摘要
目的探讨甲基转移酶样蛋白16(METTL16)对胶质母细胞瘤(GBM)细胞增殖和侵袭能力的影响,并揭示其潜在的分子生物学机制。方法体外培养U87及U251人GBM细胞株,通过转染METTL16过表达载体获得过表达METTL16基因的U87及U251细胞株,并将实验细胞分为阴性对照(NC)、过表达阴性对照(OE-NC)及METTL16过表达(OE-METTL16)组;实时荧光定量PCR(qPCR)和蛋白质免疫印迹实验(WB)检测METTL16基因过表达情况;CCK-8实验检测过表达METTL16基因对实验细胞株增殖能力的影响;细胞划痕实验及Transwell小室实验检测过表达METTL16对实验细胞株细胞迁移、侵袭能力的影响;流式细胞术检测过表达METTL16对实验细胞株凋亡及细胞周期的影响;WB实验检测过表达METTL16对实验细胞株焦亡标志物蛋白表达水平的影响。结果WB及qPCR实验表明,过表达METTL16基因的实验细胞株构建成功;CCK-8实验显示,与NC、OE-NC组相比,OE-METTL16组实验细胞株的细胞增殖能力均降低(均P<0.05);细胞划痕及Transwell实验表明,在U87、U251实验细胞株中,OE-METTL16组的细胞划痕愈合率及迁移、侵袭细胞数量均低于NC、OE-NC组(均P<0.05);流式细胞术结果显示,与NC、OE-NC组相比,OE-METTL16组实验细胞株细胞凋亡率均增加(均P<0.01)。此外,OE-METTL16组较NC、OE-NC组的G2/M期细胞占比均增加(均P<0.01),过表达METTL16可以诱导实验细胞株的细胞周期在G2/M期停滞。WB实验可见OE-METTL16组实验细胞株中的焦亡相关标志物白细胞介素1β、核因子κB、髓样分化因子88、核苷酸结合寡聚化结构域样受体3、消皮素D(GSDMD)和含半胱氨酸的天冬氨酸蛋白水解酶1的表达水平较NC、OE-NC组均增加(均P<0.05)。结论METTL16可通过调节GSDMD介导的细胞焦亡抑制GBM细胞的增殖、迁移、侵袭;其可作为潜在的靶向分子,为GBM治疗提供新的思路。
ObjectiveTo investigate the effects of METTL16 on the proliferation and invasion of glioblastoma(GBM)cells and to reveal the underlying molecular mechanisms.MethodsHuman glioblastoma cell lines U87 and U251 were cultured in vitro,and METTL16 stable overexpression cell lines were obtained by transfection of METTL16 overexpression vector.According to the cell intervention method,the experimental cell lines were divided into negative control(NC)group,overexpression negative control(OE-NC)group and METTL16 overexpression(OE-METTL16)group.METTL16 gene overexpression was detected by the Western blot(WB)and quantitative PCR(qPCR)assay.The effect of overexpression of METTL16 on the proliferation of experimental cell lines was detected by CCK-8 assay.The effects of overexpression of METTL16 on migration and invasiveness of experimental cell lines were investigated by cell scratch and Transwell assay.The effect of METTL16 overexpression on apoptosis and cell cycle was detected by flow cytometry.WB assay was used to detect the effect of overexpression of METTL16 on the expression levels of the pyroptosis markers in the experimental cell lines.ResultsWB and qPCR experiments showed that the experimental cell lines with METTL16 overexpression were successfully constructed.The results of CCK-8 experiment showed that compared with NC or OE-NC group,the cell proliferation capacity of OE-METTL16 group was significantly inhibited(both P<0.05).Cell scratch and Transwell tests showed that both cell mobility and number of invaded cells in OE-METTL16 group were significantly lower than those in NC and OE-NC groups(both P<0.05).Flow cytometry showed that compared with either NC or OE-NC group,the apoptosis rate of OE-METTL16 group was significantly increased(both P<0.01).In addition,the proportion of G2/M phase cells in the OE-METTL16 group increased compared with the NC or OE-NC group(both P<0.01);overexpression of METTL16 could induce cell cycle arrest in G2/M phase.WB assay showed that the expression levels of pyroptosis-related marker interleukin 1β,nuclear factorκB,myeloid differentiation factor 88,nucleotide-binding oligomerization domain-like receptor protein 3,gasdermin D(GSDMD)and cysteinyl aspartate specific proteinase-1 in OE-METTL16 group were all significantly increased compared with those in NC and OE-NC groups(both P<0.05).ConclusionMETTL16 gene can effectively inhibit the proliferation,invasion and migration of GBM cells by regulating GSDMD-mediated pyroptosis,which could provide new ideas and direction as a potential molecular target for GBM therapy.
作者
包洪恩
白慧茹
刘凯
窦长武
姜粉军
高乃康
任佳星
王飞
Bao Hongen;Bai Huiru;Liu Kai;Dou Changwu;Jiang Fenjun;Gao Naikang;Ren Jiaxing;Wang Fei(Department of Neurosurgery,Affiliated Hospital of Inner Mongolia Medical University,Hohhot 010050,China;Verification Centye of Forenstic Medicine,Basic Medicine College of Inner Mongolia Medical University,Hohhot 010050,China;Department of Neurosurgery,Shaoxing People′s Hospital,Shaoxing 312030,China)
出处
《中华神经外科杂志》
2024年第10期1056-1064,共9页
Chinese Journal of Neurosurgery
基金
内蒙古自治区自然科学基金(2024QN08088)
内蒙古自治区科技计划(2019GG9044)
内蒙古医科大学附属医院院级项目——科研联合基金内蒙古自治区神经系统疾病临床医学研究中心项目
首府地区高质量发展优势临床重点专科神经系统疾病项目(2023NYFY LHQN001)。
