JSH-23联合Stattic靶向NF-κB和STAT3双信号转导通路抑制胶质瘤细胞增殖和迁移实验研究

JSH-23 combined with Stattic inhibits proliferation and migration of glioma cells by targeting NF-κB and STAT3 signaling pathways

目的探讨JSH-23联合Stattic靶向抑制核因子-κB(NF-κB)和信号转导与转录激活因子3(STAT3)双信号转导通路对间质型胶质母细胞瘤细胞增殖和迁移能力以及NF-κB通路和STAT3通路相关蛋白表达的影响。方法自肿瘤基因组学图谱计划官网下载529例胶质瘤患者转录组测序结果,生物信息学分析RelA/P65和STAT3与间质型胶质母细胞瘤标志物的相关性,以及NF-κB通路和STAT3通路相关蛋白在间质型、经典型和前神经型胶质母细胞瘤中的表达。体外培养的人胶质瘤细胞系U87MG和U251MG分别经JSH-23、Stattic、JSH-23和Stattic联合处理,细胞毒性实验计算两种药物的半数抑制浓度(IC_(50)),药物协同实验评估两种药物的协同作用;CCK-8法和平板克隆实验检测肿瘤细胞增殖能力,细胞划痕实验和Transwell实验检测肿瘤细胞迁移能力,Western blotting法检测NF-κB通路和STAT3通路相关蛋白P65、磷酸化P65(p-P65)、STAT3、磷酸化STAT3(p-STAT3)以及CD44的相对表达量。结果(1)生物信息学分析显示,RelA/P65 mRNA和STAT3 mRNA与间质型胶质母细胞瘤标志物CD44、CXCR4、CHI3L1、IL-4R、TRADD呈正相关(r=0.206~0.605,均P<0.01),且间质型胶质母细胞瘤NF-κB通路和STAT3通路相关蛋白表达水平最高、经典型其次、前神经型最低。(2)细胞毒性实验显示,JSH-23对U87MG和U251MG细胞的IC_(50)为59.39和56.21μmol/L,Stattic为0.96和1.08μmol/L。药物协同实验显示,JSH-23为40~80μmol/L以及Stattic为0.50~1μmol/L时对U87MG细胞的协同效应最高,JSH-23为40μmol/L以及Stattic为1μmol/L时对U251MG细胞的协同效应最高,并且最终确定JSH-23的终浓度为60μmol/L,Stattic为1μmol/L。(3)经JSH-23、Stattic、JSH-23和Stattic联合处理后,U87MG和U251MG细胞增殖活性降低(均P=0.000),集落形成率减少(均P=0.000),细胞迁移率降低(均P<0.05),结晶紫染色阳性细胞数目减少(均P=0.000),以及P65(均P=0.000)、p-P65(均P=0.000)、STAT3(均P=0.000)、p-STAT3(均P=0.000)和CD44(均P=0.000)相对表达量降低,尤以JSH-23和Stattic联合处理后降低最显著(均P<0.01)。结论JSH-23联合Stattic靶向抑制NF-κB和STAT3双通路可以有效抑制间质型胶质母细胞瘤细胞的增殖和迁移能力,并下调NF-κB通路和STAT3通路相关蛋白的表达。

Objective To investigate the effects of JSH-23 combined with Stattic targeting inhibition of nuclear factor-κB(NF-κB)and signal transducer and activator of transcription factor 3(STAT3)signaling pathways on the proliferation and migration ability of mesenchymal glioblastoma cells and the expressions of NF-κB pathway and STAT3 pathway-related proteins.Methods The mRNA-seq results of 529 glioma patients were downloaded from The Cancer Genome Atlas(TCGA).Bioinformatics was used to analyze the correlation between RelA/P65 and STAT3 and the markers of mesenchymal glioblastoma,as well as the expression of NF-κB pathway and STAT3 pathway-related proteins in mesenchymal,classical and pro neural glioblastoma.The human glioma cell lines U87 MG and U251 MG in vitro were treated with JSH-23,Stattic,JSH-23 and Stattic,respectively.The half-inhibitory concentration(IC_(50))of the two drugs was calculated by cytotoxicity assay,and the synergistic effect of the two drugs was observed by drug synergistic assay.CCK-8 assay and colony-forming assay were used to detect the proliferation activity of tumor cells.Wound healing assay and Transwell assay were used to detect the migration ability of tumor cells.Western blotting was used to detect the relative expressions of NF-κB pathway and STAT3 pathwayrelated proteins P65,phosphorylated P65(p-P65),STAT3,phosphorylated STAT3(p-STAT3)and CD44.Results 1)Bioinformatics analysis showed that RelA/P65 mRNA and STAT3 mRNA were positively correlated with mesenchymal glioblastoma markers CD44,CXCR4,CHI3 L1,IL-4 R and TRADD(r=0.206-0.605;P<0.01,for all);the expressions of NF-κB pathway and STAT3 pathway-related proteins in mesenchymal glioblastoma were the highest,followed by classical glioblastoma,and the lowest in proneural glioblastoma.2)The cytotoxicity assay showed the IC_(50)of JSH-23 on U87 MG and U251 MG cells were59.39 and 56.21μmol/L,and Stattic were 0.96 and 1.08μmol/L.The drug synergistic assay showed that the synergistic effect of JSH-23 at 40-80μmol/L and Stattic at 0.50-1μmol/L on U87 MG was the highest,and the synergistic effect of JSH-23 at 40μmol/L and Stattic at 1μmol/L on U251 MG was the highest.The final concentration of JSH-23 was 60μmol/L and Stattic was 1μmol/L.3)After the combined treatment of JSH-23,Stattic,JSH-23 and Stattic,the proliferation activity of U87 MG and U251 MG cells decreased(P=0.000,for all),the colony forming efficiency decreased(P=0.000,for all),the cell mobility decreased(P<0.05,for all),and the positive cell numbers of crystal violet staining decreased(P=0.000,for all).The relative expressions of P65(P=0.000,for all),p-P65(P=0.000,for all),STAT3(P=0.000,for all),pSTAT3(P=0.000,for all)and CD44(P=0.000,for all)were decreased,especially after JSH-23 combined with Stattic treatment(P<0.01,for all).Conclusions JSH-23 combined with Stattic targeting inhibition of NF-κB and STAT3 signaling pathways can effectively inhibit the proliferation and migration ability of mesenchymal glioblastoma cells,and down-regulate the relative expressions of NF-κB pathway and STAT3 pathway-related proteins.