Stable G6PD knockdown inhibits the migration of renal cell carcinoma cells
10.16571/j.cnki.1008-8199.2018.07.006
- VernacularTitle: 葡萄糖-6-磷酸脱氢酶稳定敲低对肾透明细胞癌细胞迁移的抑制作用
- Author:
Hong-gang BAI
1
;
Qiao ZHANG
1
;
Ying-min KUANG
2
;
Zhe YANG
;
Qiao-qiao HAN
1
;
Yan-ling WANG
1
;
Yue-chun ZHU
1
Author Information
1. Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Kunming Medical University, Kunming 650500, Yunnan, China
2. Department of Organ Transplantation, 3
- Publication Type:Journal Article
- Keywords:
glucose-6-phosphate dehydrogenase;
clear cell renal cell carcinoma;
stable cell line
- From:
Journal of Medical Postgraduates
2018;31(7):697-702
- CountryChina
- Language:Chinese
-
Abstract:
Objective Clear cell renal cell carcinoma (ccRCC) accounts for more than 80% of malignant kidney tumors and its pathogenesis has not been elucidated. Our previous studies showed a positive correlation of Glucose-6-phosphate dehydrogenase (G6PD) with the development, progression and poor prognosis of ccRCC. In this study, we first established a G6PD defect ccRCC stable cell line, detected the influence of G6PD knockdown on ccRCC migration, and provided a cell model for further studies on the functional and molecular mechanisms of G6PD in ccRCC.Methods Using the OligoEngine RNAi software, we designed siRNA targeting the human G6PD gene 3′ non-coding region and negative control siRNA sequences, inserted the double-stranded siRNA into the pSR-GFP/Neo expression vector through Bgl Ⅱ and Hind Ⅲ enzyme loci, and constructed Caki-1-G6PD siRNA and Caki-1-negative control cell lines, followed by transfection and G418 screening of the Caki-1 cells. We measured the expression and enzyme activity of G6PD in the cells by real-time RT-PCR, determined the cell migration phenotypes by Transwell assay, and detected the expressions of p-STAT3 and STAT3 by Western blot.Results Morphologically normal Caki-1-G6PD siRNA and Caki-1-negative control cells were seen under the fluorescence microscope. With GFP expression as a marker, the transfection efficiency rate of the cells was 45－55%. The density of the adherent cells at 48 hours was 90% and their transfection efficiency rate was over 60%. Compared with the Caki-1-negative control cells, the Caki-1-G6PD siRNA cells showed significant decreases in the expressions of Caki-1-G6PD mRNA and protein (P<0.01), enzyme activity (P<0.05), relative count of migratory cells (64.0±4.2 vs 30.0±2.9, P<0.01), and the ratio of p-STAT3/STAT3 (0.45±0.05 vs 0.24±0.01, P<0.01).Conclusion The Caki-1-G6PD siRNA cell line with stable G6PD knockdown and a lower migration ability was first successfully constructed, and the decreased migration ability induced by G6PD knockdown is associated with the STAT3 signal, which is contributive to an insight into the functional and molecular mechanisms of G6PD in the development and progression of ccRCC as well as to finding intervention targets for the treatment of ccRCC.
