Effects of Silencing G6PD Expression on The Growth and Apoptosis in Human Skin Melanoma

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 0.05), compared with A375-WT cell. FCM analysis indicated that apoptosis cell in A375-G6PD△ was 2.86 times as that of A375-WT(P 0.01) with 33.8 % increase of SPF(P 0.05), 59.7 % raise of PI(P 0.01), and 27.7 % decrease of G0/G1 phase(P 0.01). Furthermore, apoptosis-associated protein check showed that Caspase-3 was 2.86 times as that of A375-WT(P 0.01) with 54.7% descend of P53(P 0.01). It is proposed that G6PD can maintain the growth and proliferation of A375 cell. G6PD deficiency probably restrains the change proceeding of G2/M phase to G0/G1 phase in A375△ cell cycle through down-regulation P53 expression and up-regulation Caspase-3 expression. The role and mechanism of G6PD in cell growth, proliferation, and differentiation of tumor cells need to be further investigated.