Screening The MicroRNA Associated With FXR1P And Target Verifating Of MiR-323a-3p

Fragile X syndrome (FXS) is a common inherited mental retardation disease. Forthis disease, there are three causative genes---Fragile X mental retardation1(FMR1),Fragile X-related gene1(FXR1) and Fragile X-related gene2(FXR2), and theyconstitute the fragile X gene family. Fragile X mental retardation protein(FMRP),Fragile X related protein1(FXR1P) and Fragile X related protein2(FXR2P) wasencoded by these genes respectively, which are also the member of the fragile Xprotein family. These protein shares some RNA binding domains such as RGG boxand KH domain, so they are the member of the RNA binding protein (RBP).MicroRNA(miRNA) is a kind of small molecular non-code RNA and its length isusually about18～25nt. They can play a role in some biological progress byspecifically binding to the3’UTR of its target mRNAs. This study about theinteraction between FXR1P，miRNA and its target mRNA can help to determine thedetailed regulation and pathologenesis mechanism of FXR1P in FXS.Objective:1. To screen some miRNA which was associated with FXR1P.2. Toverify the relationship between the screened miRNA and its predicted target.Method:1. Some miRNAs band with FXR1P were captured by RNA-bindingprotein immunoprecipitation (RIP) in Human neuroblastoma cells (SH-SY5Y cells).Then they were added with head and tail, reversely transcripted, PCR amplified,connected and transfected to determine which is the miRNA.2. The possible targetmRNA of the screened miRNA associated with FXR1P was predicted by someprediction database, such as TargetScanHuman, microRNA and miRwalk.3. The dualluciferase reporter gene vectors psiCheck-2-report was constructed, which includedthe3’UTR of the predicted target mRNA, then it was verified by double digesting andsequencing.4. The recombined vector psiCheck-2-mRNA3’UTR and miRNA mimicswere co-transfected into Human embryonic kidney epithelial cells (293T cells), andthe relative luciferase activity was detected to determine the relationship between miRNA and its predicted target.Result:1. Three miRNAs associated with FXR1P---miR-323a-3p, miR-19b-3pand miR-1252-5p were screened by RIP.2. By prediced with some database, Cytidinemonophosphate-N-acetylneuraminic acid synthetase (CMAS) was determined as apotential target for miR-323a-3p.3. It is successfully to constructed the dualluciferase reporter gene vector psiCheck-2-CMAS3’UTR from the result of digestionand sequencing.4. After co-transfected the recombined vector psiCheck-2-CMAS3’UTR and miRNA-323a-3p mimic into293T cells, the relative luciferase value ofthe miR-323a-3p overexpression gourp was significantly lower than the negativecontrol group (P＜0.05).Conclusions:1.There are three screened miRNAs--miRNA-323a-3p, miR-19b-3p and miR-1252-5p associated with FXR1P.2. CMAS was the target gene ofmiR-323a-3p.