FXR1P相关microRNA的筛选与靶标鉴定及其在脆性X综合征发病中的分子机制研究

FXR1 is the crucial related gene of fragile X syndrom（FXS）, the specific pathogenic mechanism of which remains not clear. As reading some related references and the previous study of our group, the hypothesis that FXR1P is an RNA-binding protein that binds to miRNAs and mRNAs, then FXR1P and the interactive miRNA co-regulate the expression of mRNA was proposed.Our previous research indicates that FXR1P regulates the normal development of nervous and musclar system by the interaction between protein and RNA or protein and protein. Based on the previous research of applicant, by using the RNA-binding protein immunoprecipitation (RIP) technology, Solexa high throughput sequencing and bioinformatics assaying ,we plan to screen miRNAs and mRNAs binding to FXR1P; then luciferase report gene system will be used to identify the targeted relationship between the significant miRNA and mRNA; meanwhile, we can use Quantitative Real-Time PCR to further reasearch the mechanism of the changed expression of miRNA, which was regulated by FXR1P. This study of the mechanism of FXR1P-miRNA interaction is expected to help to elucidate the pathogenesis of FXS provides a new thinking.

FXR1基因是脆性X综合征（FXS）重要的致病相关基因，但其具体致病机制仍不清楚。通过研读文献并结合课题组的前期研究，我们推测FXR1P可以同时结合miRNAs和mRNAs，FXR1P与miRNAs共同调控靶mRNAs的表达。本课题组前期研究表明：FXR1P是一种RNA结合蛋白，通过蛋白与RNA、蛋白与蛋白之间的相互作用影响神经、肌肉系统的正常发育。以申请者前期取得的研究成果为基础，课题组拟利用RNA结合蛋白免疫沉淀技术、Solexa 高通量测序和生物信息学手段分析，筛选FXR1P相关的miRNAs和mRNAs,并通过双荧光素酶报告基因系统鉴定候选miRNAs与mRNAs之间的靶标关系；同时运用实时荧光定量PCR技术深入研究FXR1P调控侯选miRNAs表达变化的机制。本项目对FXR1P-miRNA相互作用机制的研究，有望为阐明FXS的发病机制提供新的思路。

本项目利用RNA结合蛋白免疫沉淀技术(RIP)结合生物信息学分析与实验验证，成功筛选到8个与FXR1相关的miRNAs，且FXR1从转录后水平（pre-miR-132）调控hsa-miR-132-3p的表达；hsa-miR-132-3p和has-miR-19b-3p转染36h后均能够抑制SH-SY5Y细胞增殖，促进凋亡，而FXR1的过表达抑制PC12细胞的凋亡。采用报告基因系统、QRT-PCR和Western blot实验证实cmas是hsa-miR-323a-3p的靶标，USP32等3个基因是hsa-miR-19b-3p的靶标，SPAST等7个基因是hsa-miR-132-3p的靶标，IQCE等6个基因的mRNA是FXR1的靶标，hsa-miR-323a-3p、hsa-miR-19b-3p、hsa-miR-132-3p和FXR1对其靶mRNA均呈现翻译负调控或正调控作用，共同调控神经系统的发育、分化等多种生命过程。 通过免疫共沉淀和荧光共定位技术证实FXR1P与CMAS存在相互作用，两者在胞浆和胞核均存在共定位，且FXR1P使 CMAS表达从胞核向胞浆迁移，FXR1可通过提高PC12和SH-SY5Y细胞中cmas的活性，进而提高细胞中神经节苷脂GM1的浓度，对PC12和SH-SY5Y细胞的生长产生保护作用；FXR1P与BTF存在相互作用，且FXR1P与BTF在胞浆中存在共定位。 通过本研究筛选到FXR1相关的miRNAs，揭示FXR1/miRNA/靶基因的调控途径，证实了本课题组的推测——FXR1P与miRNAs相互作用共同调控靶标的表达；筛选到FXR1的靶mRNAs，是对现有FXR1靶RNAs库的有益补充；同时证实FXR1P在轴突生长、轴突分支形成、神经元和肌肉组织的兴奋性维持、神经细胞迁移、细胞信号传导及细胞凋亡中发挥着重要作用，这些为明确FXR1的作用及其机制及阐明FXS的发病机制提供新的思路。