Bhardwaj Sachin, Yadav Ajay Kumar
Molecular Cancer Genetics and Signal Transduction Laboratory, Dr. B.R Ambedkar Center for Biomedical Research, University of Delhi, North Campus, Gate No. 1, Vishwavidyalaya Marg, Mall Road, 44, AH2, Delhi, 110007, India.
J Neurooncol. 2025 Jan;171(1):47-63. doi: 10.1007/s11060-024-04831-y. Epub 2024 Nov 25.
Gliblastoma is a malignant brain tumor; despite available treatment modalities, the tumor reoccurrence rate persist in the currently prescribed Temozolomide chemotherapy. Study aimed to study the inquisitive role of RNA binding splice factor protein hnRNPA1 in promoting glioma resistance against Temozolomide drug and therapeutic insights.
In this study two non-expressing O-methylguanine-DNA methyltransferase (MGMT) glioma cell lines U87MG & LN229. U87MG cells were grown in Temozolomide from 50μM upto 400μM & LN229 cells grown upto 200μM, till then both these cells acquired Temozolomide resistance. Both of these cells were grown & maintained continously in its highest dose of Temozolomide (TMZ). Splice factor protein SF2/ASF1 was functionally correlated with abundance of hnRNPA1 protein in Temozolomide (TMZ) resistant cells using its specific siRNA transfection approach, in detrmining SF2/ASF1 mediated hnRNPA1 splicing and Temozolomide resistant reversal.
U87MG TMZ resistance, results an increase in the expression of pre mRNA-splicing factor SF2/ASF1, Heterogeneous Ribonucleoprotein A1 (hnRNPA1) and O-methylguanine-DNA methyltransferase (MGMT) protein. MGMT expression was not observed in LN229 TMZ resistant cells. Further, mRNA sequencing of hnRNPA1 confirmed the exclusive abundance of its higher isoform in TMZ- resistant cells along with increase in SF2/ASF1 expression. Knocking down of SF2/ASF1 using its specific siRNA reverted the higher isoform of hnRNPA1 isoform Var2 to its lower isoform hnRNPA1 Var1 in U87 TMZ resistant cells, reveals hnRNPA1 alternative higher isoform abundance is SF2/ASF1 splice factor dependent. Additionally, selective knock down of hnRNPA1 higher isoform Var2 in TMZ resistant U87MG & LN229 promotes apoptosis, was further specfically enhanced on Wortmannin (PI3Kinase inhibitor) treatment.
Targeting higher isoform Var2 of hnRNPA1 specifically induces chemosensitization in MGMT expressed Temozolomide resistant U87MG as well as in MGMT non-expressed LN229 TMZ resistant cells.
胶质母细胞瘤是一种恶性脑肿瘤；尽管有多种可用的治疗方式，但在目前规定的替莫唑胺化疗中，肿瘤复发率仍然居高不下。本研究旨在探讨RNA结合剪接因子蛋白hnRNPA1在促进胶质瘤对替莫唑胺耐药中的作用及治疗意义。
在本研究中，使用了两种不表达O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）的胶质瘤细胞系U87MG和LN229。U87MG细胞在浓度从50μM到400μM的替莫唑胺中培养，LN229细胞在浓度高达200μM的替莫唑胺中培养，直到这两种细胞都获得了对替莫唑胺的耐药性。这两种细胞都在其最高剂量的替莫唑胺（TMZ）中持续培养和维持。通过特异性siRNA转染方法，研究剪接因子蛋白SF2/ASF1与替莫唑胺（TMZ）耐药细胞中hnRNPA1蛋白丰度的功能相关性，以确定SF2/ASF1介导的hnRNPA1剪接和替莫唑胺耐药逆转情况。
U87MG对替莫唑胺耐药，导致前体mRNA剪接因子SF2/ASF1、异质性核糖核蛋白A1（hnRNPA1）和O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）蛋白的表达增加。在LN229替莫唑胺耐药细胞中未观察到MGMT表达。此外，hnRNPA1的mRNA测序证实其更高异构体在替莫唑胺耐药细胞中特异性丰富，同时SF2/ASF1表达增加。在U87替莫唑胺耐药细胞中，使用其特异性siRNA敲低SF2/ASF1可将hnRNPA1异构体Var2的更高异构体恢复为其较低异构体hnRNPA1 Var1，表明hnRNPA1更高异构体的丰富是SF2/ASF1剪接因子依赖性的。此外，在替莫唑胺耐药的U87MG和LN229细胞中选择性敲低hnRNPA1更高异构体Var2可促进细胞凋亡，在渥曼青霉素（PI3激酶抑制剂）处理后进一步特异性增强。
靶向hnRNPA1的更高异构体Var2可特异性诱导MGMT表达的替莫唑胺耐药U87MG以及MGMT未表达的LN229替莫唑胺耐药细胞的化学增敏作用。
