miR-505-3p在小鼠胚胎成纤维细胞中抑制可变剪接因子以调控运动神经元存活基因可变剪接  

miR-505-3p regulates the alternative splicing of survival motor neuron gene by inhibiting the alternative splicing factor in mouse embryonic fibroblasts

作　　者：杨侃[1] 李晓宁[2] 马骁骁[2] 李凯[2] 周宇荀[2] 肖君华[1,2] Yang Kan Li Xiaoning Ma Xiaoxiao Li Kai Zhou Yuxun Xiao Junhua(College of Environmental Science and Engineering College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China)

机构地区：[1]东华大学环境科学与工程学院 [2]东华大学化学化工与生物工程学院,上海201620

出　　处：《中国组织化学与细胞化学杂志》2016年第6期487-493,共7页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金(3131257);上海市科委重点项目(14140900502);东华大学研究生创新基金(DH-D-2014049)

摘　　要：目的探究微小RNA 505-3p(miR-505-3p)对于运动神经元存活基因(survival motor neuron gene,SMN)可变剪接的影响。方法利用生物信息学数据库预测miR-505-3p靶基因,构建双荧光素酶报告载体,在小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)中验证靶基因为可变剪接因子(alternative splicing factor,ASF)基因,运用免疫印迹和免疫荧光染色验证miR-505-3p对ASF的抑制效果,利用实时定量PCR技术检测miR-505-3p对具有正常功能的全长SMN(SMN-FL)以及含有毒性的第7号外显子缺失的SMN(SMN-D7)表达量的影响及ASF在其中的作用。结果 miR-505-3P直接结合ASF3'端调控区并抑制ASF mRNA及蛋白表达量,抑制ASF蛋白在细胞核中的表达和分布。过表达miR-505-3p促进SMN-FL向SMND7的转化,而过表达靶基因ASF或者抑制miR-505-3p促进SMN-FL的保留。结论 ASF对SMN基因正常剪接形成SMNFL是必需的,miR-505-3p通过抑制ASF,从而干扰ASF对于SMN可变剪接的调控,导致正常的SMN-FL向毒性的SMN-D7转化。Objective To investigate the role of mieroRNA 505-3p （miR-505-3p） in modulating the alternative splicing of survival motor neuron （SMN） gene. Methods A prediction about the target gene of miR-5OS-3p was made with bio-information databases. Then a dual-luciferase reporter vector was constructed to verify in mouse embryonic fibroblasts （MEFs） that the target gene was an alternative splicing factor （ASF） gene. The inhibition of ASF by miR-5OS-3p was verified by immunoblotting and immunofluorescent staining. Real-time PCR coupled with agarose gel electrophoresis was used to detect the effect of miR-505-3p on the expression of normal full-length SMN （SMN-FL） and toxic exon-7 excluded SMN （SMN-DT） as well as the role of ASF in the process. Results miR-505-3p directly bonded with the 3＇UTR of ASF and inhibited the expression of ASF mRNA and protein, especially the expression and distribution of ASF protein in the nucleus. The over-expression of miR-5OS-3p promoted the transition from SMN-FL to SMN-D7, while the over-expressing of ASF or inhibition of miR-505-3p facilitated the retention of SMN-FL. Conclusion ASF is indispensable for the normal splicing of SMN to form SMN-FL. By inhibiting ASF, miR-505-3p interferes with the regulation of the alternative splicing of SMN by ASF and leads to the transition of normal SMN-FL to toxic SMN-D7.

关 键 词：miR-505-3p 可变剪接因子 运动神经元存活基因 鼠胚胎成纤维细胞 

分 类 号：Q74[生物学—分子生物学]