沉默高迁移率族蛋白B1对胶质母细胞瘤细胞恶性生物学行为的抑制

Silencing high mobility group protein B1 inhibits malignant biological behavior of glioblastoma cells

目的探讨高迁移率族蛋白B1(high mobility group proteinB1,HMGB1)在胶质母细胞瘤样本中的高表达对于胶质母细胞瘤侵袭能力及替莫唑胺治疗的影响。方法利用癌症和肿瘤基因图谱(the cancer genome atlas,TCGA)数据库中319例样本的HMGB1测序数据进行差异表达及生存分析。采用免疫组织化学(immunohistochemistry,IHC)检测HMGB1在本院生物样本库中收集的4例不同WHO分级胶质瘤样本中的表达。3例胶质瘤样本(分别为WHOⅡ、Ⅲ和Ⅳ级)和1例瘤旁脑组织(作为对照,其胶质瘤组织为WHOⅡ级)均来自于陆军军医大学第一附属医院生物样本库(2019年5-10月的男性住院患者,年龄40~88岁,中位年龄58.5岁;患者行胶质瘤组织切除术前未行放化疗,术后标本经病理组织学检查证实为胶质瘤)。采用慢病毒转染法构建沉默HMGB1的细胞株,Western blot检测HMGB1蛋白表达。CCK-8和Transwell检测HMGB1对细胞增殖和侵袭能力的影响。CCK-8检测沉默HMGB1对替莫唑胺的敏感性。替莫唑胺粉末溶于二甲基亚砜中制成100 mmol/L的储存液,使用时终浓度为250、500、1000、1500μmol/L。结果TCGA数据库显示HMGB1 mRNA在肿瘤组织中表达显著上调(P<0.01),且随WHO级别的增加而增加,并与患者的预后显著负相关(P<0.01)。沉默HMGB1的胶质母细胞瘤细胞株增殖能力降低(P<0.05),侵袭能力下降(P<0.01),且对替莫唑胺治疗更敏感(P<0.05)。结论HMGB1在胶质母细胞瘤中显著上调,高表达的HMGB1促进了胶质母细胞瘤细胞的侵袭能力和替莫唑胺耐药。

Objective To investigate the effects of high expression of high mobility group protein B1(HMGB1)on the invasion ability of glioblastoma(GBM)and the efficacy of temozolomide(TMZ)therapy.Methods The HMGB1 sequencing data of 319 GBM samples from TCGA database were gained for differential expression and survival analysis.Immunohistochemistry(IHC)was adopted to assess the expression of HMGB1 in 4 samples(3 glioma samples,with WHO gradeⅡ,ⅢandⅣ,respectively,and 1 para-tumoral brain tissue as control)which were collected from our hospital’s biobank(inpatients from May to October 2019,male,aged 40~88 years,median 58.5 years,being confirmed as glioma by histopathological examination after tissue resection,without radiotherapy or chemotherapy prior to surgery).Moreover,lentiviral vector was transfected to construct HMGB1 silenced GBM cell lines(U251 and LN229).Western blotting was performed to detect HMGB1 expression;CCK-8 and transwell assays were adopted to analyze the effect of HMGB1 on cell proliferation and invasion ability.Finally,the role of HMGB1 in TMZ therapy was evaluated by CCK-8 assay(TMZ powder was dissolved in dimethyl sulfoxide to prepare 100 mmol/L storage solution,and the final concentration used was 250,500,1000 and 1500μmol/L,respectively).Results TCGA database showed that HMGB1 mRNA level was significantly upregulated in glioma tissue(P<0.01),increasing with WHO grade,and was negatively correlated with the prognosis of patients(P<0.01).HMGB1 silenced GBM cells had decreased proliferation(P<0.05)and inhibited invasion ability(P<0.01),and were more sensitive to TMZ therapy.Conclusion HMGB1 is remarkably up-regulated in GBM cells,and its high expression contributes to the enhanced invasion ability and temozolomide resistance of GBM cells.