关键词: Hax1, siRNA:293细胞, 凋亡

Abstract: Objective To construct the Hax1 gene specific siRNA (sihax), to detect sihax mediated Hax1 gene repression, and to study the influence of Hax1 and Hax1 siRNAs on cell apoptosis. Method The oligo sequences of Hax1 siRNAs are selected, and the constructed eukaryotic expression vectors are transfected into 293 cells. The influence of Hax1 and its siRNAs on the cell apoptosis is evaluated by a number of approaches such as inverted fluorescence microscope, flow cytometric analysis, annexinV-EGFP/PI double staining, and standard RT-PCR. Results Hax1 specific siRNAs, pBS/U6-sihaxA and pBS/U6-sihaxB, have been successfully constructed. Analysis by RT-PCR and inverted fluorescence microscope demonstrate that the inhibitory effect induced by sihaxB is stronger than that of sihaxA. Over-expression of Hax1 significantly inhibits the early apoptosis of 293 cells. Inhibiting the endogenous Hax1 gene expression by by sihaxA and sihaxB markedly increases late apoptotic cells from 9.03?0.473% to10.8?0.513% (p<0.05) and 16.6?0.858% (p<0.01), respectively. In the same time, sihaxB also can reduce the early apoptotic cells from 37.3?0.5% to 32?1.77% (p<0.01). Conclusion The sequence conservation of the targeting site might influence the inhibitory effect induced by Hax1 siRNA. Hax1 gene might actively involve in the multiple regulatory processes related to cell apoptosis. This study provides some valuable information for the functional study on Hax1 gene in the future.