摘要
目的:靶向干预血管内皮生长因子(VEGF)mRNA前体(pre-mRNA)第8外显子选择性剪接,重建抗血管生成因子的优势表达,高效抑制肾细胞癌血管生成。方法:针对VEGF pre-mRNA的剪接特点,分别在第7内含子和8a外显子交接附近、交接点和下游设计了与之特异性结合的3条小干扰RNA(siRNA),探讨靶向不同区域的siRNA对VEGF pre-mRNA选择性剪接的影响,并进一步观察其调控肾癌细胞增殖、迁移、克隆形成及血管生成等功能的作用。结果:促血管生成的VEGF剪接体(VEGF165a)在肾细胞癌中表达明显上调,而抗血管生成的VEGF165b在肾细胞癌中表达明显下调;针对剪接位点设计的靶向siRNA能够有效干预VEGF选择性剪接,使VEGF165a表达下降,VEGF165b表达显著上升;与此同时,肾癌细胞增殖、迁移、促血管生成能力显著下降。结论:本研究为进一步利用siRNA调控基因选择性剪接奠定理论基础,并为肾细胞癌的靶向治疗提供了更具针对性的新策略。
Objective:Targeting the splicing of exon 8 of vascular endothelial growth factor(VEGF)gene mRNA precursor to reconstruct the predominant expression of anti-angiogenic factor,and effectively inhibit angiogenesis in renal cell carcinoma.Method:Three specific binding siRNAs were designed near the junction of intron 7 and exon 8 a,at the junction point and downstream,respectively.The effects of siRNA targeting different regions on selective splicing of VEGF pre-mRNA were explored,and the regulation of proliferation,migration,cloning formation of renal cell carcinoma cells and angiogenesis was further observed.Result:In this study,we confirmed that the pro-angiogenic variant VEGF165 awas highly expressed in renal cell carcinoma tissue compared with the normal renal tissue.On the contrary,the anti-angiogenic variant VEGF165 bis lowly expressed.When treated with siRNA that specially targeting the site around the junction of intron 7 and exon 8 aof VEGF pre-mRNA,VEGF165 awas downregulated and VEGF165 bwas upregulated.Meanwhile,cell proliferation,cell migration,as well as angiogenesis were suppressed.Conclusion:This work established a solid theoretical foundation for using siRNA to regulate alternative splicing,and developed a more selective strategy for targeted therapy in renal cell carcinoma.
作者
陶丹
蒋国松
黄超
TAO Dan;JIANG Guosong;HUANG Chao(Department of Oncology,Fifth Hospital of Wuhan,Wuhan,430050,China;Department of Urology,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology)
出处
《临床泌尿外科杂志》
2019年第8期591-597,601,共8页
Journal of Clinical Urology
基金
国家自然科学基金(编号81202018、81702524)
