<i>PEA3</i>和<i>EPHA2</i>在脑胶质母细胞瘤中的表达及在Wnt/β-catenin通路的作用

doi:10.3877/cma.j.issn.2095-123X.2024.02.002

目的

探讨多瘤病毒增强活化子3（PEA3）及红细胞生成素产生肝细胞受体2（EPHA2）在脑胶质母细胞瘤中的表达及在Wnt/β-catenin信号通路的作用。

方法

构建脑胶质瘤U87细胞基因转染模型，将细胞系分为5组：空白组、PEA3干扰组、PEA3干扰空载组、EPHA2干扰组、EPHA2干扰空载组，Western blotting实验检测细胞中EPHA2、PEA3的蛋白表达水平，细胞活力检测（CCK-8）实验检测PEA3、EPHA2对细胞增殖能力的影响，定量反转录聚合酶连锁反应（qRT-PCR）实验检测Wnt/β-catenin通路下游基因转录因子4（TCF-4）、淋巴细胞增强结合因子1（LEF1）表达水平。

结果

Western blotting实验结果显示：与空白组相比，EPHA2在EPHA2干扰组中表达量降低，PEA3在PEA3干扰组和EPHA2干扰组中表达量降低，差异均具有统计学意义（P<0.05）；5组细胞Wnt1、β-catenin蛋白表达量比较，差异无统计学意义(P>0.05)。CCK-8实验结果显示，与空白组相比，PEA3干扰组和EPHA2干扰组的细胞增殖率明显下降，差异均有统计学意义（P<0.05）。qRT-PCR结果显示，TCF-4、LEF1基因在PEA3干扰组、EPHA2干扰组中表达量下降，差异均有统计学意义（P<0.05）。

结论

通过干扰PEA3和EPHA2基因可降低胶质母细胞瘤的增殖能力，但PEA3和EPHA2是否通过Wnt/β-catenin通路促进胶质母细胞瘤的增殖能力值得进一步研究证实。

关键词: 多瘤病毒增强活化子3, 红细胞生成素产生肝细胞受体2, 胶质母细胞瘤, Wnt/β-catenin信号通路

Objective

To explore the expression of polyomavirus enhancer activator 3 (PEA3) and erythropoietin-producting hepatocellular receptor 2 (EPHA2) in glioblastoma and their expression in Wnt/β-catenin pathway.

Methods

A gene transfection model of U87 glioma cells was constructed, and the cell lines were divided into 5 groups: blank group, PEA3 interference group, PEA3 interference empty group, EPHA2 interference group, and EPHA2 interference empty group. The protein expression levels of EPHA2 and PEA3 in U87 cells were detected through Western blotting experiments; The proliferation rate was detected by CCK-8 test, and the the expression levels of T cytokines factor-4 (TCF-4) and lymph enhancer factor 1 (LEF1) genes, the downstream genes of Wnt/β-catenin pathway, were detected by quantitative real time polymerase chain reaction (qRT-PCR) assay.

Results

The Western blotting experiment showed that compared with the blank group, the expression level of PEA3 was reduced in the PEA3 interference group and EPHA2 interference group, while the expression level of EPHA2 was reduced in the EPHA2 interference group, and the differences were statistically significant (P<0.05); There was no statistically significant difference in the expression levels of Wnt1 and β-catenin proteins among 5 groups (P>0.05). The CCK-8 experiment showed that compared with the blank group, the cell proliferation rates of the PEA3 interference group and the EPHA2 interference group were significantly reduced, and the differences were statistically significant (P<0.05). The qRT-PCR results showed that the expression levels of TCF-4 and LEF1 genes decreased in the PEA3 interference group and EPHA2 interference group, and the differences were statistically significant (P<0.05).

Conclusion

Interference with PEA3 and EPHA2 genes can reduce the proliferation ability of glioblastoma; whether PEA3 and EPHA2 promote the proliferative capacity of glioblastoma through the Wnt/β-catenin pathway deserves further investigation.

Key words: Polyomavirus enhancer activator 3, Erythropoietin-producting hepatocellular receptor 2, Glioblastoma, Wnt/β-catenin pathway

表1 引物序列

Tab.1 Primer sequence

基因	序列
β-actin	F-CCCATCTATGAGGGTTACGC
β-actin	R-TTTAATGTCACGCACGATTTC
PEA3	F-GATGAAAGCCGGATACTTGGAC
PEA3	R-TTCGCGCAAGCTCCCATTT
EPHA2	F-TGGCTCACACACCCGTATG
EPHA2	R-GTCGCCAGACATCACGTTG
TCF-4	F-GCAGCAAGTCCGAGAAAGGA
TCF-4	R-GTCTCCCATTCCAGGGTGTG
LEF1	F-CTTCCATGTCCAGGTTTTCCC
LEF1	R-GGCTCCTGCTCCTTTCTCTG

表1 引物序列

Tab.1 Primer sequence

基因	序列
β-actin	F-CCCATCTATGAGGGTTACGC
β-actin	R-TTTAATGTCACGCACGATTTC
PEA3	F-GATGAAAGCCGGATACTTGGAC
PEA3	R-TTCGCGCAAGCTCCCATTT
EPHA2	F-TGGCTCACACACCCGTATG
EPHA2	R-GTCGCCAGACATCACGTTG
TCF-4	F-GCAGCAAGTCCGAGAAAGGA
TCF-4	R-GTCTCCCATTCCAGGGTGTG
LEF1	F-CTTCCATGTCCAGGTTTTCCC
LEF1	R-GGCTCCTGCTCCTTTCTCTG

图1 U87细胞质粒转染图（×400）A：空白组；B：PEA3干扰组；C：PEA3干扰空载组；D：EPHA2干扰组；E：EPHA2干扰空载组

Fig.1 Plasmid transfection map of U87 cells (×400)

图2 PEA3、EPHA2基因转染效率检测结果与空白组比较，^aP<0.05

Fig.2 Detection of transfection efficiency of PEA3 and EPHA2 genes

图3 5组细胞中EPHA2、PEA3、Wnt1、β-catenin蛋白Western blotting结果

Fig.3 Western blotting results of EPHA2, PEA3, Wnt1, β-catenin of 5 groups

图4 5组细胞中EPHA2、PEA3、Wnt1、β-catenin蛋白表达水平比较A：EPHA2；B：PEA3；C：Wnt1；D：β-catenin；与空白组比较，^aP<0.05

Fig.4 Expression levels of EPHA2, PEA3, Wnt1, β-catenin protein in 5 groups

图5 5组细胞增殖率比较与空白组比较，^aP<0.05

Fig.5 Comparison of cell proliferation rates among 5 groups

图6 5组细胞TCF-4、LEF1基因表达量比较A：TCF-4；B：LEF1；与空白组比较，^aP<0.05

Fig.6 Comparison of expression levels of TCF-4 and LEF1 genes in 5 groups

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Expression of PEA3 and EPHA2 in glioblastoma and role in the Wnt/β-catenin pathway

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Expression of PEA3 and EPHA2 in glioblastoma and role in the Wnt/β-catenin pathway