Tadayoni Nia Amin, Bazi Zahra, Khosravi Ayyoob, Oladnabi Morteza
Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran.
Department of Medical Genetics, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran.
J Mol Neurosci. 2021 Aug;71(8):1696-1702. doi: 10.1007/s12031-021-01849-z. Epub 2021 May 6.
Glioblastoma is a very invasive and prevalent brain tumor that affects 15 in 100,000 persons over the age of 70 years. Studies have shown that the expression of the WD repeat domain 81 (WDR81) gene, which is effective in vesicular transport and inhibition of autophagy, is increased in glioblastoma. The decreased autophagy was found to be related to the increased production of exosomes, which is a major factor in the pathogenesis of glioblastoma. The PI-3kinase complex is a pre-autophagic complex that is highly active in the absence of WDR81. The WDR81 gene, as a negative regulator of PI3K activity, prevents autophagy and increases exosome secretion by preventing the formation of the class III PI3K complex. Therefore, targeted reduction of exosomes can be considered an effective strategy for reducing the pathogenesis of glioblastoma. This study aimed to assess the effect of WDR81 gene silencing with siRNA on exosome levels in a U87-MG cell line. Culturing of U87-MG cells was carried out in Dulbecco's modified Eagle medium (DMEM) containing 5% FBS and 1% penicillin/streptomycin. Thereafter, silencing of WDR81 was performed using WDR81 siRNA, whose gene expression level was determined via real-time qRT-PCR. Cell viability was evaluated using the MTT assay. The exosomes were extracted from a cell culture using the Exocib kit. The size accuracy of the exosomes was confirmed by dynamic light scattering (DLS). Finally, the protein content and RNA of the exosomes were assessed. WDR81 gene expression of siRNA-transfected cells was decreased to 82% after 24 h compared to the non-transfected control cells. The analysis of the exosomes showed that the concentration of exosomes and their RNA and protein content in the siRNA-transfected cells decreased significantly compared to the non-transfected control cells. No considerable difference was observed in cell viability after transfection with either WDR81-specific siRNAs or scrambled control siRNAs. Our findings showed that silencing the WDR81 gene could reduce the level of exosomes in human U87-MG glioblastoma cells. Therefore, the reduced exosome content may be suggested as a new gene therapy strategy for targeted therapy of glioblastoma by increasing autophagy via activation of PI3KIII. However, more studies are needed in this regard.
胶质母细胞瘤是一种极具侵袭性且常见的脑肿瘤，在70岁以上人群中，每10万人中有15人受其影响。研究表明，WD重复结构域81（WDR81）基因在囊泡运输和抑制自噬方面发挥作用，其在胶质母细胞瘤中的表达增加。研究发现，自噬减少与外泌体产生增加有关，而外泌体产生增加是胶质母细胞瘤发病机制的一个主要因素。PI-3激酶复合物是一种自噬前体复合物，在缺乏WDR81时高度活跃。WDR81基因作为PI3K活性的负调节因子，通过阻止III类PI3K复合物的形成来阻止自噬并增加外泌体分泌。因此，靶向降低外泌体水平可被视为降低胶质母细胞瘤发病机制的有效策略。本研究旨在评估用小干扰RNA（siRNA）沉默WDR81基因对U87-MG细胞系中外泌体水平的影响。U87-MG细胞在含有5%胎牛血清和1%青霉素/链霉素的杜氏改良 Eagle培养基（DMEM）中培养。此后，使用WDR81 siRNA对WDR81进行沉默，并通过实时定量逆转录聚合酶链反应（qRT-PCR）测定其基因表达水平。使用MTT法评估细胞活力。使用Exocib试剂盒从细胞培养物中提取外泌体。通过动态光散射（DLS）确认外泌体的大小准确性。最后，评估外泌体的蛋白质含量和RNA。与未转染的对照细胞相比，转染siRNA的细胞在24小时后WDR81基因表达降低至82%。外泌体分析表明，与未转染的对照细胞相比，转染siRNA的细胞中外泌体浓度及其RNA和蛋白质含量显著降低。用WDR81特异性siRNA或乱序对照siRNA转染后，细胞活力未观察到显著差异。我们的研究结果表明，沉默WDR81基因可降低人U87-MG胶质母细胞瘤细胞中外泌体水平。因此，外泌体含量降低可能是一种新的基因治疗策略，通过激活PI3KIII增加自噬来靶向治疗胶质母细胞瘤。然而，在这方面还需要更多的研究。
