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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的顺铂处理BGC823-siNC和BGC823-siPHGDH细胞24h后,BGC823-siNC凋亡率为(28.8±2.5)%,BGC823-siPHGDH组为(45.0±5.1)%,提示PHGDH基因沉默能够增加胃癌细胞BGC823对顺铂的敏感性。结论 PHGDH基因沉默抑制胃癌细胞BGC823的增殖,引起细胞周期阻滞,增加对化疗药物顺铂的敏感性,这不仅为胃癌临床治疗提供一个新的靶点,而且为肿瘤代谢在肿瘤发生和发展中的作用提供了新的实验证据。
OBJECTIVE Phosphoglycerate dehydrogenase (PHGDH) catalyzes the glycolytic hydrolysis of 3-phosphoglycerate into 3-phosphohydroxypyruvate and introduces the glycolytic intermediate into the serine biosynthesis pathway. Colon cancer and other tumors increased expression. The purpose of this study was to investigate the effects of PHGDH on the proliferation of gastric cancer cells (BGC823) and on the chemosensitivity to cisplatin. Methods The expression of PHGDH protein in 75 matched gastric cancer tissues and adjacent normal tissues was detected by immunohistochemical staining. The expression of PHGDH protein was detected by siRNA transfection in gastric cancer cell line BGC823. The interference efficiency of siRNA on PHGDH protein expression was verified by Western blotting. The effect of PHGDH gene silencing on the proliferation and clonality of gastric cancer cell BGC823 was detected by ELISA and plate cloning assay. The effect of PHGDH gene silencing on the cell cycle of BGC823 cells was detected by flow cytometry. Flow cytometry was used to detect the apoptosis. Effect of BGC823 on Cisplatin Reactivity of Chemotherapy Drugs. Results The positive rate of PHGDH in gastric cancer tissues was 72.0% (54/75), which was significantly higher than that in normal tissues (41.3%, 31/75), the difference was statistically significant (χ ~ 2 = 14.36, P <0.01) ; The relative gray value of PHGDH / β-actin in BGC823-siPHGDH cells transfected with siRNA was 0.09 ± 0.02, significantly lower than that in negative control group (0.70 ± 0.05) and blank control group (0.74 ± 0.06) The difference was statistically significant (F = 62.35, P <0.01). The results of CCK-8 assay showed that at day 5 after plating, the growth of BGC823 cells in BGC823-Mock, BGC823-siNC and BGC823-siPHGDH cells were 11.13 ± 0.35, 11.30 ± 0.72 and 5.58 ± 0.66 respectively, F = 48.61, P <0.01. The results of plate clone formation assay showed that after cultured for 14 days, BGC823-Mock, BGC823-siNC and BGC823-siPHGDH cells The number of colony formed was 182.67 ± 11.85,173.33 ± 7.26 and 28.33 ± 10.93, respectively, the difference was statistically significant (F = 71.85, P <0.01). The results of cell cycle assay showed that the G0 / G1 phase cells of BGC823- (70.3 ± 2.4)%, the percentage of G0 / G1 phase cells in experimental group BGC823-siPHGDH was (87.5 ± 1.3)%, which indicated that the experimental group BGC82 3-siPHGDH G0 / G1 phase cells increased, PHGDH gene silencing caused G_0 / G1 cell cycle arrest in gastric cancer cells. The results of apoptosis showed that BGC823-siNC and BGC823-siPHGDH cells were treated with 1μg / mL cisplatin 24h (28.8 ± 2.5)% in BGC823-siNC group and (45.0 ± 5.1)% in BGC823-siPHGDH group, suggesting that PHGDH gene silencing can increase the sensitivity of gastric cancer cell BGC823 to cisplatin. Conclusion Silencing PHGDH gene inhibits the proliferation of gastric cancer cell line BGC823, induces cell cycle arrest and increases sensitivity to chemotherapeutic drug cisplatin, which not only provides a new target for clinical treatment of gastric cancer but also plays an important role in tumorigenesis and progression of tumor The role provided a new experimental evidence.