摘要
目的:研究沉默Kelch样家族成员7(kelch like family member 7,KLHL7)基因表达对人胃癌细胞生物学表型(细胞增殖、凋亡及迁移)的影响,及可能的作用机制。方法:采用阿拉巴马大学伯明翰分校癌症数据分析数据库(The University of Alabamaat Birmingham Cancer Data Analysis Portal,UALCAN)和基因表达谱交互分析数据库(Gene Expression Profiling Interactive Analysis,GEPIA)分析KLHL7基因在人胃癌和癌旁组织中的差异表达以及与胃癌患者临床病理特征之间的关系,并采用Human Protein Atlas和Kaplan Meier法分析KLHL7基因异常表达对胃癌患者总生存期的影响。采用实时荧光定量PCR和蛋白质印迹法检测KLHL7在正常胃黏膜上皮细胞与不同分化胃癌细胞中的表达水平,随后用慢病毒感染的方法将携带有的shKLHL7的慢病毒载体转入胃癌细胞HGC-27和MKN45,使KLHL7基因沉默表达,再用实时荧光定量PCR和蛋白质印迹法检测沉默效率。MTT法检测细胞增殖能力,细胞划痕愈合实验和Transwell小室实验检测细胞的迁移及侵袭能力,FCM法检测细胞周期及凋亡率,最后用蛋白质印迹法检测细胞周期、凋亡、上皮-间质转化及磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB,又称AKT)通路相关蛋白的表达情况。采用BALB/c裸鼠构建转入shKLHL7的胃癌MKN45细胞的移植瘤模型,观察肿瘤在体内的生长情况,并使用实时荧光定量PCR、蛋白质印迹法及免疫组织化学法验证目的基因KLHL7的表达情况。结果:生信分析结果提示,KLHL7基因在人胃癌组织中的表达水平明显高于癌旁组织(P<0.05)。KLHL7高表达者的总生存期明显短于低表达者(P<0.05),且KLHL7高表达与胃癌患者的分级、分期、幽门螺旋杆菌感染、临床病理组织亚型及淋巴结转移等密切相关。KLHL7在各组胃癌细胞中mRNA和蛋白的表达水平均高于正常胃黏膜上皮细胞GES-1(P均<0.05)。沉默KLHL7基因表达后,细胞增殖、侵袭和迁移能力明显降低(P<0.05),凋亡率提高(P<0.01),p21、Bax和E-钙黏蛋白(E-cadherin)表达水平升高,而周期蛋白依赖激酶4(cyclin dependent kinase 4,CDK4)、CDK6、Bcl-2、波形蛋白(vimentin)、PI3K、AKT、磷酸化的PI3K和AKT表达水平下调(P均<0.05)。体内研究提示,KLHL7沉默后小鼠肿瘤体积和质量均明显降低(P均<0.01),实时荧光定量PCR、蛋白质印迹法及免疫组织化学法检测结果表明,shRNA-KLHL7可使组织内KLHL7表达明显下调(P均<0.01)。结论:沉默KLHL7基因表达可抑制人胃癌细胞的增殖、侵袭迁移能力,促进细胞凋亡并将细胞周期阻滞在G1期,其作用机制可能与抑制PI3KAKT信号通路的磷酸化有关。
Objective:To investigate the effect of kelch like family member 7(KLHL7)gene silencing on the biological phenotype of human gastric cancer cells and its underlying molecular mechanism.Methods:Bioinformatics databasesThe University of Alabama at Birmingham Cancer Data Analysis Portal(UALCAN)and Gene Expression Profiling Interactive Analysis(GEPIA)were used to analyze the differential expression of KLHL7 gene in human gastric cancer and paracancerous tissues,and the relationship between KLHL7 gene expression and the clinicopathological characteristics of gastric cancer patients.Human Protein Atlas and Kaplan-Meier databases were used to analyze the effect of abnormal KLHL7 gene expression on the overall survival time of gastric cancer patients.The differential expression of KLHL7 in normal gastric epithelial cells and gastric cancer cells with different differentiation status was analyzed by quantitative reverse transcriptase-mediated PCR(qRT-PCR)and Western blotting.The expression of KLHL7gene was silenced in gastric cancer cell lines HGC-27 and MKN45by shKLHL7with a lentiviral vector,and the silencing efficiency was verified by RT-qPCR and Western blotting.The cell proliferation was analyzed by MTT assay,cell migration and invasion wasanalyzed by scratch assay and transwell assay,cell cycle and apoptosis were analyzed by FCM assay,and the expression of cell cycle,apoptosis,epithelial mesenchymal transformation and phosphoinositide 3-kinase(PI3K)/protein kinase B(PKBor AKT)pathway related proteins were analyzed by Western blotting.The mouse modelof xenotransplantation was established by inoculation of MKN45 cells into BALB/c nude mice,andthe tumor growth in vivo was recorded,and the expression of the target gene KLHL7 was verified by qRT-PCR,Western blotting and immunohistochemistry.Results:The expression of KLHL7 gene in human gastric cancer tissues was significantly higher than that in paracancerous tissues(P<0.05).Patients with high KLHL7 expression had a significant shorter overall survival time than those with low KLHL7 expression(P<0.05),and high KLHL7expression was closely related to the grade,stage,Hp infection status,clinicopathologic tissue subtypes and lymph node metastasis of gastric cancer patients.The expression levels of KLHL7 mRNA and protein in gastric cancer cell lines were significantly higher than those in the normal gastric epithelial cells GES-1(P<0.05).After KLHL7 silencing,the cell proliferation,invasion and migration were significantly suppressed(P<0.05)whereas the apoptosis rate was increased(P<0.01);the expression levels of P21,Bax and E-cherherin were increased whereas those of cyclin dependent kinase 4(CDK4),CDK6,Bcl-2,vimentin,PI3K,AKT,p-PI3K and p-AKT decreased(all P<0.05).Thein vivo experimental results showed that the volume and weight of the tumor were significantly decreased afterKLHL7silencing o(all P<0.01).qRT-PCR,Western blotting and immunohistochemistry results showed that shRNA-KLHL7 could significantly down-regulate the expression of KLHL7 in the tissue(all P<0.01).Conclusion:KLHL7 silencing can inhibit the proliferation,invasion and migration whilepromote the apoptosis of human gastric cancer cells.KLHL7 silencingcan alsoarrest the human gastric cancer cellsatthe G1 phase of cell cycle.The participation of KLHL7in the above phenotypic changes may be related to the inhibition of the phosphorylation of the PI3K-AKT signaling pathway.
作者
王转兄
李海龙
吴建军
呼永华
许丽丽
何华
韩静
WANG Zhuangxiong;LI Hailong;WU Jianjun;HU Yonghua;XU Lili;HE Hua;HAN Jing(Department of Scientific Research Management,Affiliated Hospital of Gansu University of Traditional Chinese Medicine,Lanzhou 730000,Gansu Province,China;The First Clinical College of Gansu University of Chinese Medicine,Lanzhou 730000,Gansu Province,China;Gansu Province Key Laboratory of TCM Recipe Mining and Innovation Transformation,Key Laboratory of Pharmacology and Toxicology of Traditional Chinese Medicine of Gansu Province,Lanzhou 730000,Gansu Province,China;School of Public Health,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,Gansu Province,China;Gansu Tumor Research Center of Integrated Traditional Chinese and Western Medicine,Lanzhou 730000,Gansu Province,China)
出处
《肿瘤》
CAS
CSCD
北大核心
2022年第4期265-284,共20页
Tumor
基金
甘肃省自然科学基金(20JR5RA189)
甘肃省卫生行业计划(GSWSKY2021-011)
