AB181. In vitro study on shRNA decreasing the expression of human testis-specific gene TDRG1 and affecting the proliferation, invasion and apoptosis of NTERA-2 Cell

OBJECTIVE: We have screened and cloned a novel human testis-specific gene, named Testis developmental related gene 1 (TDRG1 GenBank DQ168992), using Digital Differential Display. In our previous work, we hypothesized that this gene might be involved in the occurrence and development of testiculoma. To investigate this possibility, we have successfully established a stabilized RNA interference system to decrease the expression of TDRG1 in NTERA-2 cell line. In this study, to explore the function of TDRG1 in testiculoma further, we successfully transfected the TDRG1-shRNA486 and TDRG1-shRNA/control expression vectors described in our previous work (PubMed PMID: 23117448) into NTERA-2 cells in vitro and tested the biological behaviors of NTERA-2 cells. METHODS: Firstly, we checked the expression of TDRG1 in NTERA-2 cell line. Then we used fluorescence microscope to ensure the successful transfection. The expression of TDRG1 mRNA and protein was verified by fluorescence quantitative PCR and indirect cell immunofluorescence, respectively. Furthermore, we used such methods as MTT assay, transwell assay and flow cytometry to detect the biological behaviors of NTERA-2 cells. RESULTS: We determined that the TDRG1 mRNA and protein levels were significantly reduced by TDRG1-shRNA486expression vector. Moreover, the ability to proliferate and invade in vitro was suppressed in cells where the expression of TDRG1 was down-regulated, and a corresponding increase in the apoptotic potential was observed. CONCLUSIONS: Firstly, these results indicate that the TDRG1-shRNA486 expression vector constructed previously can interfere the TDRG1 expression effectively and stably. We construct a good cell line model in which TDRG1 expression was inhibited. Secondly, the ability of proliferation and invasion of NTERA-2 cells in vitro can be positively regulated by TDRG1, while the potential of apoptosis can be negatively regulated. This gene has some characters resembling oncogene.