运用CRISPR-dCas9-VP64基因编辑技术验证microRNA-140的独立转录启动位点  

Using CRISPR-dCas9-VP64 genome edit tool to verify microRNA-140 own transcriptional promoter regulation

作　　者：郝耀 陈丽 韩永斌[1] 原野 Hao Yao;Chen Li;Han Yongbing;Yuan Ye(Department of Orthopaedics,First Hospital of Shanxi Medical University,Taiyuan 030001,China;Shanxi Bethune Hospital,Taiyuan 030006,China)

机构地区：[1]山西医科大学第一医院骨科,太原030001 [2]山西白求恩医院,太原030006

出　　处：《中华生物医学工程杂志》2023年第1期57-64,共8页Chinese Journal of Biomedical Engineering

基　　金：山西省应用基础研究计划资助项目(201901D211481);山西省留学回国人员科技活动择优资助项目(20200037);山西省回国留学人员科研资助项目(2022-189)。

摘　　要：目的探讨microRNA-140(miR-140)是否有独立于其宿主基因WWP2的转录启动子,从而为骨性关节炎的治疗提供新的思路。方法通过共转染CRISPR-dCas9-VP64和ACAN gRNA慢病毒的SW1353细胞验证软骨细胞特征性基因ACAN的转录水平。通过构建能稳定表达CRISPR-dCas9-VP64的SW1353细胞并结合多条sgRNA(single guide RNA,sgRNA/gRNA)、人类髋关节软骨RNA测序结果验证miR-140是否与WWP2基因共表达,是否有独立的转录启动位点。结果共转染CRISPR-dCas9-VP64和ACAN gRNA慢病毒的SW1353细胞能显著提高软骨细胞特征性基因ACAN的转录水平。从基因水平、蛋白水平证实成功构建能稳定表达CRISPR-dCas9-VP64的SW1353细胞。通过人类髋关节软骨RNA测序结果设计多条WWP2 gRNA以及miR-140启动子gRNA。实时定量PCR结果显示miR-140的表达既受其宿主基因WWP2调控,同时也有其独立的转录位点。结论运用CRISPR-Cas9基因编辑技术成功构建稳定表达CRISPR-dCas9-VP64蛋白的SW1353细胞能极大减少实验中样本间的差异化,保证实验结果的一致性。结合多条gRNA证明了miR-140既与WWP2共表达,同时也受其独立的转录启动子调控转录。运用CRISPR-dCas9-VP64d-gRNA能单独调控miR-140的表达,同时其宿主基因的表达不受影响,有潜力作为新兴的基因治疗方法治疗骨性关节炎。Objective To explore whether microRNA-140(miR-140)has a transcriptional promoter independent of its host gene WWP2,so as to provide new ideas for the treatment of osteoarthritis.Methods The transcript level of chondrocyte characteristic gene ACAN was verified by co-transfecting SW1353 cells with CRISPR-dCas9-VP64 and ACAN gRNA lentivirus.By constructing SW1353 cells that can stably express CRISPR-dCas9-VP64 and combining multiple sgRNA(single guide RNA,sgRNA/gRNA)and human hip cartilage RNA sequencing results to verify whether miR-140 is co-expressed with WWP2 gene,whether there is an independent Transcription initiation site.Results SW1353 cells co-transfected with CRISPR-dCas9-VP64 and ACAN gRNA lentivirus could significantly increase the transcription level of chondrocyte characteristic gene ACAN.The successful construction of SW1353 cells that can stably express CRISPR-dCas9-VP64 was confirmed from the gene level and protein level.Multiple WWP2 gRNAs and miR-140 promoter gRNAs were designed based on the RNA sequencing results of human hip cartilage.The results of real-time quantitative PCR showed that the expression of miR-140 was not only regulated by its host gene WWP2,but also had its independent transcription site.Conclusions Using CRISPR-Cas9 gene editing technology to successfully construct SW1353 cells stably expressing CRISPR-dCas9-VP64 protein can greatly reduce the difference between samples in the experiment and ensure the consistency of the experimental results.Combining multiple gRNAs proved that miR-140 was not only co-expressed with WWP2,but also regulated by its independent transcriptional promoter.The use of CRISPR-dCas9-VP64d-gRNA can regulate the expression of miR-140 alone,while the expression of its host gene is not affected,which has the potential to be used as an emerging gene therapy method for the treatment of osteoarthritis.

关 键 词：CRISPR-dCas9-VP64 骨性关节炎 非编码小RNA-140 含WW域E3泛素蛋白连接酶2 

分 类 号：Q78[生物学—分子生物学]