Study Of The Biological Effect Of The Interaction Between Mental Retardation Related Protein FXR1P And CMAS

Fragile X syndrome, which is called FXS for short, is one of the most commonhereditary mental retardation disease, and99%of the disease was resulted fromabnormal amplification of CGG trinucleotide repeats in5’-UTR of the FMR1gene orover-methylation of CpG island in the upstream of the gene, which can result in thedecreased expression or absence of FMRP-the product of the FMR1. FXR1(Fragile Xrelated gene1) that located in3q28and FMR1were both belonged to the same genefamily, and the proteins which were coded by the two gene respectively can bindproteins and RNA specifically. Based on the interaction between FXR1P and CMASidentified by preliminary works of our laboratory, this study refers to the biologicaleffect after the interaction between FXR1P and CMAS furtherly.Objective: To study the biological effect after the interaction between FXR1Pand CMAS, in order to provide some experimental evidence to demonstrate the genefunction of FXR1.Method:1. Amplified FXR1gene by PCR, then the fragment was connectedwith vector pcDNA3.1(-), and connection was identified by digestion and sequencing.2. Transfected the recombinant eukaryotic expression vector into PC12and VSMC byLipofectamine2000, and to assay the efficiency of transfection by western blot.3.To observe the change in cell form; and to detect the change of theGM1(ganglioside) concentration that was resulted from the interaction betweenFXR1P and CMAS by using Rat anti-ganglioside antibody(GM1)， ELISA Kit, andthe change of the activity of Na+-K+~ATPase and Ca2+-Mg2+~ATPase by using ultramicro-ATPase Kit; the rate of apoptosis by using FCM (flow cytometry).Result:1. The recombinant eukaryotic expression vector pcDNA3.1(-)/FXR1wassuccessfully constructed, and construction of the vector was identified by digestionand sequencing.2. pcDNA3.1(-)/FXR1was successfully transfected into PC12andVSMC.3.The overexpression of FXR1couldnot change the PC12cell form, butcould result in irregular of VSMC form; the overexpression of FXR1could increasethe concentration of GM1significantly in PC12but not in VSMC; increased theactivity of Na+-K+~ATPase and Ca2+-Mg2+~ATPase significantly in PC12but not inVSMC; decrease the rate of apoptosis dramatically in PC12but not in VSMC.Conclusions:1. The overexpression of FXR1gene may increase the activity of CMAS in PC12cell, then increase the concentration of GM1, and showed growth protection for PC12cell.2. FXR1P tissue-specific regulates the concentration of some cell, such as PC12but not VSMC.