新型miRNA表达框架下调端粒酶活性并抑制视网膜母细胞瘤细胞生长  

Novel miRNA Expression Cassette Downregulates Telomerase Reverse Transcriptase and Inhibits Cell Growth in Retinoblastoma Cells

作　　者：赵丹丹 丁彩云[2] 李文涛 ZHAO Dan-Dan;DING Cai-Yun;LI Wen-Tao(Shanxi Eye Hospital,Affiliate of Shanxi Medical University,Taiyuan 030002,China;Central Laboratory,Shanxi Provincial People’s Hospital,Affiliate of Shanxi Medical University,Taiyuan 030001,China;Taiyuan University of Science and Technology,Taiyuan 030024,China)

机构地区：[1]山西医科大学附属山西眼科医院,太原030002 [2]山西医科大学附属山西省人民医院中心实验室,太原030001 [3]太原科技大学,太原030024

出　　处：《生物化学与生物物理进展》2022年第12期2428-2439,共12页Progress In Biochemistry and Biophysics

基　　金：山西省自然科学基金青年项目(201801D221256);山西省眼科医院青年基金(Q201803)资助项目。

摘　　要：目的本研究致力于设计一种新的miRNA表达框架(MEC),其以hTERT和hTR的特异性序列为靶点。该框架能够有效改善传统RNAi方法中miRNA易降解和细胞毒性问题,为miRNA的合成提供一种简便的方法。方法采用重叠PCR方法构建hTERT和hTR特异的miRNA表达框架。采用TRAP银染色和TRAP实时荧光定量PCR检测端粒酶活性。实时荧光定量PCR法测定端粒长度,MTT法测定细胞活力。annexin V/PI双染色法检测细胞凋亡,PI单染色法检测细胞周期,流式细胞仪检测细胞凋亡。结果成功构建了靶向hTERT/hTR特异性miRNA表达框架。不同MEC对端粒酶活性的抑制程度不同。端粒酶的沉默可引起视网膜母细胞瘤(RB)细胞G0/G1期生长阻滞,导致细胞凋亡。结论miRNA介导的端粒酶沉默是一种有效抑制RB细胞生长的策略,开发一个强大的系统来充分探索miRNA的作用是必要的。本文构建的MEC显示了强大的RNAi效应,可成为筛选用于RB基因治疗的RNAi靶向序列的有效工具。Objective The present study attempted to design novel miRNA expression cassettes(MEC)targeting specific consensus sequences of hTERT and hTR,which remedied the degradability and cytotoxicity and provided a convenient method for miRNA synthesis.Methods The MECs specific to hTERT and hTR were constructed by overlap polymerase chain reaction(PCR).Telomeric repeat amplification protocol(TRAP)-silver staining and TRAP real-time PCR analysis were used to determine the telomerase activity.The telomere length was determined by real-time PCR,whereas cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.The cell apoptosis rate and cell cycle were assessed using the annexin V/propidium iodide(PI)double staining and PI single staining assays,respectively,coupled with flow cytometry.Results The telomerase-specific MECs were successfully constructed.Each MEC inhibited the telomerase activity differently.Telomerase silencing could induce immediate growth arrest in the G0/G1 phase and led to retinoblastoma(RB)cell apoptosis.Conclusion miRNA-mediated telomerase silencing is an efficient strategy to impair RB cell growth.A robust system must be developed to fully explore the efficacy of miRNAs.The constructed MECs exhibited a strong RNAi effect and thus may be utilized to effectively screen RNAi-targeted sequences for RB gene therapy.

关 键 词：端粒酶 MIRNA RNAI 视网膜母细胞瘤 基因治疗 

分 类 号：R73-3[医药卫生—肿瘤]