PHGDH对胃癌细胞BGC823增殖和化疗药物敏感性的影响  

The role of PHGDH on cell proliferation and chemotherapy sensitivity in gastric cancer

作　　者：李婷宇 郭昊[1] 刘海明[2] 易晓芳[1] 聂勇战[1] 樊代明[1] 

机构地区：[1]第四军医大学西京消化病医院.肿瘤生物学国家重点实验室,陕西西安710032 [2]吉林大学计算机与技术学院生物识别与信息安全技术研究室,吉林长春130012

出　　处：《中华肿瘤防治杂志》2017年第8期518-523,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81430072;61471181)

摘　　要：目的磷酸甘油酸脱氢酶(phosphoglycerate dehydrogenase,PHGDH)催化糖酵解中间产物3-磷酸甘油酸生成3-磷酸羟基丙酮酸,将糖酵解中间产物引入丝氨酸生物合成途径,它在包括乳腺癌和结肠癌等多种肿瘤中表达升高。本研究旨在探讨PHGDH的表达对胃癌细胞(BGC823)增殖以及对化疗药物顺铂敏感性的影响。方法免疫组织化学染色检测PHGDH蛋白在75例配对的胃癌和癌旁正常组织中的表达情况;siRNA转染胃癌细胞BGC823干扰PHGDH表达,蛋白质印迹法验证siRNA对PHGDH蛋白表达的干扰效率;CCK-8法和平板克隆实验检测PHGDH基因沉默对胃癌细胞BGC823增殖和克隆形成能力的影响;流式细胞术检测PHGDH基因沉默对BGC823细胞周期的影响;流式细胞术检测凋亡评估PHGDH基因沉默对胃癌细胞BGC823对化疗药物顺铂反应性的影响。结果 PHGDH在胃癌组织中的阳性表达率为72.0%(54/75),显著高于癌旁正常组织的41.3%(31/75),差异有统计学意义,χ~2=14.36,P<0.01;在转染siRNA后,实验组BGC823-siPHGDH细胞中的PHGDH/β-actin蛋白表达相对灰度比值为0.09±0.02,显著低于阴性对照组的0.70±0.05和空白对照组的0.74±0.06,差异有统计学意义,F=62.35,P<0.01;CCK-8实验结果显示,细胞铺板后5d,BGC823-Mock、BGC823-siNC和BGC823-siPHGDH细胞各组在第5天时BGC823细胞增长倍数分别为11.13±0.35、11.30±0.72和5.58±0.66,差异有统计学意义,F=48.61,P<0.01;平板克隆形成实验结果显示,细胞培养14d后,BGC823-Mock、BGC823-siNC和BGC823-siPHGDH细胞形成的克隆数分别为182.67±11.85、173.33±7.26和28.33±10.93,差异有统计学意义,F=71.85,P<0.01;细胞周期检测结果显示,阴性对照组BGC823-siNC的G_0/G_1期细胞百分比为(70.3±2.4)%,实验组BGC823-siPHGDH的G_0/G_1期细胞百分比为(87.5±1.3)%,说明实验组BGC823-siPHGDH的G_0/G_1期细胞增多,PHGDH基因沉默引起胃癌细胞发生G_0/G_1细胞周期阻滞;细胞凋亡检测结果显示,经1μg/mL的顺铂处�OBJECTIVE To investigate the expression and function of PHGDH in gastric cancer（GC）cell line BGC823.METHODS Immunohistochemistry（IHC）was used to detect the expression of PHGDH in GC specimens.SiRNA-mediated PHGDH knockdown efficiency in BGC823 cells was examined by western blotting.The proliferation rates of BGC823 were detected by CCK8 assay and colony formation assay.Cell cycle and apoptosis rate analysis were performed by flow cytometry.RESULTS IHC analysis showed PHGDH to be significantly overexpressed in 54 of 75GC tissues compared with 31 of 75 non-tumor gastric tissues（χ~2=14.36,P 0.01）.PHGDH siRNA was transfected into BGC823 cells,and significantly reduced PHGDH expression at protein levels（0.09±0.02,P〈0.01）,compared with negative control（0.70±0.05）and untreated control（0.74±0.06）.Cell proliferation of BGC823 was inhibited at 5dafter PHGDH knockdown（5.58±0.66,P〈0.01）,compared with negative control（11.30±0.72）and untreated control（11.13±0.35）.Plate colony formation assay showed that BGC823-siPHGDH cells formed fewer colonies（28.33±10.93,P〈0.01）,compared with negative control（173.33±7.26）and untreated control（182.67±11.85）.Cell cycle distribution analysis showed that the percentage of BGC823-siPHGDH cells at G_0/G_1 phase was（87.5±1.3）%（P〈0.01）,higher than（70.3±2.4）%in negative control BGC823-siNC cells,indicating that PHGDH knockdown induced G_0/G_1 phase cell cycle arrest in BGC823 cells.In addition,apoptosis rate assays showed that apoptosis rates of BGC823-siPHGDH cells were significantly increased [（45.0±5.1）%,P〈0.05]compared with negative control [（28.8±2.5）%]after 24 htreatment of cisplatin.CONCLUSIONS PHGDH is up-regulated in GC specimens.SiRNA-mediated PHGDH knockdown can suppress proliferation,induce cell cycle arrest and promote sensitivity to cisplatin in BGC823 cells.Our findings suggest that PHGDH might serve as a potential target for GC treatment.

关 键 词：胃癌 PHGDH基因 细胞增殖 顺铂 肿瘤代谢 

分 类 号：R735.2[医药卫生—肿瘤]