LPCAT1调节表皮生长因子受体表达影响胶质母细胞瘤生长和侵袭的实验研究  

Experimental study on the effect of LPCAT1 on the growth and invasion of glioblastomas by regulating the expression of epidermal growth factor receptor

作　　者：王敬 刘金秋 邓宇轩 苗亚洲 张绍东[1] 吕一帆 郝淑煜[2] 季楠[2] 万虹[1] 冯洁[1] Wang Jing;Liu Jinqiu;Deng Yuxuan;Miao Yazhou;Zhang Shaodong;Lyu Yifan;Hao Shuyu;Ji Nan;Wan Hong;Feng Jie(Beijing Neurosurgical Institute,Capital Medical University,Beijing 100070,China;Neurosurgery Center,Beijing Tiantan Hospital,Capital Medical University,Beijing 100070,China)

机构地区：[1]首都医科大学,北京市神经外科研究所,北京100070 [2]首都医科大学附属北京天坛医院神经外科学中心,北京100070

出　　处：《中华神经外科杂志》2023年第4期395-400,共6页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金(81872052)。

摘　　要：目的探讨溶血磷脂酰胆碱酰基转移酶1(LPCAT1)对人脑胶质母细胞瘤(GBM)增殖、侵袭和细胞凋亡的作用及其作用机制。方法基于中国胶质瘤基因组图谱(CGGA)数据库分析LPCAT1在不同级别[世界卫生组织(WHO)Ⅱ、Ⅲ级和Ⅳ级]胶质瘤中的表达情况。通过蛋白质免疫印迹法(WB)检测LPCAT1在来源于首都医科大学附属北京天坛医院神经外科学中心行手术治疗的低级别胶质瘤(LGG)(WHOⅡ级,4例)和GBM(4例)患者组织样本中的表达情况。采用LPCAT1 siRNA转染GBM细胞株LN18,将转染的细胞分为阴性对照组、LPCAT1 si1组和LPCAT1 si2组。采用实时荧光定量PCR(qPCR)和WB检测LPCAT1 siRNA的敲减效率。分别通过CCK-8实验、Transwell实验和流式细胞术明确LPCAT1敲减对各组细胞增殖、侵袭能力和细胞凋亡的影响。采用qPCR和WB检测LPCAT1敲减对表皮生长因子受体(EGFR)的表达及其磷酸化Tyr1068的影响。结果CGGA数据库分析结果表明,GBM中LPCAT1 mRNA的表达水平显著高于WHOⅡ、Ⅲ级胶质瘤(均P<0.05)。对临床样本的WB结果显示,GBM组织中LPCAT1蛋白的表达显著高于LGG(分别为1.167±0.123和0.561±0.081,t=4.13,P<0.001)。qPCR和WB结果显示,LPCAT1 si1和LPCAT1 si2组LPCAT1基因和蛋白表达水平均明显低于阴性对照组(均P<0.01)。CCK-8实验结果显示,LPCAT1 si1和LPCAT1 si2组的吸光度值分别为1.043±0.025、0.875±0.040,均显著低于阴性对照组(1.202±0.028)(均P<0.01)。Transwell实验结果显示,LPCAT1 si1和LPCAT1 si2组细胞的迁移能力均低于阴性对照组(均P<0.001)。LPCAT1 si1和LPCAT1 si2组的凋亡细胞比率分别为(49.5±0.3)%和(38.7±0.3)%,均高于阴性对照组[(17.1±0.1)%](均P<0.001)。qPCR结果显示,LPCAT1 si1和LPCAT1 si2组的EGFR mRNA表达水平均低于阴性对照组(均P<0.001)。WB结果显示,LPCAT1 si1和LPCAT1 si2组的EGFR Tyr1068磷酸化水平均显著低于阴性对照组(均P<0.05)。结论初步结果表明,通过抑制LPCAT1的表达可降Objective To investigate the effect of lysophosphatidylcholine acyltransferase 1(LPCAT1)on the tumor proliferation,invasion and apoptosis of human glioblastomas(GBMs)and to explore its mechanisms.Methods Based on the Chinese Glioma Genome Atlas(CGGA)database,the expression of LPCAT1 was analyzed among lower grade gliomas[LGG,World Health Organization(WHO)gradeⅡandⅢ]and GBMs.The expression of LPCAT1 was validated through Western blot(WB)in 4 LGG and 4 GBM tissue samples from patients undergoing surgical resection at the Neurosurgery Center,Beijing Tiantan Hospital,Capital Medical University.LPCAT1 was knocked down by siRNAs,and the transfected LN18 cells were divided into three experimental groups including negative control group,LPCAT1 si1 group and LPCAT1 si2 group.The knockdown efficiency of LPCAT1 siRNA was detected by real-time quantitative PCR(qPCR)and WB.The effects of LPCAT1 knockdown on tumor cell proliferation,invasion and apoptosis in three experimental groups were detected by cell counting Kit-8(CCK8)test,transwell assay and flow cytometry respectively.The effect of LPCAT1 knockdown on the expression of epidermal growth factor receptor(EGFR)and phosphorylation Tyr1068 of EGFR in the three experimental groups was also detected by qPCR and WB.Results Based on the CGGA database analysis,LPCAT1 mRNA expression in GBMs was significantly higher than that in WHO gradeⅡandⅢgliomas(both P<0.05).WB results based on clinical samples showed that the expression of LPCAT1 in GBM was significantly higher than that in LGG(1.167±0.123 vs.0.561±0.081,t=4.13,P<0.001).The qPCR results showed that the gene expression level of LPCAT1 in LPCAT1 si1 and LPCAT1 si2 groups were significantly lower than that in negative control group(both P<0.001).WB results revealed the same results(both P<0.01).The results of CCK8 showed that the cell proliferation in LPCAT1 si1 and LPCAT1 si2 groups(absorbance value:1.043±0.025 and 0.875±0.040 respectively)were significantly lower than that in negative control group(1.202±0.028)(

关 键 词：胶质母细胞瘤 细胞增殖 肿瘤侵润 细胞凋亡 受体 表皮生长因子 溶血磷脂酰胆碱酰基转移酶1 

分 类 号：R739.41[医药卫生—肿瘤]