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

Stable G6PD knockdown inhibits the migration of renal cell carcinoma cells

目的肾透明细胞癌(ccRCC)是一种代谢性疾病,葡萄糖-6-磷酸脱氢酶(G6PD)与ccRCC的发生发展及患者的不良预后呈正相关。文章首次构建了G6PD缺陷型ccRCC稳定细胞株,检测其迁移表型。方法 Oligo Engine RNAi设计针对人G6PD基因3'端非编码区的siRNA及无关siRNA序列,合成双链后通过BglⅡ和HindⅢ酶切位点插入p SP-GFP/Neo表达载体,构建Caki-1-G6PD siRNA细胞株及Caki-1-Negative control细胞株,进而转染并经G418筛选。应用Real-time RT-PCR、Western blot及酶活性方法,检测细胞株的G6PD表达和酶活性;Transwell方法检测细胞迁移表型、Western blot检测p-STAT3/STAT3表达。结果荧光显微镜下可见Caki-1-G6PD siRNA细胞与Caki-1-Negative control细胞形态良好,通过GFP表达量计算细胞转染效率约为45%~55%;48 h后细胞贴壁密度达90%,荧光表达略有增强,转染效率可达60%。光镜和荧光显微镜下亦可见细胞状态良好,GFP阳性率达95%。与Caki-1-Negative control细胞相比,Caki-1-G6PD siRNA细胞的G6PD mRNA表达量蛋白表达量和酶活性均显著降低(P<0.05)。与Caki-1-Negative control细胞相比,Caki-1-G6PD siRNA迁移细胞的相对数量明显降低[(64.0±4.2)个vs(30.0±2.9)个,P<0.01],p-STAT3/STAT3值亦明显下降[(0.45±0.05)vs(0.24±0.01),P<0.01]。结论文章首次成功构建了G6PD稳定敲低和迁移能力低下的Caki-1细胞系,其G6PD敲低所致ccRCC细胞迁移能力降低与STAT3信号相关。

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（G6 PD）with the development,progression and poor prognosis of ccRCC. In this study,we first established a G6 PD defect ccRCC stable cell line,detected the influence of G6 PD knockdown on ccRCC migration,and provided a cell model for further studies on the functional and molecular mechanisms of G6 PD in ccRCC. Methods Using the OligoEngine RNAi software, we designed siRNA targeting the human G6 PD gene 3＇ non-coding region and negative control siRNA sequences,inserted the double-stranded siRNA into the p SR-GFP/Neo expression vector through Bgl Ⅱ and Hind Ⅲ enzyme loci,and constructed Caki-1-G6 PD 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 G6 PD 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-G6 PD 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-G6 PD siRNA cells showed significant decreases in the expressions of Caki-1-G6 PD 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-G6 PD siRNA cell line with stable G6 PD knockdown and a lower migration ability was first successfully constructed,and the decreased migration ability induced by G6 PD knockdown is associated with the STAT3 signal,which is contributive to an insight into the functional and molecular mechanisms of G6 PD in the development and progression of ccRCC as well as to finding intervention targets for the treatment of ccRCC.