthermore, possible correlation between ZEB1 and AR was evaluated to elucidate a novel prognostic marker using Kaplan-Meier method and Cox regression model. Finally, molecular interaction of ZEB1 and AR was assessed using a potent AR antagonist in GC cells. Results Among GC patients, 70.2% (40/57) overexpressed ZEB1 and 64.91% (37/57) overexpressed AR relative to normal gastric tissues. ZEB1 overexpression was significantly correlated with the AR overexpression in GC patients. Moreover, ZEB1 overexpression was remarkably associated with lower overall survival; however, it was not an independent prognostic factor. Evidence shows that simultaneous evaluation of ZEB1 and AR expression could independently predict survival of GC patients (HR= 2.193, p=0.047). Conclusion These findings have clinical importance suggesting simultaneous evaluation of ZEB1 and AR expression as a potential prognostic marker. Moreover, AR may regulate ZEB1 expression in GC cells proposing a possible promising targeted therapy for GC patients. Copyright© 2020 Avicenna Research Institute.Background The delivery of exogenous genes into cells for functional expression is required for development of DNA vaccine and gene therapy in medicine and pharmacology. Cell Penetrating Peptides (CPPs) were considered to mediate gene and drug delivery into living cells. In this study, an attempt was made to evaluate the efficiency of an arginine-rich CPP, HR9, in HCV NS3 gene delivery compared to TurboFect cationic polymer and supercharged +36 GFP into HEK-293T cells. Methods The recombinant pEGFP-NS3 was constructed and their accuracy was confirmed by digestion and sequencing. Then, the recombinant plasmid was transfected into HEK-293T cells by TurboFect, +36 GFP and HR9 gene delivery systems. The expression of NS3 protein was assessed by fluorescent microscopy, flow cytometry and western blotting. Results Our data indicated that HR9 peptide was able to form stable complexes with plasmid DNA and increased its delivery into HEK-293T cells in a non-covalent manner. Furthermore, treatment of cells with HR9 and HR9/DNA complexes resulted in a viability of 90-95% indicating this CPP was not cytotoxic. The analysis of zeta potential and size showed the importance of interactions between positively-charged HR9/pEGFP-NS3 complexes and negatively-charged plasma membranes. Conclusion The non-toxic HR9 CPP can be considered an effective carrier for delivering plasmid DNA harboring Hepatitis C virus (HCV) gene in therapeutic vaccine design. Copyright© 2020 Avicenna Research Institute.Background Despite the ease of conventional splicing by overlap-extension (SOEing) PCR technique in theory, when splicing more than two fragments, and especially if one of the complementary sequences is A-T rich, the attachment of the fragments would be challenging. A new rapid and highly efficient SOEing PCR assay was developed for simultaneous splicing of multiple DNA fragments and induction of site-directed mutagenesis in a single tube. Methods The method was adapted for splicing human beta-globin UTRs to OCT4, SOX2, KLF4, C-MYC, LIN28A, and destabilized GFP for the construction of chimeric DNA fragments for in vitro transcription. In addition, the native Kozak sequence of beta-globin (K1) was replaced by the strongest Kozak sequence (K2) using site-directed mutagenesis to enhance the expression of target genes. Results ChimericGFPd2/K1, GFPd2/K2, OCT4, and KLF4 were created by the optimized conventional SOEing PCR. The single tube method was able to create the chimeric SOX2, C-MYC, and LIN28A in high quality and quantity in comparison with the conventional SOEing PCR. Moreover, using single tube SOEing PCR, the reaction time and materials that are required in the conventional SOEing PCR were significantly reduced. Fluorescent microscopy and flow cytometry examinations indicated highly efficient translation of K2 sequence in comparison with the K1sequence. Conclusion Single tube SOEing PCR is a valuable method to construct more multiple fragments with high yield. The method can successfully be applied for construction of various kinds of complex chimeric genes. Copyright© 2020 Avicenna Research Institute.Background The Secretory Leukocyte Protease Inhibitors (SLPI) has many biological functions including anti-bacterial, anti-fungal, anti-viral, anti-inflammatory, and immuno-modulatory. Previous studies have shown that gene-encoding human SLPI have successfully been expressed in Escherichia coli (E. coli) with a C-terminal poly-histidine tag (His-tag). The aim of this research was to investigate the inhibition activity of N-terminal His-tag position (NSLPI) and C-terminal His-tag position (CSLPI). We hypothesized that a His-tag close to an active site SLPI domain may interfere with the inhibition activity of SLPIs. Methods A NSLPI and CSLPI were constructed with polymerase chain reaction (PCR) amplification. The PCR products were then ligated into pET-30a plasmid and transformed into E. coli TOP10. Recombinant plasmids were verified by using restriction analysis and nucleotide sequence analysis. pET-NSLPI and pET-CSLPI were then subcloned in E. coli BL21(DE3) for its expression. The SLPI protein was expressed using Isopropyl β-D-1-thiogalactopyranoside induction (IPTG). The inhibition effect of both SLPI against Porcine Pancreatic Elastase (PPE) enzyme was tested using the N-succinyil-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-4-nitroanalide (NPN) substrate. Results The SLPI gene was successfully cloned and expressed in E. coli BL21. GS9674 Fusion proteins of NSLPI and CSLPI were generated with His-tag in the N-terminal and C-terminal position, respectively. The inhibition effect of NSLPI and CSLPI on PPE indicated that both SLPI were active. The inhibition activity of NSLPI was 66.7%, higher than CSLPI by 44.4%. Conclusion The His-tag position on the C-terminal of SLPI reduced the inhibition activity of SLPI. Copyright© 2020 Avicenna Research Institute.