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通过RNA干扰技术沉默端粒酶关键组分WDR79的表达,采用PCR法、Western blot、免疫荧光共聚焦、Southern blot、MTT、流式细胞术(FCM)分别检测WDR79 siRNA瞬时转染A549细胞后WDR79基因和蛋白的表达水平、端粒酶和端粒的结合情况和端粒长度变化、细胞生长增殖变化、细胞周期的变化及细胞凋亡率,探讨该基因的沉默表达对A549细胞的抑制作用。结果表明,WDR79 siRNA瞬时转染A549细胞显著降低WDR79 mRNA及蛋白水平(P<0.05),端粒和端粒酶的结合水平明显减少(P<0.05),细胞增殖的活力被抑制(P<0.05),干扰后停留在S期的细胞减少(P<0.05),停留在G1期的细胞增多(P<0.05)。提示WDR79 siRNA的瞬时转染可显著降低WDR79基因及蛋白的表达,并有抑制肺腺癌A549细胞的生物学效应,进一步探索了WDR79基因在肿瘤发生发展中的重要作用,并在为肺癌的治疗中寻找更有效的靶点提供了实验证据。
The expression of WDR79, a key component of telomerase, was silenced by RNA interference. The WDR79 siRNA was transiently transfected into A549 cells by PCR, Western blot, immunofluorescence confocal, Southern blot, MTT and flow cytometry (FCM) Gene and protein expression level, telomerase and telomere binding and telomere length changes, cell proliferation and proliferation changes, cell cycle changes and apoptosis rate, to explore the gene silencing expression of A549 cells. The results showed that WDR79 siRNA transiently transfected A549 cells significantly reduced the WDR79 mRNA and protein levels (P <0.05), the binding of telomere and telomerase was significantly decreased (P <0.05), and the cell proliferation activity was inhibited (P <0.05 ), The number of cells staying in S phase decreased after interference (P <0.05), and the number of cells staying in G1 phase increased (P <0.05). These results suggest that the transient transfection of WDR79 siRNA can significantly reduce the expression of WDR79 gene and protein and inhibit the biological effect of A549 cells. Further explore the important role of WDR79 gene in tumorigenesis, and in the treatment of lung cancer In finding more effective target provides experimental evidence.