HIPK2在脑胶质瘤中的表达及前神经元-间质亚型转化中作用的研究

Expression and proneural-to-mesenchymal transition function of HIPK2 in gliomas

目的探讨同源结构域相互作用蛋白激酶2(HIPK2)在人脑胶质瘤中的表达及前神经元-间质亚型转化(PMT)中的作用。方法分析美国国立生物信息技术中心基因表达数据库(NCBI-GEO:GSE16011)中HIPK2 mRNA在低级别和高级别胶质瘤中的表达。基于GSE16011数据库进一步分析HIPK2 mRNA在前神经元型、间质型和经典型胶质瘤样本中的表达。根据HIPK2 mRNA的表达量将胶质瘤患者分为HIPK2低表达组和高表达组,并绘制Kaplan-Meier生存曲线,采用log-rank检验比较HIPK2低表达组与高表达组患者的生存差异。体外实验以人星形胶质细胞(NHA)为对照,通过蛋白质免疫印迹法(WB)验证HIPK2蛋白在胶质瘤细胞株H4、A172、U87和U251中的表达。在U87细胞中采用携带HIPK2 cDNA序列的过表达慢病毒载体构建过表达HIPK2的细胞株,以空载慢病毒载体构建对照细胞株。在H4细胞中采用siRNA转染敲低HIPK2的表达。通过划痕实验和Transwell实验探讨HIPK2表达改变对胶质瘤细胞迁移和侵袭能力的影响。以WB实验检测HIPK2表达改变对FN1、CD44和SNAI2蛋白表达的影响。采用Pearson相关分析探讨HIPK2与FN1、CD44和SNAI2表达的相关性。结果HIPK2 mRNA在高级别胶质瘤中的表达水平低于低级别胶质瘤(t=6.86,P<0.001),且HIPK2 mRNA在间质型和经典型胶质瘤中的表达水平均低于前神经元型胶质瘤(均P<0.001)。对GSE16011数据库数据的分析显示,HIPK2高表达组患者的总体生存期长于低表达组[分别为(40.8±3.9)个月和(24.9±3.2)个月,P<0.001]。WB结果显示,与NHA比较,H4、A172、U87和U251细胞中HIPK2蛋白的表达水平均降低(均P<0.05)。U87细胞HIPK2过表达组HIPK2蛋白的表达较对照组显著上调(t=24.88,P<0.05),H4细胞HIPK2敲低组的HIPK2蛋白表达水平较对照组明显下调(t=26.72,P<0.001)。划痕实验结果显示,U87细胞HIPK2过表达组的细胞划痕修复率低于对照组[分别为(18.1±2.0)%和(45.6±4.2)%,t=10.23,P<0.001];而H4细胞HIPK2敲低组的细胞划痕修复率高于对照组[分别为(60.5±5.4)%和(37.9±4.0)%,t=5.83,P=0.004]。Transwell实验结果显示,U87细胞HIPK2过表达组的穿膜细胞数少于对照组[分别为(92.0±11.9)个和(193.1±16.2)个,t=8.75,P<0.001];而H4细胞HIPK2敲低组的穿膜细胞数高于对照组[分别为(204.0±16.9)个和(135.1±15.0)个,t=5.27,P=0.006]。WB实验结果表明,U87细胞HIPK2过表达组细胞中FN1、CD44和SNAI2蛋白的表达水平均低于对照组(均P<0.001);H4细胞HIPK2敲低组中FN1、CD44和SNAI2蛋白的表达水平均高于对照组(均P<0.001)。Pearson相关分析结果显示,胶质瘤中HIPK2的表达与FN1、CD44和SNAI2表达均呈负相关(均P<0.05)。结论HIPK2在高级别胶质瘤和间质型胶质瘤中呈低表达,低表达的HIPK2可能通过影响胶质瘤PMT促进胶质瘤细胞的迁移和侵袭。

Objective To investigate the expression and proneural-to-mesenchymal transition(PMT)function of homeodomain-interacting protein kinase-2(HIPK2)in gliomas.Methods We first analyzed the expression of HIPK2 in the low-grade gliomas(LGGs)and high-grade gliomas(HGGs)in National Center for Biotechnology Information Gene Expression Omnibus(NCBI-GEO)dataset GSE16011.Based on the GSE16011 dataset,we further analyzed the expression of HIPK2 in proneural subtype,mesenchymal subtype and classical subtype gliomas.According to the HIPK2 expression,the testees were divided into low expression group and high expression group,and Kaplan-Meier survival curve was drawn.Log-rank test was used to compare the survival of glioma patients in the HIPK2 low expression group with that in the HIPK2 high expression group.Human astrocytes(NHA)were set as the control group and Western blot was performed in vitro to assess the expression of HIPK2 in H4,A172,U87 and U251 glioma cell lines.In U87 cells,overexpressed lentiviral vectors carrying the HIPK2 cDNA sequence were used to construct cells overexpressing HIPK2.Construction of control cells was made with lentiviral vectors.H4 cells were transfected with siRNA to knock down the expression of HIPK2.We then studied the effects of HIPK2 on the migration and invasion ability of glioma cells through cell scratch experiment and transwell experiment.We further performed Western blot to detect the effect of HIPK2 on the expression of FN1,CD44 and SNAI2.Finally,Pearson correlation analysis was used to analyze the correlation between HIPK2 expression and FN1,CD44 and SNAI2 expression in GSE16011 dataset.Results In the GSE16011 dataset,HIPK2 mRNA expression levels of HGGs was significantly lower than that of LGGs(t=6.86,P<0.001).Lower expression of HIPK2 mRNA was found in mesenchymal subtype and classical subtype gliomas(both P<0.001).Among glioma patients or HGGs,the overall survival of the HIPK2 low expression group was shorter than that of the high expression group(40.8±3.9 months vs.24.9±3.2 months,P<0.001).In further in vitro experiments,Western blot results showed that the protein level of HIPK2 in H4,A172,U87 and U251 cells was decreased compared with that of NHA group(all P<0.05).The protein level of HIPK2 in U87 overexpression group was upregulated compared with that of the control group(t=24.88,P<0.05).The protein level of HIPK2 in the H4 knockdown group was downregulated compared with the control group(t=26.72,P<0.001).The cell scratch experiments results showed that the cell scratch repair rate of U87 overexpression group was lower than that of U87 control group(18.1%±2.0%vs.45.6%±4.2%,t=10.23,P<0.001).On the contrary,the cell scratch repair rate of H4 knockdown group was higher than that of H4 control group(60.5%±5.4%vs.37.9%±4.0%,t=5.83,P=0.004).Transwell experiments results showed that the number of membrane-penetrating cells in the U87 overexpression group was lower than that of U87 control group(92.0±11.9 vs.193.1±16.2,t=8.75,P<0.001).On the contrary,the number of membrane-penetrating cells in the H4 knockdown group was higher than that of H4 control group(204.0±16.9 vs.135.1±15.0,t=5.27,P=0.006).Western blot results showed that the protein levels of FN1,CD44 and SNAI2 in the U87 overexpression group were lower than those of U87 control group(all P<0.001).On the contrary,the protein levels of FN1,CD44 and SNAI2 in the H4 knockdown group were higher than those of H4 control group(all P<0.001).Pearson correlation analysis results showed that the expression of FN1,CD44 and SNAI2 was negatively correlated with HIPK2 expression in GSE16011 dataset(all P<0.05).Conclusions HIPK2 is lowly expressed in HGGs and mesenchymal subtype gliomas.Low expression of HIPK2 may promote glioma cell migration and invasion via enhancing PMT process.