B7-H3促进胶质母细胞瘤增殖侵袭和迁移的实验研究

The experimental study of B7-H3 promoting the proliferation,invasion and migration of glioblastoma

目的研究B7-H3对胶质母细胞瘤(GBM)增殖、侵袭、迁移能力的影响。方法采集GEPIA数据库中GBM、低级别胶质瘤(LGG)及正常脑组织中B7-H3的表达量数据,并分析差异及其与患者生存期的关系。通过转染短发夹RNA(shRNA)构建B7-H3低表达的GBM细胞株A172和TG-905(shRNA-B7-H3组),并通过实时定量PCR(qRT-PCR)、流式细胞学技术和蛋白质免疫印迹法(WB)分别检测B7-H3的表达量,验证敲低效率。对照组为未转染的野生型细胞(wt组)和转染对照shRNA的细胞(shRNA-ctl组)。通过羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)染色掺入法、CCK-8法、克隆形成实验分别检测B7-H3敲低后细胞增殖能力;通过划痕愈合实验及Transwell小室法检测B7-H3敲低后细胞迁移和侵袭能力;采用WB检测B7-H3敲低后下游信号分子的表达情况。结果GEPIA数据库分析结果显示,B7-H3在LGG和GBM组织中表达均高于正常脑组织(均P<0.01),且低表达B7-H3的患者总生存期较高表达者长(P<0.001)。A172细胞株中,qRT-PCR和流式细胞学技术检测结果显示,shRNA-B7-H3组的B7-H3表达量均低于shRNA-ctl组和wt组(均P<0.01);WB结果显示,shRNA-B7-H3组的B7-H3蛋白表达低于shRNA-ctl组和wt组;CFSE结果显示,shRNA-B7-H3组24、48、72 h的细胞增殖能力均较shRNA-ctl组降低(均P<0.001);CCK-8法检测和克隆形成实验结果均显示,shRNA-B7-H3组的细胞增殖能力低于shRNA-ctl组和wt组(均P<0.05)。细胞划痕愈合实验结果显示,与shRNA-ctl组和wt组比较,shRNA-B7-H3组48、72 h的划痕愈合能力均降低(均P<0.01)。Transwell小室结果显示,B7-H3敲低后,迁移和侵袭能力均下降(均P<0.001)。WB结果显示,B7-H3敲低后细胞周期蛋白依赖性激酶4(CDK4)和磷酸化Janus激酶2(p-JAK2)表达下调,而Bcl-2相关性蛋白(Bax)表达上调。上述实验在TG-905细胞中得到相似的结果。结论B7-H3通过上调CDK4、p-JAK2和下调Bax促进GBM的增殖、侵袭和迁移。

Objective To study the effect of B7-H3 on the proliferation,invasion and migration of glioblastoma(GBM).Methods The expression data of B7-H3 in GBM,low-grade glioma(LGG)and normal brain tissue in GEPIA database were collected,and the differences and their relationship with patient survival were analyzed.GBM cell lines A172 and TG-905 with low expression of B7-H3(shRNA-B7-H3 group)were constructed by transfecting short hairpin RNA(shRNA),and real-time quantitative PCR(qRT-PCR),flow cytometry and Western blotting(WB)were used to detect the expression of B7-H3 to verify the knockdown efficiency.The control groups were untransfected wild-type cells(wt group)and transfected with control shRNA(shRNA-ctl group).Carboxyfluorescein diacetate succinimidyl ester(CFSE)staining assay,cell counting kit-8(CCK-8),and colony formation assay were used to detect cell proliferation ability after B7-H3 knockdown.Scratch healing experiment and Transwell chamber method were used to detect cell migration and invasion ability after B7-H3 knockdown.WB was used to detect the expression of downstream signal molecules after B7-H3 knockdown.Results The GEPIA database analysis results showed that the expression of B7-H3 in LGG and GBM tissues was higher than that in normal brain tissues(both P<0.01),and patients with low expression of B7-H3 had a longer survival time(P<0.001).In the A172 cell line,the results of qRT-PCR and flow cytometry showed that the expression level of shRNA-B7-H3 group was lower than that of shRNA-ctl group and wt group(both P<0.01).WB results showed that the expression level of shRNA-B7-H3 group was lower than that of shRNA-ctl group and wt group.CFSE results showed that the cell proliferation ability of the shRNA-B7-H3 group was lower than that of the shRNA-ctl group at 24,48,and 72 h(all P<0.001).The results of both CCK-8 and colony formation assay showed that the proliferation ability of shRNA-B7-H3 group was lower than that of shRNA-ctl group and wt group(both P<0.05).The results of the scratch healing experiment showed that the scratch healing ability of the shRNA-B7-H3 group at 48 h and 72 h was lower compared with that of the shRNA-ctl group and the wt group(all P<0.01).The results of Transwell chamber experiments showed that after B7-H3 knockdown,the migration and invasion abilities were decreased(both P<0.001).WB results showed that the expression of cyclin-dependent kinase 4(CDK4)and phosphorylated Janus kinase 2(p-JAK2)was down-regulated after B7-H3 knockdown,while the expression of Bcl-2-related protein(Bax)was up-regulated.The above experiments revealed similar results in TG-905 cells.Conclusion B7-H3 promotes the proliferation,invasion and migration of GBM by up-regulating CDK4,p-JAK2 and down-regulating Bax.