RESUMO
Clear cell renal cell carcinoma (ccRCC) has been proved to be a metabolic disease with high
RESUMO
Acetyl-CoA carboxylase (ACC) is the rate limiting enzyme of fatty acid synthesis pathway. Studies have shown that ACC1 is implicated in a variety of metabolic diseases and cancer. However, the role and mechanism of action of ACC1 in clear cell renal cell carcinoma (ccRCC) have not been reported. In this study, 786-O and Caki-1 clear cell renal carcinoma cells were used as research objects to investigate the effect of abnormal expression of ACC1 on their proliferation and unravel the underlying mechanism. Red oil-O-staining results showed that the lipid content of 786-O and Caki-1 cells was significantly higher than that of human kidney 2 (HK2) cells. By searching TCGA database, we found that the expression of ACC1 proteins in ccRCC was significantly higher than that in normal renal tissues (P < 0.001). Plus, ACC1 protein expression in all clinical TNM stages was significantly higher than that in normal tissues, and the higher the expression of ACC1, the higher the pathological grade. Furthermore, high expression of ACC1 mRNA is positively correlated with poor prognosis in ccRCC patients. Western blotting analysis showed that the expression of ACC1 in 786-O and Caki-1 cells was significantly higher than that in HK2 cells. The results of red oil-O-staining showed that knocking down ACC1 could significantly reduce the lipid content of 786-O and Caki-1 cells. The results of CCK-8 assays and clonogenicity analysis showed that knocking down ACC1 could significantly reduce the proliferation and colony forming ability of 786-O and Caki-1 cells. Flow cytometry analysis showed that after knocking down ACC1, the cell cycle was blocked at the G
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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.