Stemness genes Oct and Nanog were recently suggested by our and other studies to be involved in development of teratomas belonging to testicular germ cell tumours (TGCT). Teratomas can be experimentally obtained by cultivating gastrulating mouse embryos in vitro. EsiRNAs are constructs able to partake RNA interference pathway of the cell to induce mRNA degradation i.e. inhibit the expression of the genes of interest. The aim of this study was to determine whether the stemness genes Oct and Nanog have a role in teratoma development. Therefore, esiRNAs targeting the coding regions of mouse Nanog (transcript XM_132755.3), mouse Oct4 (transcript NM_013633.1) or GFP (132-591) were synthesized. 7, 5–days-old C3H mouse embryos were microsurgically isolated under the steromicroscope and cultivated at the air-liquid interface for 7 days in Eagle’s MEM with 50% rat serum. Specific esiRNA (5pmol esiRNA) in transfecting agent Lipofectamine 2000 (0, 25μl) were added to the culture medium. Embryos/teratomas were measured at the beginning of cultivation and for consequent 7 days. Data were analyzed by the One sample t-test. EsiGFP treated group served as a negative control since no esiGFP target in a mouse cell exists. Indeed, esiGFP group showed no statistically significant difference in growth when compared to controls. Teratomas developed from esiOct and especially esiNanog treated embryos showed statistically significant reduction in growth when compared to controls. By this experiment we confirmed crucial role of stemness genes Oct and Nanog in teratoma development and the utility of intervening in tumour growth by esiRNA treatment.

Temeljne medicinske znanosti