Ectopic Expression of Human DPPA2 Gene in ESCC Cell Line Using Retroviral System.

BACKGROUND: Cancer/Testis Antigens (CTAs) are a sub-group of tumor-associated antigens which are expressed normally in germ line cells and trophoblast, and aberrantly in a variety of malignancies. One of the most important CTAs is Developmental Pluripotency Associated-2(DPPA2) with unknown biological function. Considering the importance of DPPA2 in developmental events and cancer, preparing a suitable platform to analyze DPPA2 roles in the cells seems to be necessary. METHODS: In this study, the coding sequence of DPPA2 gene was amplified and cloned into the retroviral expression vector to produce recombinant retrovirus. The viral particles were transducted to Esophageal Squamous Cell Carcinoma (ESCC) cell line (KYSE-30 cells) and the stable transducted cells were confirmed for ectopic expression of DPPA2 gene by real-time PCR. RESULTS: According to the critical characteristics of retroviral expression system such as stable and long time expression of interested gene and also being safe due to deletion of retroviral pathogenic genes, this system was used to induce expression of DPPA2 gene and a valuable platform to analyze its biological function was prepared. Transduction results clearly showed efficient overexpression of the gene in target cells in protein level due to high level of GFP expression. CONCLUSION: Such strategies can be used to produce high levels of desired protein in target cells as a therapeutic target. The produced recombinant cells may present a valuable platform to analyze the effect of DPPA2 ectopic expression in target cells. Moreover, the introduction of its potential capacity into the mouse model to evaluate the tumorigenesis of these cancer cells in vivo leads to an understanding of the biological importance of DPPA2 in tumorigenesis. In addition, our purified protein can be used in a mouse model to produce specific antibody developing a reliable detection of DPPA2 existence in any biological fluid through ELISA system.

TWIST1 upregulates the MAGEA4 oncogene.

Overexpression of MAGEA4 oncogene has been demonstrated in different malignancies; however, little is known about its exact mechanism for overexpression. TWIST1, as a bHLH transcription factor, activates a cell migration-invasion program involved in both embryonic and tumor development. Since MAGEA4 overexpression was statistically correlated to TWIST1, we aimed to elucidate the probable regulatory role of TWIST1 on MAGEA4 expression in KYSE30 cells. METHODS: Expression pattern of MAGEA4 and TWIST1 was analyzed in 55 ESCC patients using relative comparative real-time PCR. In silico analysis of the MAGEA4 gene was performed. Methylation status of MAGEA4 promoter was determined by quantitative methylation specific PCR (qMSP). Using a retroviral system, KYSE30 cells were transduced to ectopically express TWIST1, followed by qRT-PCR, Western blot analysis, chromatin immunoprecipitation (ChIP), and luciferase assays to elucidate the regulatory role of TWIST1 on MAGEA4 gene expression. RESULTS: Concomitant overexpression of MAGEA4 and TWIST1 was detected in ESCC in significant correlation with each other in different clinicopathological indices of poor prognosis (P < 0.05). The TWIST1-expressing cells showed significantly higher MAGEA4 expression compared to control cells. ChIP and luciferase assays results confirmed indirect binding of TWIST1 to the E-boxes of MAGEA4 promoter sequence and revealed a novel regulatory role of TWIST1 in MAGEA4 upregulation. CONCLUSION: Since MAGEA4 is a highly expressed oncogene in a variety of malignancies in significant correlation with tumor cell invasiveness and aggressiveness, our finding may help understand one regulatory mechanism of increased expression in tumor cells. © 2016 Wiley Periodicals, Inc.

Cloning and Sequence Analysis of LipL32, a Surface-Exposed Lipoprotein of Pathogenic Leptospira Spp.

BACKGROUND: Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira species. A major challenge of this disease is the application of basic research to improve diagnostic methods and related vaccine development. Outer membrane proteins of Leptospira are potential candidates that may be useful as diagnostic or immunogenic factors in treatment and analysis of the disease. OBJECTIVES: To develop an effective subunit vaccine against prevalent pathogenic Leptospira species, we sequenced and analyzed the LipL32 gene from three different Leptospira interrogans (L.interrogans) vaccinal serovars in Iran. MATERIALS AND METHODS: Following DNA extraction from these three serovars, the related LipL32 genes were amplified and cloned in the pTZ57R/T vector. Recombinant clones were confirmed by colony- PCR and DNA sequencing. The related sequences were subjected to homology analysis by comparing them to sequences in the Genbank database. RESULTS: The LipL32 sequences were >94% homologous among the vaccinal and other pathogenic Leptospira serovars in GenBank. This result indicates the conservation of this gene within the pathogenic Leptospires. CONCLUSIONS: The cloned gene in this study may provide a potentially suitable platform for development of a variety of applications such as serological diagnostic tests or recombinant vaccines against leptospirosis.