同源盒基因A11反义RNA在脑胶质瘤中的表达及生物学功能

Expression and biological function of Homebox gene A11 antisense RNA in gliomas

目的探讨长链非编码RNA同源盒基因A11反义RNA(HOXA11-AS)在脑胶质瘤中的表达及生物学功能。方法首先分析HOXA11-AS在癌症基因组图谱(TCGA)计划数据库(689例)的低级别脑胶质瘤、胶质母细胞瘤以及GTEx数据库(255例)的正常脑组织样本中的表达情况。基于TCGA数据库,进一步分析HOXA11-AS在世界卫生组织(WHO)Ⅱ~Ⅳ级的脑胶质瘤样本中的表达情况。根据HOXA11-AS表达量的中位数分为高表达组和低表达组,并绘制Kaplan-Meier生存曲线,采用log-rank检验比较HOXA11-AS低表达组与高表达组脑胶质瘤患者的生存差异。进一步的体外实验以人星形胶质细胞为对照组,通过实时荧光定量聚合酶链式反应(qRT-PCR)实验验证HOXA11-AS在脑胶质瘤U251、U87及LN229细胞株中的表达。U87和LN229细胞株中分别使用携带HOXA11-AS cDNA序列的过表达慢病毒载体构建过表达HOXA11-AS的细胞株,使用空载慢病毒载体构建对照细胞株。U87和LN229细胞株中分别使用siRNA转染敲低HOXA11-AS的表达。进一步通过EdU增殖实验、细胞划痕实验和Transwell实验研究HOXA11-AS对胶质瘤细胞增殖、迁移以及侵袭能力的影响。结果HOXA11-AS在正常脑组织、低级别脑胶质瘤及胶质母细胞瘤中表达量的差异具有统计学意义(P<0.001)。TCGA数据库中,WHOⅢ、Ⅳ级脑胶质瘤的HOXA11-AS表达量均高于Ⅱ级脑胶质瘤(P<0.001)。全级别脑胶质瘤患者中,HOXA11-AS高表达组(n=344)的总生存期短于低表达组(n=345,P<0.001)。进一步的体外实验中,qRT-PCR检测结果显示,与对照组比较,U87组、LN229组及U251组HOXA11-AS的表达均升高(均P<0.001)。U87和LN229过表达组HOXA11-AS的表达均较其对照组上调(均P<0.001)。U87敲低组、LN229敲低组的HOXA11-AS表达均较其对照组下调(均P<0.001)。EdU增殖实验结果显示,U87和LN229过表达组的EdU阳性率分别高于U87和LN229对照组(均P<0.05);相反,U87敲低组较U87无义序列组、LN229敲低组较LN229无义序列组的EdU阳性率均下降(均P<0.001)。细胞划痕实验结果显示,U87和LN229过表达组的细胞迁移率分别高于U87和LN229对照组(均P<0.05);相反,U87敲低组较U87无义序列组、LN229敲低组较LN229无义序列组的细胞迁移率均下降(均P<0.001)。Transwell实验结果显示,U87和LN229过表达组的穿胶细胞数分别多于U87对照组和LN229对照组(均P<0.05);相反,U87敲低组较U87无义序列组、LN229敲低组较LN229无义序列组的穿胶细胞数均低(均P<0.05)。结论HOXA11-AS在脑胶质瘤中呈高表达,并可促进胶质瘤细胞的增殖、迁移和侵袭。

Objective To investigate the expression and biological function of long non-coding RNA Homeobox gene A11 antisense RNA(HOXA11-AS)in gliomas.Methods We first analyzed the expression of HOXA11-AS in the low-grade glioma and glioblastoma in The Cancer Genome Atlas(TCGA)database(689 cases)and normal brain tissue samples from the GTEx database(255 cases).Based on the TCGA database,we further analyzed the expression of HOXA11-AS in World Health Organization(WHO)gradeⅡtoⅣglioma samples.According to the median of HOXA11-AS expression,the testees were divided into high expression group and low expression group,and Kaplan-Meier survival curve was drawn.Log-rank test was used to compare the survival of glioma patients in the HOXA11-AS low expression group with that in the HOXA11-AS high expression group.Human astrocytes were set as the control group and real-time fluorescence quantitative polymerase-linked chain reaction(qRT-PCR)was performed in further in vitro experiments to assess the expression of HOXA11-AS in glioma U251,U87 and LN229 cell lines.In U87 and LN229 cell lines,overexpressed lentiviral vectors carrying the HOXA11-AS cDNA sequence were used to construct cell lines overexpressing HOXA11-AS.Construction of control cell lines was made with lentiviral vector.U87 and LN229 cell lines were transfected with siRNA to knock down the expression of HOXA11-AS.We then further studied the effects of HOXA11-AS on the proliferation,migration and invasion ability of glioma cells through EdU experiment,cell scratch experiment and Transwell experiment.Results The difference in HOXA11-AS expression among normal brain tissue,low-grade glioma and glioblastoma was statistically significant(P<0.001).In the TCGA database,the HOXA11-AS expression levels of WHO gradeⅢandⅣgliomas were higher than that of gradeⅡgliomas(P<0.001).Among glioma(all grades)patients,the overall survival of the HOXA11-AS high expression group(n=344)was shorter than that of the low expression group(n=345,P<0.001).In further in vitro experiments,the qRT-PCR test results showed that the expression of HOXA11-AS in the U87 group,LN229 group and U251 group was increased compared with the control group(all P<0.001).The expression of HOXA11-AS in the U87 and LN229 overexpression groups was up-regulated compared with the control group(both P<0.001).The expression of HOXA11-AS in U87 knockdown groups and LN229 knockdown groups were all down-regulated compared with the control group(all P<0.001).The EdU experiment results showed that the EdU positive rates of U87 and LN229 overexpression groups were higher than those of U87 and LN229 control groups(all P<0.05).On the contrary,the EdU positive rates of U87 knockdown groups were lower than that of U87 nonsense sequence group,and the EdU positive rates of LN229 knockdown groups were lower than that of LN229 nonsense sequence group(all P<0.001).The results of cell scratch experiments showed that the cell migration rates of U87 and LN229 overexpression groups were higher than those of U87 and LN229 control groups(all P<0.05).On the contrary,the cell migration rates of U87 knockdown groups were lower than that of U87 nonsense sequence;compared with the LN229 nonsense sequence group,the cell migration rates of LN229 knockdown groups were both decreased(P<0.001).The results of the Transwell experiment showed that the numbers of gel-penetrating cells in the U87 and LN229 overexpression groups was higher than those in the U87 control group and the LN229 control group(all P<0.05).On the contrary,the numbers of gel-penetrating cells in U87 knockdown groups were lower than that of U87 nonsense sequence group(all P<0.05);the number of gel-penetrating cells in LN229 knockdown groups were lower than that of LN229 nonsense sequence group(all P<0.05).Conclusion HOXA11-AS is highly expressed in brain gliomas and can promote the proliferation,migration and invasion of glioma cells.