摘要
目的:探讨磷酸肌醇3激酶的p110α催化亚基(PIK3CA)对中性粒细胞抗子宫内膜癌(UCEC)细胞增殖的影响及其机制。方法:将PI3KCA过表达载体(pcDNA-PI3KCA)转染至UCEC细胞MFE-296后,与中性粒细胞dHL-60共培养24 h。取出培养下室的MFE-296细胞,CCK-8法检测MFE-296细胞增殖能力,RT-qPCR法检测MFE-296细胞内CEACAM1、β-catenin、血管内皮生长因子A(VEGF-A)、基质金属蛋白酶-9(MMP-9)和白细胞介素-8(IL-8)mRNA表达,Western blotting法检测PIK3CA、CEACAM1和β-catenin蛋白表达。应用癌症基因组图谱(TCGA)和UCEC mRNA-seq数据分析PIK3CA在UCEC中的表达。免疫荧光检测UCEC患者组织中PIK3CA和CEACAM1的定位,Spearman法分析74例UCEC组织中PIK3CA表达与CEACAM1表达的相关性。结果:转染pcDNA-PI3KCA后,MFE-296细胞PIK3CA mRNA及蛋白表达水平升高(P<0.05);与dHL-60共培养24 h后,过表达PIK3CA的MFE-296细胞中VEGF-A、MMP9和IL-8 mRNA表达及CEACAM1和β-catenin蛋白表达上调(均P<0.05)。MFE-296与dHL-60共培养24 h后MFE-296细胞增殖率降低,而过表达PIK3CA减弱了dHL-60细胞对MFE-296细胞的增殖抑制作用(均P<0.05),且anti-CEACAM1处理后,过表达PIK3CA的MFE-296细胞增殖率降低(P<0.05)。TCGA数据库分析显示,PIK3CA在UCEC组织中的表达水平低于正常组织(P<0.0001);Kaplan-Meier生存分析显示,PIK3CA高表达组生存率低于PIK3CA低表达组(P<0.05)。免疫荧光结果显示CEACAM1与PIK3CA蛋白在UCEC中的共定位,两者表达呈正相关关系(P<0.05)。结论:PIK3CA通过上调CEACAM1/β-catenin表达减弱中性粒细胞对UCEC细胞的增殖抑制作用。
Objective:To investigate the effects of P110αcatalytic subunit of phosphoinositol 3 kinase(PIK3CA)on the proliferation of neutrophil-resistant Uterine Corpus Endometrial Cancer(UCEC)cells and its mechanism.Methods:PI3KCA overexpression vector(pcDNA-PI3KCA)was transfected into UCEC cells MFE-296 and co-cultured with neutrophils dHL-60 for 24 h.PI3KCA overexpression vector(pcDNA-PI3KCA)was transfected into UCEC cells MFE-296 and co-cultured with neutrophils dHL-60 for 24 h.MFE-296 cells in the lower chamber of the culture were removed and the proliferation ability of MFE-296 cells was detected by CCK-8 assay,and RT-qPCR assay was performed to detect MFE-296 cells intracellular CEACAM1,β-catenin,vascular endothelial growth factor A(VEGF-A),and the mRNA expressions of matrix metalloproteinase-9(MMP-9)and interleukin-8(IL-8).Western blotting was used to detect PIK3CA,CEACAM1 andβ-catenin protein expressions.The Cancer Genome Atlas(TCGA)and UCEC mRNA-seq data were applied to analyze the expression of PIK3CA in UCEC.Immunofluorescence was used to detect the localization of PIK3CA and CEACAM1 in tissues of UCEC patient,and Spearman method was used to analyze the correlation between the expressions of PIK3CA and CEACAM1 in 74 cases of UCEC tissues.Results:After transfection with pcDNA-PI3KCA,PIK3CA mRNA and protein expression levels were elevated in MFE-296 cells(P<0.05);after co-culture with dHL-60 for 24 h,the expressions of VEGF-A,MMP9 and IL-8 mRNA in MFE-296 cells of overexpression PIK3CA and protein expressions of CEACAM1 and β-catenin were upregulated(all P<0.05).The proliferation rate of MFE-296 cells was reduced after 24 h co-culture with dHL-60,while overexpression of PIK3CA attenuated the inhibitory effect of dHL-60 cells on the proliferation of MFE-296 cells(P<0.05),and the proliferation rate of MFE-296 cells overexpressing PIK3CA was reduced after anti-CEACAM1 treatment(P<0.05).TCGA database analysis showed that the expression level of PIK3CA in UCEC tissues was lower than that in normal tissues(P<0.0001);Kaplan-Meier survival analysis showed that the survival rate of PIK3CA high expression group was lower than that of PIK3CA low expression group(P<0.05).Immunofluorescence results showed co-localization of CEACAM1 with PIK3CA protein in UCEC,and their expressions were positively correlated(P<0.05).Conclusions:PIK3CA attenuates the proliferation inhibitory effect of neutrophils on UCEC cells by upregulating CEACAM1/β-catenin expression.
作者
王发辉
邓青春
林佳佳
陈春妃
Wang Fahui;Deng Qingchun;Lin Jiajia;Chen Chunfei(Department of Gynecology,Second Affiliated Hospital of Hainan Medical College,Haikou 570311)
出处
《广西医科大学学报》
CAS
2022年第7期1086-1092,共7页
Journal of Guangxi Medical University
基金
海南省科协青年科技英才创新计划项目(No.QCXM202018)。
