MAML1 promotes ESCC aggressiveness through upregulation of EMT marker TWIST1.

BackgroundMastermind-like 1 (MAML1) is the main transcriptional co-activator of Notch signaling pathway. It plays essential roles in several pathways including MEF2C, p53, Nf-кB and Wnt/β-catenin. TWIST1 is known as a regulator of epithelial mesenchymal transition (EMT), which is considered as a primary step in promotion of tumor cell metastasis. Since concomitant expression of these genes was observed in tumors, our aim in this study was to elucidate the linkage between MAML1 and TWIST1 co-overexpression in esophageal squamous cell carcinoma (ESCC).ResultsWhile MAML1 silencing significantly down-regulated TWIST1, its ectopic expression up-regulated TWIST1 expression in both mRNA and protein levels in KYSE-30 cells. Expression of mesenchymal markers was increased significantly after MAML1 and TWIST1 ectopic expression, while epithelial markers expression was significantly decreased after silencing of both genes. Concomitant protein expression of MAML1 and TWIST1 was significantly observed in ESCC patients. Enforced expression of TWIST1 had no impact on MAML1 gene expression in KYSE-30 cells.ConclusionThe results clearly suggest transcriptional regulation of TWIST1 by MAML1 transcription factor in ESCC cells KYSE-30. Since TWIST1 is known as an EMT inducing marker, our results may revealed the mastermind behind TWIST1 function and introduced MAML1 as an upstream master regulator of TWIST1 and EMT in KYSE-30 cells

Ectopic Expression of Embryo/Cancer Sequence A (ECSA) in KYSE-30 Cell Line Using Retroviral System

Background Human preimplantation embryonic cells share many similarities with cancer cells such as ability to self-renew, unlimited proliferation and maintenance of the undifferentiated state. Embryo-cancer sequence A (ECSA), also known as developmental pluripotency associated-2 (DPPA2), is a cancer testis antigen (CTA) with unclear biological function yet. Objective: CTAs are expressed normally in germ line cells and trophoblast, and aberrantly in a variety of cancers. According to the importance of ECSA in developmental events and cancer, preparing a suitable platform to analyze its roles seems necessary. Methods The coding sequence of the gene was amplified and sub-cloned in pRUF retroviral expression vector. pRUF- ECSA vector was cotransfected with pVSV-G to GP293 cells and with pVSV-G and pGP to HEK293 packaging cell lines. Then the viral particles were transducted to KYSE-30 cells and the concentration of retroviral particles was determined by Real time PCR. Results  The coding sequence of ECSA gene was successfully subcloned in pRUF expression vector and transfected to packaging cells that the efficiency of transfection to GP293 was higher than the HEK293 cells. The enriched virus particles were obtained at a final concentration of 105 TU/ml. Conclusion  Considering the critical characteristics of retroviral expression system such as stable and longtime expression of interested gene, being safe due to the deletion of retroviral pathogenic genes, and since the function of ECSA gene is not clear, we used this system to induce expression of ECSA and prepared a valuable platform to analyze the biological function of the gene. Also the recombinant ECSA protein can be used in production of recombinant vaccines and serological tests. Keywords: DPPA2, ECSA, Embryo/cancer gene, KYSE cell line, Retroviral expression system

Detection of heat-labile toxin in enterotoxigenic Escherichia coli using PCR-ELISA technique

Background and Objective: Enterotoxigenic Escherichia coli (ETEC) are the most common agent which causes diarrhea, worldwide. ETEC is colonized along the cells and then producing heat-labile (LT) and heat-stable enterotoxigenic which enter into intestinal epithelial cells and causes water and electrolyte loss from intestinal epithelial cells and eventually cause diarrhea.This study was done to detect the heat-labile toxin in Enterotoxigenic Escherichia coli using PCR-ELISA technique. Methods: In this descriptive study, DIG-labeled PCR products were bounded to streptoavidin-coated wells of a microtiter plate and detected by anti-DIG–peroxidase conjugate. The biotin-labeled internal probe was used for verification of PCR products. Results: Heat-labile toxin was detected by PCR-ELISA method. The sensitivity of heat-labile toxin was 1.9 ng. This method did not cross-react with bacteria from this variety. Conclusion: PCR-ELISA method is 100 times more sensitive than conventional PCR method and due to lack of agarose gel and electrophoresis device it can be a good alternative to traditional method