摘要
目的探讨胶质瘤组织中Bcl-2相关抗凋亡基因3(BAG3)表达及其对U87细胞增殖和侵袭能力的影响。方法选取2011年4月至2017年4月于新乡市中心医院手术切除的胶质瘤组织及癌旁组织(距瘤旁边缘>2 cm)标本各73例,采用免疫组织化学法检测胶质瘤组织和癌旁组织中BAG3蛋白表达;培养人胶质瘤U87细胞,待细胞融合度达70%以上时随机分为siRNA-BAG3组、siRNA-对照序列组和空白对照组,siRNA-BAG3组细胞转染BAG3干扰序列,siRNA-对照序列组细胞转染阴性对照序列,空白对照组细胞不作任何处理,转染后继续培养48 h。采用实时荧光定量聚合酶链反应检测U87细胞中BAG3 mRNA表达,Western blot法检测U87细胞中BAG3蛋白表达,细胞计数试剂盒-8检测U87细胞增殖情况,克隆形成实验检测U87细胞克隆能力,流式细胞仪检测U87细胞凋亡情况,Transwell小室检测U87细胞侵袭能力。结果胶质瘤组织中BAG3蛋白高表达率为73.97%(54/73),低表达率为26.03%(19/73);癌旁组织中BAG3蛋白高表达率为36.99%(27/73),低表达率为63.01%(46/73);胶质瘤组织中BAG3蛋白高表达率显著高于癌旁组织(χ~2=20.215,P<0.05)。胶质瘤组织中BAG3蛋白表达与患者的年龄、性别、肿瘤位置、肿瘤直径及Karnofsky评分无关(P>0.05),而与肿瘤分级有关(P<0.05)。siRNA-BAG3组、siRNA-对照序列组和空白对照组U87细胞中BAG3 mRNA相对表达量分别为1.42±0.11、2.17±0.12和2.31±0.18;siRNA-BAG3组、siRNA-对照序列组和空白对照组U87细胞中BAG3蛋白相对表达量分别为0.20±0.12、0.84±0.13和0.92±0.12;siRNABAG3组U87细胞中BAG3 mRNA及蛋白相对表达量显著低于siRNA-对照序列组和空白对照组(P<0.05);siRNA-对照序列组与空白对照组U87细胞中BAG3 mRNA及蛋白相对表达量比较差异无统计学意义(P>0.05)。培养24、48、72、96 h时,siRNA-BAG3组U87细胞增殖能力显著低于siRNA-对照序列组和空白对照组(P<0.05);siRNA-对照序列组与空白对照组U87细胞增殖能力比较差异无统计学意义(P>0.05)。siRNA-BAG3组、siRNA-对照序列组和空白对照组U87细胞克隆形成数分别为30.76±6.19、81.32±3.55、85.07±3.05;siRNA-BAG3组U87细胞克隆形成数显著少于siRNA-对照序列组和空白对照组(P<0.05);siRNA-对照序列组与空白对照组U87细胞克隆形成数比较差异无统计学意义(P>0.05)。siRNA-BAG3组、siRNA-对照序列组和空白对照组细胞凋亡率分别为(21.43±6.89)%、(5.50±2.95)%、(7.37±3.31)%;siRNA-BAG3组细胞凋亡率显著高于siRNA-对照序列组和空白对照组(P<0.05);siRNA-对照序列组与空白对照组细胞凋亡率比较差异无统计学意义(P>0.05)。siRNA-BAG3组、siRNA-对照序列组和空白对照组侵袭细胞数分别为116.92±8.82、179.00±11.64、172.48±18.16,siRNA-BAG3组侵袭细胞数显著少于siRNA-对照序列组和空白对照组(P<0.05),siRNA-对照序列组与空白对照组侵袭细胞数比较差异无统计学意义(P>0.05)。结论 BAG3蛋白在胶质瘤组织中呈高表达,且与肿瘤恶性程度有关,特异性沉默人胶质瘤U87细胞中BAG3基因可有效抑制细胞增殖及侵袭能力。
Objective To investigate the expression of Bcl-2-associated anti-apoptotic gene 3（BAG3） in glioma tissues and its effect on proliferation and invasion of U87 cells.Methods The glioma tissues and adjacent tissues（from the edge of the tumor 〉 2 cm） from 73 patients with glioma were selected in Xinxiang Central Hospital from April 2011 to April 2017.The expressions of BAG3 protein in gliomas tissues and adjacent tissues were detected by immunohistochemistry.The human glioma U87 cells were cultured,and the U87 cells were randomly divided into siRNA-BAG3 group,siRNA-control sequence group and blank control group when the cell fusion degree was over 70%.The cells in the siRNA-BAG3 group were transfected with BAG3 interference sequence,and the cells in the siRNA-control sequence group were transfected with the negative control sequence,while the cells in the blank control group were not treated.The U87 cells were cultured for 48 hours after transfection.The expression of BAG3 mRNA in U87 cells was detected by real time fluorescence quantitative polymerase chain reaction.The expression of BAG3 protein in U87 cells was detected by Western blot.The proliferation of U87 cells was detected by cell counting kit-8.The cloning ability of U87 cells was detected by clone formation assay.The apoptosis of U87 cells was detected by flow cytometry.The invasion ability of U87 cells was detected by Transwell chamber.Results The high expression rate of BAG3 protein in glioma tissues was 73.97%（54/73）,and the low expression rate was 26.03%（19/73）.The high expression rate of BAG3 protein in adjacent tissues was 36.99%（27/73）,and the low expression rate was 63.01%（46/73）.The high expression rate of BAG3 protein in glioma tissues was significantly higher than that in adjacent tissues（ χ 2=20.215, P 〈0.05）.The expression of BAG3 protein in glioma tissues was not correlated with age,gender,tumor location,tumor diameter and Karnofsky score（ P 〉0.05）;but the expression of BAG3 protein in glioma tissues was correlated with tumor grade（ P 〈0.05）.The relative expression levels of BAG3 mRNA in U87 cells of siRNA-BAG3 group,siRNA-control sequence group and blank control group were 1.42±0.11,2.17±0.12 and 2.31±0.18,respectively;the relative expression levels of BAG3 proteins in U87 cells of siRNA-BAG3 group,siRNA-control sequence group and blank control group were 0.20±0.12,0.84±0.13 and 0.92±0.12,respectively.The relative expression levels of BAG3 mRNA and protein in U87 cells in siRNA-BAG3 group were significantly lower than those in the siRNA-control sequence group and the blank control group（ P 〈0.05）;there was no significant difference in relative expression level of BAG3 mRNA and protein between siRNA-control sequence group and the blank control group（ P 〉0.05）.When cultured for 24,48,72 and 96 hours,the proliferation ability of U87 cells in siRNA-BAG3 group was significantly higher than that in the siRNA-control sequence group and the blank control group（ P 〈0.05）,but there was no significant difference in the proliferation ability of U87 cells between siRNA-control sequence group and blank control group（ P 〉0.05）.The number of clone of U87 cells in siRNA-BAG3 group,siRNA-control sequence group and blank control group was 30.76±6.19,81.32±3.55 and 85.07±3.05,respectively.The number of clone of U87 cells in siRNA-BAG3 group was significantly less than that in the siRNA-control sequence group and the blank control group（ P 〈0.05）.There was no significant difference in the number of clone of U87 cells between the siRNA-control sequence group and the blank control group（ P 〉0.05）.The apoptosis rates of U87 cells in the siRNA-BAG3 group,siRNA-control sequence group and blank control group were （21.43±6.89）%,（5.50±2.95）%and （7.37±3.31）%,respectively.The apoptosis rate of U87 cells in the siRNA-BAG3 group was significant higher than that in the siRNA-control sequence group and the blank control group （ P 〈0.05）.There was no significant difference in apoptosis rate of U87 cells between the siRNA-control sequence group and blank control group （ P 〉0.05）.The number of invasive cells in the siRNA-BAG3 group,siRNA-control sequence group and blank control group was 116.92±8.82,179.00±11.64 and 172.48±18.16,respectively.The number of invasive cells in the siRNA-BAG3 group was significantly less than that in the siRNA-control sequence group and the blank control group （ P 〈0.05）.There was no significant difference in the number of invasive cells between the siRNA-control sequence group and the blank control group（ P 〉0.05）.Conclusion BAG3 protein is highly expressed in glioma tissues,and it was associated with malignancy.Specific silencing of BAG3 gene in human glioma U87 cells can effectively inhibit cell proliferation and invasion.
作者
赵岗
杜伟
魏新亭
王进忠
程振国
ZHAO Gang;DU Wei;WEI Xin-ting;WANG Jin-zhong;CHENG Zhen-guo(Department of Neurosurgery,Xinxiang Central Hospital,Xinxiang 453000,Henan Province,China;Department of Neurosurgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 451000,Henan Province,China)
出处
《新乡医学院学报》
CAS
2018年第10期843-848,共6页
Journal of Xinxiang Medical University
基金
河南省科技攻关计划项目(编号:201602083)
