摘要
目的探讨SPON2对胃癌细胞的影响及其机制。方法收集2019-11-16-2021-2-24,在深圳市人民医院行胃癌根治术的7例原发性胃癌患者标本。蛋白质印迹法检测胃癌样本中SPON2、Cyclin D1和PCNA的表达,并对蛋白相对水平进行线性回归分析。以胃癌细胞株BGC-823和AGS为模型,转染SPON2 shRNA或SPON2 cDNA,检测SPON2对细胞增殖、细胞周期进程和DNA复制的影响。BGC-823细胞随机分成shNC组、shSPON2组、pcDNA-CCND1组、shSPON2+pcDNA-CCND1组,AGS细胞随机分成pcDNA-Vector组、pcDNA-SPON2组、shCTNNB1组、pcDNA-SPON2+shCTNNB1组、shCCND1组、pcDNA-SPON2+shCCND1组。蛋白质印迹法检测Cyclin D1蛋白表达水平,实时定量PCR检测Cyclin D1的mRNA水平,双荧光素酶报告基因实验检测转录因子与Cyclin D1启动子的结合。通过转染Cyclin D1 shRNA或Cyclin D1 cDNA,检测Cyclin D1对SPON2调控胃癌细胞增殖的影响。结果在BGC-823细胞中,shSPON2组细胞相对增殖能力为0.58±0.07,低于shNC组细胞的相对增殖能力(1.00±0.09),差异有统计学意义,t=5.061,P=0.007。在AGS细胞中,pcDNA-SPON2组细胞相对增殖能力为1.58±0.19,高于pcDNA-Vector组细胞的相对增殖能力(1.00±0.13),差异有统计学意义,t=3.463,P=0.026。此外,与shNC组相比,shSPON2组Cyclin D1蛋白表达降低,而与pcDNA-Vector组相比,pcDNA-SPON2组Cyclin D1蛋白表达增加。实时定量PCR实验结果显示,Cyclin D1 mRNA相对表达量在shSPON2组中(0.47±0.12)比shNC组中(1.00±0.11)低,差异有统计学意义,t=4.595,P=0.010。而Cyclin D1 mRNA相对表达量在pcDNA-SPON2组中(2.80±0.19)比pcDNA-Vector组中(1.00±0.17)高,差异有统计学意义,t=10.010,P<0.001。与shNC组[(100.00±7.38)%]相比,shSPON2组[(47.94±5.43)%]中Cyclin D1的转录活性降低,差异有统计学意义,t=8.036,P=0.001。而pcDNA-SPON2组[(216.92±26.51)%]与pcDNA-Vector组[(100.00±5.80)%]相比Cyclin D1的转录活性升高,差异有统计学意义,t=6.093,P=0.004。结论SPON2通过上调Cyclin D1的转录水平和蛋白表达促进胃癌细胞增殖。
Objective To explore the effects of SPON2 on gastric cancer cells and the related mechanisms.Methods Gastric cancer cells BGC-823 and AGS were transfected with SPON2 shRNA or SPON2 cDNA to detect the effects of SPON2 on cell proliferation,cell cycle progression and DNA replication.The protein expression level of Cyclin D1 was detected by Western Blot,the mRNA level of Cyclin D1 was detected by real-time quantitative PCR,and the binding of transcription factor and Cyclin D1 promoter was detected by double luciferase reporter gene experiment.BGC-823 cells were randomly divided into shNC group,shSPON2 group,pcDNA-CCND1 group,and shSPON2 plus pcDNA-CCND1 group;AGS cells were randomly divided into pcDNA-Vector group,pcDNA-SPON2 group,shCTNNB1 group,pcDNA-SPON2 plus shCTNNB1 group,shCCND1 group,and pcDNA-SPON2 plus shCCND1 group.Cyclin D1 shRNA or Cyclin D1 cDNA transfection was used to detect the effect of Cyclin D1 on SPON2 regulation of gastric cancer cell proliferation.Finally,western blot was used to detect the expression of SPON2,Cyclin D1 and PCNA in 7 patients with primary gastric cancer undergoing radical gastrectomy in Shenzhen People’s Hospital,and the relative protein levels were analyzed by linear regression analysis.Results In BGC-823cells,the relative proliferation capacity of shSPON2group cells were 0.58±0.07,which was lower than that of shNC group cells(1.00±0.09),and the difference was statistically significant,t=5.061,P=0.007.In AGS cells,the relative proliferation capacity of pcDNA-SPON2 group cells were 1.58±0.19,which was higher than that of the pcDNA-Vector group(1.00±0.13),and the difference was statistically significant,t=3.463,P=0.026.In addition,Cyclin D1 protein expression was decreased in the shSPON2 group compared with the shNC group,while Cyclin D1 protein expression was increased in the pcDNA-SPON2 group compared with the pcDNA-Vector group.Real-time quantitative PCR assay showed that the relative expression of Cyclin D1 mRNA was lower in shSPON2 group(0.47±0.12)than in the shNC group(1.00±0.11),with statistical significance,t=4.595,P=0.010.The relative expression of Cyclin D1 mRNA in pcDNA-SPON2 group(2.80±0.19)was higher than that in the pcDNA-Vector group(1.00±0.17),and the difference was statistically significant,t=10.010,P<0.001.Compared with the shNC group[(100.00±7.38)%],the transcriptional activity of Cyclin D1 in shSPON2 group[(47.94±5.43)%]was decreased,and the difference was statistically significant,t=8.036,P=0.001.The transcriptional activity of Cyclin D1 in pcDNASPON2 group[(216.92±26.51)%]was higher than that in the pcDNA-Vector group[(100.00±5.80)%],with statistical significance,t=6.093,P=0.004.Conclusion SPON2 can promote the proliferation of gastric cancer cells by upregulating the transcription of Cyclin D1.
作者
徐正磊
王立生
洪英财
廖碧红
俞志超
郭栗良子
XU Zheng-lei;WANG Li-sheng;HONG Ying-cai;LIAO Bi-hong;YU Zhi-chao;GUO Li-liang-zi(Department of gastroenterology,Shenzhen People’s Hospital,Shenzhen 518000,China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2022年第7期481-487,共7页
Chinese Journal of Cancer Prevention and Treatment
基金
广东省自然科学基金(2018A0303130278)。
