PO.ET03.05 · 实验与分子治疗
研究PRMT5作为EGFR TKI耐药NSCLC的治疗靶点，并评估黏蛋白作为NSCLC血液生物标志物的作用
Investigating PRMT5 as a therapeutic target in EGFR TKI resistant NSCLC and assessing the role of mucins in NSCLC as blood biomarkers
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摘要 Abstract
中文摘要
肺癌预计将在2025年造成美国超过124,000例死亡，而美国所有肺癌中约10-15%涉及EGFR耐药性NSCLC。EGFR突变NSCLC患者最初对TKI有反应，但常在19个月内复发。PRMT5是一种II型精氨酸甲基转移酶，主要通过PI3K-Akt信号通路驱动致癌和炎症信号传导，而这可能因香烟烟雾而增强。我们假设通过使用siRNA抑制PRMT5将调节这些通路并恢复EGFR-TKI敏感性。在香烟烟雾提取物（CSE）暴露和使用DharmaFECT进行siRNA敲低后，采用qPCR和Western印迹分析EGFR突变NSCLC亲本细胞系（H3255P、H1975P、PC9）和耐药细胞系（H3255OR、H1975OR、PC9OR）中的PRMT5表达。使用qPCR对细胞因子表达进行分析，以评估下游炎症效应。对来自吸烟者和非吸烟者的正常组织和肿瘤组织进行免疫组织化学（IHC）以评估PRMT5水平。使用Keyence显微镜软件（BZX-800分析仪）分析PRMT5水平，并使用Fisher精确检验进行统计分析。CSE使EGFR突变细胞系的PRMT5 mRNA在48小时时增加1.2-11.5倍（p<0.01-0.001）。免疫印迹证实耐药细胞在24-48小时时PRMT5上调1.3-2.5倍（p<0.01-0.001）。在H3255-OR和PC9-OR细胞中，siPRMT5将48小时CSE诱导的细胞因子激增（170-760%）降低了10-20%。与Mock加CSE相比，siPRMT5加CSE降低了IL-8（60-75%）、TNF-alpha（30-50%）和IL-1beta（35-55%）。在奥希替尼与siPRMT5联合治疗后也观察到这些细胞因子的下调。在PC9-P/OR细胞中，与Mock相比，siPRMT5使PRMT5转录本降低35-50%，而与Mock加CSE相比，siPRMT5加CSE降低15-45%。对21例吸烟者和20例非吸烟者肺癌患者肺组织进行的IHC显示，吸烟者的PRMT5表达高于非吸烟者（p<0.05）。还对来自25例吸烟者和19例非吸烟者的正常肺组织进行了研究，使用Fisher精确检验发现吸烟者的PRMT5表达高于非吸烟者（p<0.001）。总之，在奥希替尼耐药的EGFR突变细胞中，PRMT5被CSE上调，且在吸烟者中上调。PRMT5敲低抑制炎症细胞因子，支持抑制PRMT5可能有助于恢复EGFR-TKI敏感性的理论依据。
查看英文原文 English abstract
Lung cancer is on track to cause over 124,000 U.S. deaths in 2025, and about 10-15% of all lung cancers in the United States involve EGFR-resistant NSCLC. Patients with EGFR-mutant NSCLC are initially responsive to TKIs but frequently relapse within 19 months. PRMT5, a type II arginine methyltransferase, drives oncogenic and inflammatory signaling primarily through P13K-Akt signaling, which may be increased by cigarette smoke. We hypothesize that inhibiting PRMT5 by using siRNA will modulate these pathways and restore EGFR-TKI sensitivity. PRMT5 expression was analyzed in EGFR-mutant NSCLC parental cell lines (H3255P, H1975P, PC9) and resistant cell lines (H3255OR, H1975OR, PC9OR) using qPCR and Western blotting following cigarette smoke extract (CSE) exposure and siRNA knockdown using DharmaFECT. Cytokine expression was profiled using qPCR to assess downstream inflammatory effects. Immunohistochemistry (IHC) was performed on normal and tumor tissues from smokers and non-smokers to evaluate PRMT5 levels. The PRMT5 levels were analyzed using a Keyence microscopic software (BZX-800 Analyzer) and statistical analysis was done using Fisher's Exact test. CSE increased PRMT5 mRNA across EGFR-mutant lines by 1.2-11.5 fold at 48 h (p<0.01-0.001). Immunoblotting confirmed PRMT5 upregulation at 24-48 h in resistant cells by 1.3-2.5 fold (p<0.01-0.001). In H3255-OR and PC9-OR cells, siPRMT5 reduced the 48-h CSE-induced cytokine surge (170-760%) by 10-20%. Compared to Mock plus CSE, siPRMT5 plus CSE reduced IL-8 (60-75%), TNF-alpha (30-50%), and IL-1beta (35-55%). Downregulation of these cytokines was also seen after combinatory treatment with osimertinib and siPRMT5. In PC9-P/OR cells, siPRMT5 lowered PRMT5 transcripts by 35-50% compared to Mock and siPRMT5 plus CSE reduced by 15-45% relative to Mock plus CSE. IHC on 21 smoker and 20 non-smoker lung tissues from lung cancer patients showed PRMT5 expression was higher in smokers versus non-smokers (p<0.05). Studies were also conducted on normal lung tissue from 25 smokers and 19 non-smokers and PRMT5 expression was found to be higher in smokers compared to non-smokers using Fisher's exact test (p<0.001). In conclusion, PRMT5 is upregulated by CSE in osimertinib-resistant EGFR-mutant cells and is upregulated in smokers. PRMT5 knockdown suppresses inflammatory cytokines and supports the rationale that inhibiting PRMT5 could help restore EGFR-TKI sensitivity.
利益披露 Disclosure
A. Krishnan, None..
J. Jin, None..
U. Altayeh, None..
J. Hindenburg, None.