Department of Biomedical and Biotechnological Sciences-Section of Biology and Genetics, University of Catania, 95123 Catania, Italy.
Department of Medical, Surgical Sciences and Advanced Technologies and Biotechnological Sciences G.F. Ingrassia, University of Catania, 95123 Catania, Italy.
Int J Mol Sci. 2018 Feb 6;19(2):480. doi: 10.3390/ijms19020480.
Circular RNAs (circRNAs) have recently emerged as a new class of RNAs, highly enriched in the brain and very stable within cells, exosomes and body fluids. To analyze their involvement in glioblastoma multiforme (GBM) pathogenesis, we assayed the expression of twelve circRNAs, physiologically enriched in several regions of the brain, through real-time PCR in a cohort of fifty-six GBM patient biopsies and seven normal brain parenchymas. We focused on hsa_circ_0001445 (circSMARCA5): it was significantly downregulated in GBM biopsies as compared to normal brain tissues (-value < 0.00001, student's -test), contrary to its linear isoform counterpart that did not show any differential expression (-value = 0.694, student's -test). Analysis of a public dataset revealed a negative correlation between the expression of circSMARCA5 and glioma's histological grade, suggesting its potential negative role in the progression to malignancy. Overexpressing circSMARCA5 in U87MG cells significantly decreased their migration, but not their proliferation rate. In silico scanning of circSMARCA5 sequence revealed an enrichment in binding motifs for several RNA binding proteins (RBPs), specifically involved in splicing. Among them, serine and arginine rich splicing factor 1 (SRSF1), a splicing factor known to be a positive controller of cell migration and known to be overexpressed in GBM, was predicted to bind circSMARCA5 by three different prediction tools. Direct interaction between circSMARCA5 and SRSF1 is supported by enhanced UV crosslinking and immunoprecipitation (eCLIP) data for SRSF1 in K562 cells from Encyclopedia of DNA Elements (ENCODE). Consistently, U87MG overexpressing circSMARCA5 showed an increased expression of serine and arginine rich splicing factor 3 (SRSF3) RNA isoform containing exon 4, normally skipped in a SRSF1-dependent manner, resulting in a non-productive non-sense mediated decay (NMD) substrate. Interestingly, SRSF3 is known to interplay with two other splicing factors, polypyrimidine tract binding protein 1 (PTBP1) and polypyrimidine tract binding protein 2 (PTBP2), that positively regulate glioma cells migration. Collectively, our data show circSMARCA5 as a promising druggable tumor suppressor in GBM and suggest that it may exert its function by tethering the RBP SRSF1.
环状 RNA（circRNAs）是一类新型的 RNA，在大脑中高度富集，在细胞、外泌体和体液中非常稳定。为了分析它们在多形性胶质母细胞瘤（GBM）发病机制中的作用，我们通过实时 PCR 检测了 12 种在大脑多个区域生理上丰富的 circRNAs 在 56 例 GBM 患者活检和 7 例正常脑组织中的表达。我们专注于 hsa_circ_0001445（circSMARCA5）：与正常脑组织相比，它在 GBM 活检中显著下调（-值<0.00001，student's t 检验），而其线性异构体则没有表现出任何差异表达（-值=0.694，student's t 检验）。对公共数据集的分析显示，circSMARCA5 的表达与神经胶质瘤的组织学分级呈负相关，表明其在恶性进展中可能发挥负作用。在 U87MG 细胞中过表达 circSMARCA5 显著降低了它们的迁移率，但不影响它们的增殖率。circSMARCA5 序列的计算机扫描显示，它富含几种 RNA 结合蛋白（RBPs）的结合基序，这些基序专门参与剪接。其中，剪接因子 1（SRSF1）是一种剪接因子，已知其为细胞迁移的正调控因子，并且在 GBM 中过度表达，被三个不同的预测工具预测为与 circSMARCA5 结合。来自 DNA 元素百科全书（ENCODE）的 K562 细胞中的增强紫外线交联和免疫沉淀（eCLIP）数据支持 circSMARCA5 与 SRSF1 之间的直接相互作用。一致地，过表达 circSMARCA5 的 U87MG 细胞显示出含有外显子 4 的剪接因子 3（SRSF3）RNA 异构体的表达增加，该外显子 4 通常以 SRSF1 依赖性方式被跳过，导致非生产性无义介导的衰变（NMD）底物。有趣的是，SRSF3 已知与另外两个剪接因子多嘧啶 tract 结合蛋白 1（PTBP1）和多嘧啶 tract 结合蛋白 2（PTBP2）相互作用，这两个因子正向调节神经胶质瘤细胞的迁移。总的来说，我们的数据表明 circSMARCA5 是 GBM 中一种有前途的可药物治疗的肿瘤抑制因子，并表明它可能通过锚定 RBP SRSF1 来发挥其功能。
