WDR82在神经胶质瘤发生发展中的作用及其机制

Role and mechanism of WDR82 in the genesis and development of glioma

目的探讨WD重复结构域82(WDR82)蛋白在神经胶质瘤发生发展中的作用及其机制。方法利用GEPIA和UALCAN数据库分析WDR82在神经胶质瘤组织中的表达情况;采用Kaplan-Meier生存分析WDR82表达与神经胶质瘤患者预后的关系。采用组织芯片进行免疫组织化学染色检测WDR82在神经胶质瘤组织及其癌旁组织中的表达情况。取人胶质瘤A172和U251细胞,设置对照组(转染3μg pcDNA3)、p WDR82组(转染3μg pWDR82)、shR-Con组(转染3μg pSilencer2.1-U6)、shR-WDR82组(转染3μg shR-WDR82)。转染48 h后,采用q RT-PCR验证各组转染效率;CCK-8法检测转染48 h的细胞活性,克隆形成实验检测转染后14 d的细胞增殖能力,流式细胞术检测转染48 h的细胞凋亡情况;Western blotting检测转染48 h细胞中增殖相关蛋白及Akt/mTOR信号通路相关蛋白的表达。采用体内荷瘤实验检测WDR82对NOD-SCID小鼠肿瘤生长的影响。结果GEPIA和UALCAN数据库分析及免疫组织化学染色结果显示,WDR82在神经胶质瘤组织中的表达水平明显高于癌旁正常组织(P<0.05),其表达在不同性别及年龄段患者间差异无统计学意义(P>0.05)。Kaplan-Meier生存分析结果显示,高表达WDR82的神经胶质瘤患者的预后不良(log-rank P=0.029)。过表达WDR82可明显促进A172和U251细胞的增殖,抑制细胞凋亡,明显增高A172和U251细胞中MKI67、BCL2、CCND1、p-Akt和p-mTOR的表达(P<0.05)。反之,敲降WDR82则抑制A172和U251细胞的增殖,促进细胞凋亡,降低MKI67、BCL2、CCND1、p-Akt和p-mTOR的表达(P<0.05)。敲降U251细胞中的WDR82,可明显抑制荷瘤NOD-SCID小鼠肿瘤的生长(P<0.05)。结论高表达WDR82可能通过调控Akt/mTOR信号通路促进神经胶质瘤细胞的增殖及体内肿瘤的生长。

Objective To investigate the role and underlying mechanisms of WD repeat domain 82(WDR82)protein in the pathogenesis and progression of glioma.Methods We analyzed the expression level of WDR82 in glioma tissues using GEPIA and UALCAN databases and further assessed WDR82 protein expression in glioma and adjacent normal tissues through immunohistochemical staining.The correlation between WDR82 expression and the prognosis of glioma patient was evaluated using Kaplan-Meier plotter.Experiments were conducted on A172 and U251 cell lines,which were categorized into four groups:control group(transfected with 3μg pcDNA3),shR-control group(transfected with 3μg pSilencer 2.1-U6),pWDR82 group(transfected with 3μg pWDR82),and shR-WDR82 group(transfected with 3μg shR-WDR82).Post-transfection,we confirmed transfection efficiency at 48 hours using qRT-PCR and measured cell viability at the same time point with CCK-8 assay.Clone formation assay was employed to assess cell proliferation capacity after 14 days of transfection,while flow cytometry was utilized to analyze cell apoptosis after 48 hours of transfection.Additionally,Western blotting was conducted to determine the expression levels of proteins related to proliferation and Akt/mTOR signaling pathway after 48 hours of transfection.Finally,the effect of WDR82 on tumor growth in NOD-SCID mice was investigated using tumor carrying experiment in vivo.Results Analysis of WDR82 expression in glioma tissues using GEPIA and UALCAN databases,along with immunohistochemical staining,revealed significantly higher expression levels compared to normal and paracancerous tissues(P<0.05).Additionally,WDR82 expression was not associated with gender or age of patients(P>0.05).Kaplan-Meier plotter analysis indicated that elevated WDR82 expression correlated with a poor prognosis in glioma patients(log-rank P=0.029).Overexpression of WDR82 notably enhanced the proliferation and inhibited the apoptosis of A172 and U251 cells,and also significantly upregulated the expression of MKI67,BCL2,CCND1,p-Akt and p-mTOR in A172 and U251 cells(P<0.05).Conversely,WDR82 knockdown had the opposite effects,inhibiting cell proliferation,increasing apoptosis and downregulating the expression of MKI67,BCL2,CCND1,p-Akt and p-mTOR(P<0.05).WDR82 knockdown in U251 cells significantly inhibited tumor growth in NOD-SCID mice(P<0.05).Conclusion High expression of WDR82 promotes the proliferation of glioma cells and the growth of tumors in vivo by regulating the AKT/mTOR signaling pathway.