子宫平滑肌肉瘤组织EphA2/EphrinA1表达及沉默EphA2对肿瘤细胞生长与RelA/p65磷酸化的影响  

EphA2/EphrinA1 expression in uterine leiomyosarcoma and the effect of silencing EphA2 on tumor cell growth and RelA/p65 phosphorylation

作　　者：邓卉[1] 陈晓燕[1] 严园 魏征 肖凤莲 余晓 DENG Hui;CHEN Xiaoyan;YAN Yuan;WEI Zheng;XIAO Fenglian;YU Xiao(Department of Gynecology,Chongqing Hospital of Traditional Chinese Medicine,Chongqing 410000,China;Department of Obstetrics and Gynecology,Jiulongpo District Maternity and Child Health Hospital,Chongqing 400000,China)

机构地区：[1]重庆市中医院妇科,重庆400011 [2]重庆市九龙坡区妇幼保健院妇产科,重庆400051

出　　处：《西部医学》2022年第5期644-651,共8页Medical Journal of West China

基　　金：重庆市自然科学基金面上项目(cstc2019jcyj-msxmx0749)。

摘　　要：目的探究生促红素肝细胞受体A2(EphA2)及其配体(EphrinA1)在子宫平滑肌肉瘤组织中的表达情况,通过沉默EphA2探究其对肿瘤细胞生长及RelA/p65磷酸化的影响。方法以重庆市中医院28例子宫平滑肌肉瘤患者为研究对象,免疫组织化学染色和实时荧光定量PCR(qRT-PCR)检测子宫平滑肌肉瘤与瘤旁组织中EphA2、EphrinA1mRNA表达;通过脂质体介导法将EphA2-siRNA转染至SK-UT-1细胞以沉默EphA2表达,同时以正常培养的细胞作为正常组、转染NC-siRNA的细胞作为阴性对照组(NC-siRNA组);qRT-PCR和Western blot检测细胞中EphA2表达,以确定转染效率;CCK-8法、Transwell小室实验检测细胞增殖活性、迁移与侵袭;TUNEL染色和流式细胞术检测细胞凋亡情况;免疫荧光染色检测细胞内p65表达变化;Western blot检测RelA/p65磷酸化位点蛋白表达水平。结果子宫平滑肌肉瘤组织中EphA2、EphrinA1 mRNA与蛋白表达水平均显著高于瘤旁组织(P<0.05);与空白对照组比较,EphA2-siRNA组细胞在转染24、48及72 h后的增殖活性下降,细胞迁移与侵袭数目均减少,TUNEL阳性染色细胞率增加,细胞凋亡率增加,p65绿色荧光强度明显减弱,同时,p53蛋白表达升高,RelA/p65 Ser536和RelA/p65 Ser276磷酸化蛋白水平降低,差异均具有统计学意义(P<0.05)。结论EphA2与EphrinA1在子宫平滑肌肉瘤组织中呈高表达,靶向沉默EphA2表达可抑制子宫平滑肌肉瘤细胞增殖与转移,诱导细胞发生凋亡,并抑制RelA/p65的Ser536和Ser276位点磷酸化。Objective To explore the expression of erythropoietin hepatocyte receptor A2(EphA2)and its ligand(EphrinA1)in uterine leiomyosarcoma tissues,and explore its effect on tumor cell growth and RelA/p65 phosphorylation by silencing EphA2 Impact.Methods 28 cases of uterine leiomyosarcoma patients from Chongqing Hospital of Traditional Chinese Medicine as the research object,immunohistochemical staining and real-time fluorescent quantitative PCR(qRT-PCR)were used to detect the expression of EphA2 and EphrinA1mRNA in uterine leiomyosarcoma and adjacent tissues.EphA2-siRNA was transfected into SK-UT-1 cells by liposome-mediated method to silence EphA2 expression.At the same time,normal cultured cells were used as the normal group,and NC-siRNA transfected cells were used as the negative control group(NC-siRNA group).qRT-PCR and Western blot detection of EphA2 expression in cells to determine transfection efficiency;CCK-8 method,Transwell chamber experiment to detect cell proliferation activity,migration and invasion;TUNEL staining and flow cytometry to detect cell apoptosis;Immunofluorescence staining to detect changes in p65 expression in cells;Western blot was used to detect the protein expression level of RelA/p65 phosphorylation sites.Results The mRNA and protein expression levels of EphA2 and EphrinA1 in uterine leiomyosarcoma tissue were significantly higher than those in adjacent tissues(P<0.05).Compared with the blank control group,the proliferation activity of cells in the EphA2-siRNA group decreased at 24,48 and 72 h after transfection,the number of cell migration and invasion decreased,the rate of TUNEL positive staining cells increased,the rate of apoptosis increased,and the p65 green fluorescence The intensity was significantly weakened.At the same time,the expression of p53 protein increased,and the phosphorylated protein levels of RelA/p65 Ser536 and RelA/p65 Ser276 decreased,and the difference was statistically significant(P<0.05).Conclusion EphA2 and EphrinA1 are highly expressed in uterine leiomyosarcoma

关 键 词：子宫平滑肌肉瘤 EPHA2 EPHRINA1 RelA/p65磷酸化 

分 类 号：R737.33[医药卫生—肿瘤]