甘油-3-磷酸脱氢酶1-样酶通过调节细胞凋亡对肾透明细胞癌的影响

Effect of glycerol-3-phosphate dehydrogenase 1 like on clear renal cell carcinoma via regulating apoptosis

目的探讨甘油-3-磷酸脱氢酶1-样酶(GPD1L)对肾透明细胞癌的影响及其机制。方法选择来自肿瘤基因组图谱(TCGA)和基因表达综合(GEO)的两个主要的微阵列数据库(GSE16441、GSE36895、GSE53757、GSE66270、GSE68417), 探讨在肾透明细胞癌(ccRCC)肿瘤样本中低表达基因, 并进行生存分析。选择与患者生存差异最显著的基因GPD1L进行体外验证。体外培养正常人肾小管上皮细胞HK-2及ACHN、786-O、769-P 3种人肾细胞癌细胞株, 利用聚合酶链反应(PCR)和Western blot验证GPD1L在肾癌细胞株中的表达量。利用GSE16441微阵列数据进行GPD1L的单基因富集分析, 观察GPD1L参与调控的相关通路。随后利用pcDNA3.1过表达质粒, 过表达ACHN、769-P肾癌细胞中GPD1L的表达, 利用划痕、原位缺口末端标记法(TUNEL)、流式细胞术和Western blot探讨GPD1L对肾癌细胞迁移、凋亡的影响。多组间比较采用单因素方差分析, 两两比较采用LSD-t检验。结果对5个ccRCC微阵列数据集的综合分析确定了ccRCC中SLC4A1、GPD1L、CLCNKB 3个显著的低表达基因, 并且这3个基因的表达量均与患者的生存时间呈正相关(P<0.05)。PCR和Western blot结果表明, 在ACHN、786-O、769-P 3种细胞株中, GPD1L mRNA和蛋白的表达水平显著低于HK-2细胞(转录表达值:0.163±0.067比1.000±0.124, t=7.838, P<0.01;0.338±0.171比1.000±0.124, t=6.982, P<0.01;0.626±0.147比1.000±0.124, t=5.632, P<0.05)。Western blot结果显示, 在ACHN、769-P肾癌细胞中, pcDNA-GPD1L组GPD1L蛋白的表达量明显高于pcDNA-Vector组(条带相对灰度值:2.343±0.371比1.000±0.122, t=5.776, P<0.01;3.207±0.537比1.000±0.082, t=6.224, P<0.01);肾癌样本中GPD1L单基因GSEA富集分析显示凋亡相关通路被明显抑制。同时, pcDNA-GPD1L组细胞凋亡率明显高于pcDNA-Vector组[流式:(30.14±1.05)%比(5.20±1.21)%, t=7.775, P<0.01;(28.54±2.13)%比(3.44±1.34)%, t=6.862, P<0.01], 划痕结果也显示pcDNA-GPD1L组细胞增殖能力明显低于pcDNA-Vector组。结论过表达GPD1L能够通过激活肾透明细胞癌内的细胞凋亡, 而抑制肾透明细胞癌的发展。

Objective To investigate the effect of glycerol-3-phosphate dehydrogenase 1 like(GPD1L)on clear renal cell carcinoma(ccRCC)and its mechanism.Methods Two major microarray databases from the Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO,GSE16441,GSE36895,GSE53757,GSE66270,GSE68417)were selected to explore the low expressed genes in the tumor samples of ccRCC,and survival analysis was performed.Normal human renal tubular epithelial cells HK-2 and three human renal cell carcinoma cell lines ACHN,786-O and 769-P were cultured in vitro.The expression of GPD1L in renal cell carcinoma cell lines was verified by polymerase chain reaction(PCR)and Western blotting.The single gene enrichment analysis of GPD1L was performed by GSE16441 microarray data to observe the related pathways involved in the regulation of GPD1L.Subsequently,the expression of GPD1L in ACHN and 769-P kidney cancer cells was overexpressed using pcDNA3.1 overexpression plasmid,and the effect of GPD1L on migration and apoptosis of kidney cancer cells was explored using scratch,terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL),flow cytometry and Western blotting.Data were expressed as mean±standard deviation.One-way ANOVA was used for comparison between multiple groups,and LSD-t test was used for two-way comparison.P<0.05 was considered statistically significant difference.Results Comprehensive analysis of five ccRCC microarray datasets identified three significant low expression genes,SLC4A1,GPD1L and CLCNKB,and the expression levels of these three genes were positively correlated with the survival time of patients(P<0.05).The most significant difference in survival time,GPD1L,was selected for in vitro validation,and PCR and Western blotting results showed that the expression levels of GPD1L mRNA and protein were significantly lower in ACHN,786-O,and 769-P cell lines than in HK-2 cells(PCR:0.163±0.067 vs.1.000±0.124,t=7.838,P<0.01;0.338±0.171 vs.1.000±0.124,t=6.982,P<0.01;0.626±0.147 vs.1.000±0.124,t=5.632,P<0.05).Western blotting results showed that in ACHN,769-P renal cell carcinoma cells,the expression of GPD1L protein was significantly higher in the pcDNA-GPD1L group than in the pcDNA-vector group(relative grayscale value of blots:2.343±0.371 vs.1.000±0.122,t=5.776,P<0.01;3.207±0.537 vs.1.000±0.082,t=6.224,P<0.01).GPD1L single gene GSEA enrichment analysis showed that apoptosis-related pathways were significantly inhibited.Compared with pcDNA-vector group,the apoptosis rate of pcDNA-GPD1L group increased significantly[(30.14±1.05)%vs.(5.20±1.21)%,t=7.775,P<0.01;(28.54±2.13)%vs.(3.44±1.34)%,t=6.862,P<0.01].Meanwhile,the scratch results also showed that the cell proliferation ability in pcDNA-GPD1L group was significantly lower than that in pcDNA-Vector group.Conclusion Overexpression of GPD1L can inhibit the development of ccRCC via activating apoptosis pathway.