| 牟瑞姝,周雨昕,李雪东,等.采用新型微泡技术靶向AC005229.4/Cul1抑制肝癌对索拉非尼耐药的研究[J].肿瘤学杂志,2026,32(8):677-691. |
| 采用新型微泡技术靶向AC005229.4/Cul1抑制肝癌对索拉非尼耐药的研究 |
| Inhibition of Liver Cancer Resistance to Sorafenib by Targeting AC005229.4/Cul1 with Novel Microbubble Technology |
| 投稿时间：2025-03-03 |
| DOI：10.11735/j.issn.1671-170X.2026.08.B007 |
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| 中文关键词: 肝肿瘤 液态氟碳纳米粒 索拉非尼 耐药 长链非编码RNA |
| 英文关键词:liver neoplasms liquid fluorocarbon nanoparticles sorafenib drug resistance lncRNA |
| 基金项目:国家自然科学基金（82073301） |
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| 摘要点击次数: 9 |
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| 中文摘要: |
| 摘 要：［目的］ 探究索拉非尼治疗晚期肝癌的耐药机制，寻找新的分子靶点，为提升索拉非尼疗效提供新策略。［方法］ 利用美国国家生物技术信息中心（National Center for Biotechnology Information，NCBI）的基因表达综合数据库（Gene Expression Omnibus，GEO）公共数据集收集肝癌细胞和肝癌患者在索拉非尼使用前后基因差异表达数据情况，癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库分析Cul1基因在肝癌中的表达及与患者预后相关性。采用免疫组化和Western blot检测Cul1和细胞周期相关因子的表达水平。利用CRISPR/Cas9基因编译技术过表达Cul1，进行流式细胞学和免疫共沉淀明确Cul1对肝癌细胞活力、凋亡和细胞周期的影响。于Cul1基因反义链附近转录出lncRNA-AC005229.4并确定其基因序列，采用CRISPR/Cas9基因编译过表达AC005229.4，利用聚合酶链式反应（polymerase chain reaction，PCR）检测AC005229.4基因表达水平，通过Jasper生物信息学分析、RIP检测、双荧光素酶报告基因检测和ChIP检测确定AC005229.4诱导Cul1表达的作用机制。于体外通过Western blot、PCR、双荧光素酶报告基因检测进行靶向抑制AC005229.4和Cul1 miRNAs的筛选和验证。超声乳化法构建多功能液态氟碳纳米粒PFH/ICG/miR-520b-3p/PLGA-PEI，使用肝癌HepG2（R）细胞建立裸鼠皮下移植瘤模型，于饮用水中加入索拉非尼，进行肿瘤组织的活体外荧光成像，检测裸鼠瘤的体积和重量，通过肿瘤组织免疫组化、PCR检测进一步验证多功能液态氟碳纳米粒PFH/ICG/miR-520b-3p/PLGA-PEI 对裸鼠HepG2（R）细胞瘤增殖的影响。 ［结果］ TCGA数据库分析显示，Cul1基因在肝癌组织表达有上升趋势，Cul1表达水平越高，肝癌患者预后越差。免疫组化和Western blot检测显示索拉非尼耐药患者肝癌组织中Cul1和SKP2表达水平升高，P27和P21的蛋白水平降低，而CDKs/Cyclin信号下游因子p-Rb、E2F和c-myc蛋白水平出现增加。过表达Cul1后，肝癌细胞在S期的比例提升了50%，而在G1期的比例平均下降了25%。过表达Cul1抑制了细胞凋亡并使得不同泛素多聚体的水平增加。肝癌中AC005229.4与Cul1 mRNA的表达水平呈正相关（r=0.620，P<0.001）。Jasper生物信息学分析、RIP检测、双荧光检测和ChIP检测均明确转录因子CUX2是诱导Cul1表达的转录因子，而AC005229.4过表达后促进CUX2与Cul1基因启动子的结合，说明AC005229.4是通过cis机制对Cul1表达进行诱导。体外的Western blot、PCR、双荧光素酶报告基因检测实验说明了耐药细胞株HepG2和Huh7中的AC005229.4近3倍表达水平和Cul1蛋白近5倍表达量的显著上调，且发现了miR-520b-3p同时抑制Cul1和AC005229.4的表达。在体内实验中，肿瘤组织的免疫组化和PCR检测证实了PFH/ICG/miR-520b-3p/PLGA-PEI对肿瘤生长的抑制，提升了HepG2（R）细胞裸鼠瘤中miR-520b-3p的表达，下调了AC005229.4、Cul1的表达。［结论］ 采用新型微泡技术靶向AC005229.4/Cul1，可抑制Cul1表达，提升肝癌的索拉非尼耐药细胞的药物敏感性，为晚期肝癌靶向研究与治疗提供重要理论基础和有效的技术手段。 |
| 英文摘要: |
| Abstract： ［Objective］ To investigate the mechanisms underlying sorafenib resistance in advanced hepatocellular carcinoma (HCC) and identify novel molecular targets to improve sorafenib efficacy. ［Methods］ Gene expression profiles of HCC cells and patients pre- and post-sorafenib treatment were retrieved from the NCBI GEO database. The Cancer Genome Atlas (TCGA) was utilized to analyze Cul1 expression in HCC tissues and its correlation with patient prognosis. Immunohistochemistry and Western blot were employed to assess protein levels of Cul1 and cell cycle-related factors. CRISPR/Cas9-mediated Cul1 overexpression was performed to evaluate its effects on HCC cell viability， apoptosis， and cell cycle progression via flow cytometry and co-immunoprecipitation. The antisense lncRNA-AC005229.4 adjacent to Cul1 was identified and sequenced. CRISPR/Cas9 was used to overexpress AC005229.4， followed by PCR validation. Mechanistic insights into AC005229.4-mediated Cul1 regulation were explored using Jasper bioinformatics prediction， RNA immunoprecipitation， dual-luciferase assays， and chromatin immunoprecipitation. Western blot， PCR， and dual-luciferase assays were conducted to screen and validate miRNAs targeting AC005229.4 and Cul1. A multifunctional perfluorocarbon nanobubble (PFH/ICG/miR-520b-3p/PLGA-PEI) was synthesized and tested in a sorafenib-resistant HepG2(R) subcutaneous xenograft nude mouse model. Tumor growth， volume， weight， and molecular markers were analyzed via in vivo fluorescence imaging， IHC， and PCR. ［Results］ TCGA analysis revealed elevated Cul1 expression in hepatocellular carcinoma tissues， correlating with poor prognosis. Immunohistochemistry and Western blot confirmed upregulated Cul1 and SKP2， downregulated P27/P21， and increased p-Rb， E2F， and c-Myc in sorafenib-resistant hepatocellular carcinoma tissues. Cul1 overexpression increased S-phase cell proportion by 50%， reduced G1-phase cells by 25%， suppressed apoptosis， and enhanced ubiquitinated polyprotein levels. AC005229.4 expression was positively correlated with Cul1 mRNA in HCC(r=-0.620， P<0.001). AC005229.4 facilitated transcriptional factor CUX2 binding to the Cul1 promoter via cis-regulation， as validated by chromatin immunoprecipitation and RNA immunoprecipitation. The miR-520b-3p simultaneously suppressed AC005229.4 and Cul1 expression in resistant HepG2 and Huh7 cells (3- and 5- fold downregulation， respectively). PFH/ICG/miR-520b-3p/PLGA-PEI nanoparticles inhibited tumor growth in vivo， upregulated miR-520b-3p， and downregulated AC005229.4 and Cul1 in xenografts. ［Conclusion］ Targeting the AC005229.4/Cul1 axis via a novel microbubble-based strategy suppresses Cul1 expression and resensitizes sorafenib-resistant HCC cells， providing an experimental evidence for innovative therapeutic approach for advanced HCC. |
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