胰岛再生源蛋白3A对胶质瘤细胞增殖和迁移的影响及机制  

Effect of REG3A on proliferation and invasion of glioma cells by regulating PI3K/Akt signaling pathway

作　　者：全艳春 王利莹 王增勇 高伟[3] 车峰远 Quan Yanchun;Wang Liying;Wang Zengyong;Gao Wei;Che Fengyuan(Central Laboratory,Linyi People′s Hospital,Linyi 276000,China;Department of Clinical Medicine,Weifang Medical College,Weifang 261000,China;Department of Clinical Laboratory,Linyi People′s Hospital,Linyi 276000,China;Key Laboratory of Neurophysiology,Health Commission of Shandong Province,Linyi 276000,China)

机构地区：[1]临沂市人民医院中心实验室,临沂276000 [2]潍坊医学院临床医学院,潍坊261000 [3]临沂市人民医院检验科,临沂276000 [4]山东省卫生健康委员会医药卫生神经生理学重点实验室,临沂276000

出　　处：《中华肿瘤杂志》2023年第8期642-650,共9页Chinese Journal of Oncology

基　　金：中国博士后科学基金(2020M672105);山东省博士后创新项目(202002040);山东省自然科学基金(ZR2022MH119);山东省医药卫生科技发展计划(2017WS130、2019WS130);临沂市科技发展计划(201818003);徐州医科大学附属医院发展基金(XYFY202222);临沂市人民医院博士创新基金(2018LYBS07)。

摘　　要：目的探讨胰岛再生源蛋白3A(REG3A)对胶质瘤细胞增殖和迁移的影响及其分子作用机制。方法收集2017年10月17日至2018年10月18日于临沂市人民医院行手术治疗的10例胶质瘤患者的癌及癌旁组织,低级别和高级别胶质瘤各5例。体外培养不同胶质瘤细胞株(SF295、U251、TG905、A172和CRT)及原代胶质瘤细胞株PT1,采用实时荧光定量聚合酶链反应和Western blot检测组织标本及各细胞株中REG3A的mRNA和蛋白表达水平。通过脂质体转染si-REG3A至TG905细胞敲低REG3A的表达,通过慢病毒包装REG3A质粒感染SF295细胞上调REG3A的表达,使用REG3A重组蛋白和Akt抑制剂单独或联合处理胶质瘤细胞,采用细胞计数试剂盒8法检测细胞增殖能力,细胞划痕实验检测细胞迁移能力,Western blot法检测细胞中N-cadherin、vimentin和磷酸化Akt(p-Akt)的表达情况。结果实时荧光定量聚合酶链反应(RT-qPCR)结果显示,U251、TG905、CRT、A172和PT-1细胞中REG3AmRNA的表达水平分别为2.129±0.13、2.22±0.59、5.02±0.31、6.62±1.34和9.18±0.61,高于SF295细胞(1.00±0.18,均P<0.001)。高级别胶质瘤组织中REG3AmRNA的表达水平为3.18±2.92,高于癌旁组织(1.00±1.14,P=0.031)和低级别胶质瘤组织(0.90±0.67,P=0.014)。Western blot和免疫组化检测结果与RT-qPCR结果一致。si-REG3A组TG905细胞的迁移率为(60.57±5.30)%,低于空白对照组[(79.65±12.09)%,P=0.076]和si-NC组[(84.18±13.63)%,P=0.038]。REG3A质粒转染组SF295细胞的迁移率为(96.05±6.41)%,高于空载转染组[(74.47±8.23)%,P=0.021]。REG3A重组蛋白能够上调SF295细胞中N-cadherin、vimentin和p-Akt的表达。与对照组[(100.00±2.53)%]相比,REG3A重组蛋白组的增殖率[(117.70±10.24)%]明显提高,REG3A重组蛋白+Akt抑制剂组的增殖率[(98.31±3.64)%]明显低于REG3A重组蛋白组(P=0.017)。REG3A重组蛋白+Akt抑制剂组的迁移率为(63.35±4.06)%,低于REG3A重组蛋白组[(89.26±11.07)%,P=0.019]。结论REG3A能够通�Objective To investigate the effects of regenerating islet-derived protein 3A(REG3A)on the proliferation and invasion of glioma cells and its molecular mechanism.Methods Five low-grade,five high-grade glioma tissues and ten adjacent tissues from glioma patients who underwent surgery at Linyi People′s Hospital from October 17,2017 to October 18,2018 were collected.Human glioma cell lines(SF295,U251,TG905,A172,CRT)and a primary glioma cell line PT-1 were cultured in vitro.The protein and mRNA expressions of REG3A in these tissues and glioma cell lines were detected by Western blot and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR).SF295 cells were infected with lentivirus and labeled as REG3A plasmid transfection group,and the TG905 cells were transfected with si-REG3A by liposome transfection reagent and labeled as si-REG3A transfection group.At the same time,the empty transfection control and blank control groups were set up.Glioma cells were treated with REG3A recombinant protein alone or in combination with Akt1/2 inhibitors.Cell counting kit-8(CCK-8)and cell scratch assay were used to detect cell proliferation and invasion,respectively.Western blot was used to detect the protein expression of N-cadherin,vimentin and phosphorylation of Akt(p-Akt)in REG3A overexpressed and knockdown glioma cells.Results RT-qPCR results showed that the mRNA expression levels of REG3A in glioma cells in each group were U251(2.129±0.13),TG905(2.22±0.59),CRT(5.02±0.31),A172(6.62±1.34)and PT-1(9.18±0.61),respectively,higher than its expression in SF295 cells(1.00±0.18,P<0.001).The mRNA expression level of REG3A in high-grade glioma tissue samples(3.18±2.92)was higher than that in the control group(1.00±1.14,P=0.031)and low-grade glioma group(0.90±0.67,P=0.014).The results of western blot and immunohistochemical staining were consistent with that of RT-qPCR.The migration rate of cells in si-REG3A transfection group[(60.57±5.30)%]was lower than that of the empty transfection group[(84.18±13

关 键 词：胶质瘤 胰岛再生源蛋白3A 细胞增殖 细胞迁移 磷酯酰肌醇-3-激酶/Akt信号通路 

分 类 号：R739.41[医药卫生—肿瘤]