Research On The Role And Mechanism Of Fxr1p And DUSP6

Objective: Fragile X syndrome(FXS)is a common single gene x-linked dominant genetic disease,its main pathogenesis is due to the fragile X mental retardation 1(FMR1)lack of mutation and even cause the fragile X mental retardation protein(FMRP)expression is unusual,lead to the balance between inhibition and excitement in the nervous system pathways disorders.The main clinical manifestations are different degrees of mental disorders,autism,anxiety and other neurological symptoms.Fragile X-related gene 1(FXR1)and FMR1 belong to fragile X gene family,and their coding product fragile X related protein 1(FXR1P)is highly homologous with FMRP and has similar biological functions.FXR1 P is an RNA binding protein that interacts with target mRNAs and proteins,it plays an important role in all tissues of the body.Dual specificity phosphatase 6(Dusp6)is a target of FXR1 P identified in our previous study,but the specific regulatory mechanism between the two is unclear.Therefore,we studied the interaction between FXR1 P and DUSP6 in human neuroblastoma cells,identified the DUSP6-mediated regulatory pathway and biological effects,provided experimental basis for revealing the function of FXR1 in nerve cells.Methods:(1)String database was used to predict the interaction between FXR1 P and DUSP6.(2)pEGFP-N1-FXR1 and pDsRed2-N1-Dusp6 were co-transfected into SK-N-SH cells,and the expression and localization of DUSP6 and FXR1 P in the cells were observed under inverted fluorescence microscope and laser scanning confocal microscope.(3)pCMV-Flag-Dusp6 and pCMV-HA-FXR1 were constructed to detect the interaction between DUSP6 and FXR1 P proteins by immunoprecipitation.(4)Use BioGRID,Hitpredict,Genemania and other six databases to predict Dusp6 target genes,analyze their functions and screen out candidate target genes.(5)After the Dusp6 overexpression vector was constructed,it and the FXR1 overexpression vector were co-transfected into SK-N-SH cells,and the regulation of DUSP6 and FXR1 P on DUSP6 target gene transcription was detected by qRT-PCR.(6)Western blot detection of the regulation of DUSP6 and FXR1 P on DUSP6 target gene translation.(7)The effect of DUSP6 on the proliferation of SK-N-SH cells was detected by CCK8.(8)The effects of DUSP6 on apoptosis of SK-N-SH cells were detected by flow cytometry,Hoechst33342 and Giemsa staining.(9)SPSS19.0 was used for statistical analysis of all the data.Results: 1.Identification of the interaction between FXR1 P and DUSP6: Bioinformatics predicted that FXR1 P and DUSP6 may interact with each other through MAPK3.When GFP-FXR1 P and DsRed2-DUSP6 fusion protein expression plasmid were transfected into SK-N-SH cells,green and red fluorescence were observed in the cytoplasm of the cells,respectively.When the two fusion proteins expressed plasmid co-transformed cells,green and red fluorescence could be seen in the cytoplasm and cytonucleus,overlapping into yellow fluorescence.PCR amplification of Dusp6 and 1.9kb FXR1 genes were cloned into eukaryotic expression vectors pCMV-Flag and pCMV-HA respectively.DNA sequencing showed that the recombinant vectors pCMV-Flag-Dusp6 and pCMV-HA-FXR1 were successfully constructed.FXR1P-HA fusion protein expression plasmid with HA label and DUSP6-Flag fusion expression plasmid with Flag label were co-transfected into SK-N-SH cells,and the presence of FXR1P-HA and DUSP6-Flag fusion proteins was detected in anti-HA and anti-Flag precipitated protein mixtures,respectively.2.FXR1P-DUSP6-mediated signal pathway studies: Screening for MAPK1/ 3(also named ERK1/2),phosphatidylinositol 3-kinase(PI3K),tyrosine kinase(TRKA),mammalian target of rapamycin(mTOR),serine/arginine-rich protein-specific kinase 1(SRPK1)and testis expressed 11(TEX11)are candidate targets for Dusp6.The Dusp6 gene with a size of about 1.1 kb was amplified by PCR,cloned into the eukaryotic expression vector pcDNA3.1(+),and identified by DNA sequencing.The eukaryotic expression vector pcDNA3.1(+)-Dusp6 was successfully constructed.After pcDNA3.1(+)-Dusp6 and pcDNA3.1(-)-FXR1 recombinant vectors were transfected individually and co-transfected into SK-N-SH cells,the mRNA expression of Dusp6 candidate was detected by qRT-PCR.In the control group with empty plasmid,the mRNA expression level of Dusp6 increased significantly after transfection with pcDNA3.1(+)-Dusp6(P<0.001),and the mRNA expression levels of ERK1,ERK2,TRKA,SRPK1 and TEX11 decreased(P<0.05).However,the mRNA expression levels of PI3 K and mTOR did not change significantly(P>0.05).After transfection of pcDNA3.1(-)-FXR1,the mRNA expression levels of FXR1,ERK1/2,PI3 K and TRKA increased(P<0.05).The mTOR mRNA expression level did not change significantly(P>0.05).After co-transfection of pcDNA3.1(+)-Dusp6 and pcDNA3.1(-)-FXR1,the mRNA expression levels of PI3 K and mTOR decreased significantly(P<0.01).The mRNA expression levels of ERK1/2 and TRKA did not change significantly(P<0.05).Western blot showed that when the expression of DUSP6 increased,not only the expression of ERK1/2 protein decreased(P<0.05),but the expression of p-ERK1/2 protein also decreased(P<0.01);when the expression of FXR1 P increased,the expression of ERK1/2 and p-ERK1/2 protein also increased(P<0.05);when the expression of FXR1 P and DUSP6 increased at the same time,the expression of ERK1/2 protein and p-ERK1/2 protein did not change significantly(P>0.05).3.Effect of DUSP6 on the proliferation and apoptosis of SK-N-SH cells: After transfection of pcDNA3.1(+)-Dusp6 into SK-N-SH cells,CCK8 results showed that the cells proliferated at 48 and 72 hours after transfection The inhibition was significant(P<0.01);Cell apoptosis was promoted 48 h after transfection by flow cytometry,Giemsa,and Hoechst staining(P<0.05).Conclusions: 1.FXR1 P interacts with DUSP6 in SK-N-SH cells,and the two co-localize in cytoplasm and nucleus.2.FXR1 P regulates MAPK and AKT/mTOR cell signal transduction pathway in SK-N-SH cells by interacting with DUSP6.3.DUSP6 can inhibit the proliferation and promote apoptosis of SK-N-SH cells.FXR1 P may reversed the negative regulation of ERK1/2 by DUSP6 in SK-N-SH cells to some extent.