Effects of Silencing G6PD Expression on The Growth and Apoptosis in Human Skin Melanoma
- VernacularTitle:siRNA沉默G6PD表达对人皮肤黑色素瘤细胞生长及凋亡的影响
- Author:
Yuechun ZHU
;
Huiru LU
;
Danyi LI
;
Shufen TONG
- Publication Type:Journal Article
- Keywords:
siRNA interference, glucose-6-phosphate dehydrogenase, human skin melanoma, stable cell strain, cell cycle, P53, Caspase-3
- From:
Progress in Biochemistry and Biophysics
2006;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
Glucose-6-phosphate dehydrogenase (G6PD) derives from the expression of the house-keeping gene G6PD. Recent studies have indicated that G6PD is related to tumor genesis, growth, clinical phenotype, therapy, and prognosis. To elucidate the relationship between G6PD and cancer, three siRNA sequences and one negative control sequence were designed based on the 3' noncoding region of the human G6PD gene. Two complementary single-strand DNA (sense and antisense) were designed and synthesized based on siRNA sequences. The DNA fragments were annealed and ligated to the GFP expression vector pRNAT-U6.2/Lenti. One siRNA with higher interference efficiency than the other two was found after siRNA plasmid transfecting human skin A375 melanoma cells. After lentivirus particle packaging and virus production, the A375 cells were infected, and the single cell clone was acquired and cultured to establish the stable cell strain. Western blotting showed that the endogenous G6PD in the stable A375 cell strain was 0.257 ? 0.074, which was 11.17% of G6PD expression (2.301 ? 0.286) in wild type A375 cells. The final siRNA interference efficiency in this stable cell strain was 88.83%. The G6PD activity of A375-G6PD?驻 was 21.53% of A375-WT. Further study showed that A375-G6PD△ doubling generation time prolonged, and its proliferation was greatly inhibited and the cloning efficiency lowered 25%(P
