摘要
目的 探讨hsa_circ_0010697在胃癌中的表达水平及发生、发展过程中可能的分子机制。方法 从美国生物技术信息中心数据库中查询hsa_circ_0010697基因序列,合成慢病毒载体;制备胃癌细胞SGC-7901。设置3组。对照组:胃癌细胞SGC-7901,未转染慢病毒载体。shRNA-NC组:空载体(慢病毒载体)转染胃癌细胞SGC-7901。hsa_circ_0010697-shRNA组:hsa_circ_0010697慢病毒载体转染胃癌细胞SGC-7901。采用qRT-PCR检测3组hsa_circ_0010697基因在胃癌细胞SGC-7901中的表达,采用CCK-8法检测3组细胞增殖活性[以波长为450 nm处的吸光度(A_(450))值表示],Tranwell实验检测3组细胞迁移和侵袭能力(穿透基质膜到达下室的细胞数量),Western blot检测3组细胞RAS-ERK信号通路上的RAS、细胞外调节蛋白激酶(ERK)1/2、细胞周期蛋白依赖性激酶4(CDK4)、细胞周期蛋白D1(Cyclin D1)、E2F转录因子1(E2F1)的蛋白质相对表达量。取6周龄雄性SPF级裸鼠6只,随机分入shRNA-NC组、hsa_circ_0010697-shRNA组,每组3只,检测两组裸鼠的体重和种瘤体积。结果 qRT-PCR结果显示,hsa_circ_0010697-shRNA组hsa_circ_0010697基因相对表达量为1.423±0.109,显著高于对照组的0.994±0.172和shRNA-NC组的1.006±0.137(P值均<0.05)。CCK-8法检测细胞增殖结果显示,hsa_circ_0010697-shRNA组细胞A_(450)值为0.757±0.051,显著低于对照组的1.000±0.044和shRNA-NC组的0.981±0.054(P值均<0.05)。Transwell实验结果显示,hsa_circ_0010697-shRNA组穿透基质膜到达下室的细胞数量为(93±11)个,显著少于对照组的(165±16)个和shRNA-NC组的(152±15)个(P值均<0.05)。Western blot结果显示,hsa_circ_0010697-shRNA组RAS-ERK信号通路上的RAS、ERK1/2、CDK4、Cyclin D1、E2F1蛋白质相对表达量显著高于对照组和shRNA-NC组(P值均<0.05)。裸鼠移植瘤模型实验结果显示,饲养12、18、22、24 d时,hsa_circ_0010697-shRNA组裸鼠的体重均显著大于shRNA-NC组同时间(P值均<0.05);饲养22、24 d时,hsa_circ_0010697-shRNA组裸鼠种瘤体积均显著小于shRNA-NC同时间(P值均<0.05)。结论 hsa_circ_0010697可以在胃癌诊治中作为一种潜在生物标志物和治疗靶点,为胃癌的药物靶向治策略提供了新思路。
Objective To investigate the expression of hsa_circ_0010697 in gastric cancer and its possible molecular mechanism in the development of gastric cancer.Methods The gene sequence of hsa_circ_0010697 was retrieved from the National Center for Biotechnology Information database,a lentiviral vector was synthesized,and gastric cancer cell line SGC-7901 was prepared.The cells were divided into 3 groups:control group(gastric cancer cell line SGC-7901 was not transfected with lentivirus vector),shRNA-NC group(gastric cancer cell line SGC-7901 was transfected with empty lentiviral vector),and hsa_circ_0010697-shRNA group(hsa_circ_0010697 lentivirus vector was transfected into gastric cancer cell line SGC-7901).qRT-PCR was used to detect the expression of hsa_circ_0010697 in gastric cancer cell line SGC-7901.The CCK-8 method was used to detect the cell proliferation(absorbance value at a wavelength of 450 nm,A_(450)).The Tranwell experiment was used to detect the migration and invasion of cells(the number of cells penetrating the matrix membrane to reach the lower chamber).Western blot was used to detect the relative expression of RAS,extracelluar regulated protein kinases(ERK)1/2,cyclin-dependent kinase 4(CDK4),Cyclin D1,and recombinant E2F transcription factor 1(E2F1)proteins in the RAS-ERK signaling pathway.Six 6-week-old male SPF nude mice were randomly divided into the shRNA-NC group and hsa_circ_0010697-shRNA group,with three mice in each group.The body weight and tumor volume of nude mice were measured.Results The relative expression level of genes in the hsa_circ_0010697 shRNA group was 1.423±0.109,which was significantly higher than that in the control group and shRNA-NC group(0.994±0.172,1.006±0.137,both P<0.05).The cell A_(450) value of the hsa_circ_0010697-shRNA group was 0.757±0.051,which was significantly lower than that in the control group and shRNA-NC group(1.000±0.044,0.981±0.054,both P<0.05).The number of cells penetrating the matrix membrane to reach the lower chamber in the hsa_circ_0010697-shRNA group was 93±11,which was significantly less than that in the control group and shRNA-NC group(165±16,152±15,both P<0.05).The relative expression levels of RAS,ERK1/2,CDK4,Cyclin D1,and E2F1 proteins in the RAS-ERK signaling pathway in the hsa_circ_0010697 shRNA group were significantly higher than those in the control group and shRNA-NC group(all P<0.05).On the day 12,18,22,and 24 of feeding,the body weight of nude mice in the hsa_circ_0010697 shRNA group was significantly higher than that in the shRNA-NC group(all P<0.05).On the day 22 and 24 of feeding,the tumor volume of nude mice in the hsa_circ_0010697 shRNA group was significantly smaller than that in the shRNA-NC(all P<0.05).Conclusion hsa_circ_0010697 can serve as a potential biomarker and therapeutic target in the diagnosis and treatment of gastric cancer,providing new ideas for targeted therapy for gastric cancer.
作者
谢富佳
翟娜娜
习城
孙亮
罗华友
XIE Fujia;ZHAI Nana;XI Cheng;SUN Liang;LUO Huayou(Department of Gastrointestinal Surgery,The First Affiliated Hospital of Kunming Medical University,Kunming 650000,Yunnan,China;不详)
出处
《上海医学》
CAS
2024年第4期238-244,共7页
Shanghai Medical Journal
基金
云南省科技厅昆医科技计划项目昆医联合专项(202001AY070001-146)
云南省教育厅科学研究基金项目(2019J1244)。
