Kafka Anja, Bukovac Anja, Brglez Emilija, Jarmek Ana-Marija, Poljak Karolina, Brlek Petar, Žarković Kamelija, Njirić Niko, Pećina-Šlaus Nives
Laboratory of Neuro-Oncology, Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Šalata 12, 10 000 Zagreb, Croatia.
Department of Biology, School of Medicine, University of Zagreb, Šalata 3, 10 000 Zagreb, Croatia.
Cancers (Basel). 2021 May 21;13(11):2530. doi: 10.3390/cancers13112530.
In the present study, we investigated genetic and epigenetic changes and protein expression levels of negative regulators of Wnt signaling, , , and as well as glycogen synthase kinase 3 (GSK3β) and β-catenin in 64 human astrocytomas of grades II-IV. Methylation-specific PCR revealed promoter methylation of , , and in 38%, 43%, and 18% of samples, respectively. Grade IV comprised the lowest number of methylated cases and highest of . Evaluation of the immunostaining using H-score was performed for β-catenin, both total and unphosphorylated (active) forms. Additionally, active (pY216) and inactive (pS9) forms of GSK3β protein were also analyzed. Spearman's correlation confirmed the prevalence of β-catenin's active form (r = 0.634, < 0.001) in astrocytoma tumor cells. The Wilcoxon test revealed that astrocytoma with higher levels of the active pGSK3β-Y216 form had lower expression levels of its inactive form ( < 0.0001, Z = -5.332). Changes in exon 11 were observed in 44.44% of samples by PCR/RFLP. Astrocytomas with changes of had higher H-score values of total β-catenin compared to the group without genetic changes (t = -2.264, = 0.038). Furthermore, a positive correlation between samples with methylated promoter and the expression of active pGSK3β-Y216 (r = 0.356, = 0.011) was established. Our results emphasize the importance of methylation for the regulation of Wnt signaling. Large deletions of the gene associated with increased β-catenin levels, together with oncogenic effects of both β-catenin and GSK3β, are clearly involved in astrocytoma evolution. Our findings contribute to a better understanding of the etiology of gliomas. Further studies should elucidate the clinical and therapeutic relevance of the observed molecular alterations.
在本研究中，我们调查了64例II - IV级人类星形细胞瘤中Wnt信号通路负调节因子、糖原合酶激酶3（GSK3β）和β-连环蛋白的基因和表观遗传变化以及蛋白质表达水平。甲基化特异性PCR显示，分别有38%、43%和18%的样本中、和启动子发生甲基化。IV级中甲基化的病例数最少，而甲基化的病例数最多。使用H评分对β-连环蛋白的总形式和未磷酸化（活性）形式进行免疫染色评估。此外，还分析了GSK3β蛋白的活性（pY216）和非活性（pS9）形式。Spearman相关性证实了β-连环蛋白活性形式在星形细胞瘤肿瘤细胞中的普遍性（r = 0.634，<0.001）。Wilcoxon检验显示，活性pGSK3β-Y216形式水平较高的星形细胞瘤其非活性形式的表达水平较低（<0.0001，Z = -5.332）。通过PCR/RFLP在44.44%的样本中观察到外显子11的变化。与无基因变化的组相比，发生变化的星形细胞瘤总β-连环蛋白的H评分值更高（t = -2.264，= 0.038）。此外，启动子甲基化的样本与活性pGSK3β-Y216的表达之间建立了正相关（r = 0.356，= 0.011）。我们的结果强调了甲基化对Wnt信号通路调节的重要性。与β-连环蛋白水平升高相关的基因大片段缺失，以及β-连环蛋白和GSK3β的致癌作用，显然都参与了星形细胞瘤的演变。我们的发现有助于更好地理解胶质瘤的病因。进一步的研究应阐明所观察到的分子改变的临床和治疗相关性。
