Effects And Mechanism Of Hsa-miR-19a/b-3p In Fragile X Syndrome

Background: FXS(Fragile X Syndrome),a common hereditarily cognitive disorder,as well as a ASDs-related disease,mainly results from FMR1(Fragile X Mental Retardation 1)gene abnormally expanded and methylated and thereby the absence of FMRP protein(Fragile X mental retardation protein).FMRP is an RNA-binding protein and relates with miRNA regulation pathways in biochemistry and genetics.FXR1(Fragile X related gene 1),the homologous gene of FMR1,guides the synthesis of FXR1P(Fragile X related protein 1)protein,which is highly homology with FMRP protein in amino acid sequences and function,and both FXR1 P and FMRP make up the Fragile X protein family.Hsa-miR-19b-3p is related to FXR1 in our previous study and belongs to the same miRNA family with hsa-miR-19a-3p,but the regulatory mechanism between FXR1 and them is unclear.Therefore,the purpose of this study is to elucidate the regulatory miRNA network involved in FXR1,reveal its role and molecular mechanism in FXS.Objects/Aims: Research on the regulation relationships between FXR1 and hsa-miR-19a/b-3p?hsa-miR-19a/b-3p and its targeted mRNA? FXR1 and hsa-miR-19a/b-3p targeted m RNA,hoping to discover the regulation network pathway among FXR1,hsa-miR-19a/b-3p and targeted mRNA;At the same time,study the influence of SH-SY5 Y cell growth by hsa-miR-19a/b-3p and FXR1 respectively.Methods:(1)Using ten databases,including TargetScan ?miRWalk?miRanda,and online service platforms,such as STRING?GeneCards?NCBI and so on to predict and screen candidated targeted genes of hsa-miR-19a-3p and hsa-miR-19b-3p;(2)Detect the transcriptional regulation of targeted mRNAs in SH-SY5 Y cells when transfected with hsa-miR-19a/b-3p and FXR1 over-expressed constructed vector respectively by QRT-PCR technology;(3)Detect the protein level of targeted genes in SH-SY5 Y cells when transfected with hsa-miR-19a/b-3p and FXR1 respectively by Western Blot;(4)Detect the influence of hsa-miR-19a/b-3p and FXR1 on the proliferation capacity of SH-SY5 Y cells by MTT;(5)Detect the influence of hsa-miR-19a/b-3p and FXR1 on the apoptosis and cell cycle of SH-SY5 Y cells by FACS and Hoechst 33342 respectively;(6)Construct recombinant vector p DsRed2-N1-Dusp6 and detect the expression and localization in SH-SY5 Y cells of DUSP6 and FXR1 P by Fluorescence microscopy.Results:Part 1:Study on the Biological Function of hsa-miR-19a/b-3p in SH-SY5 Y Cells:(1)Preliminary screening and determining seven candidate targeted genes for hsa-miR-19a/b-3p by bioinformatics methods : FXR1?RAB18?USP32?CD47,Cdkn1a?Rhob and Dusp6;(2)QRT-PCR indicated that when transfected with hsa-miR-19a-3p mimics and hsa-miR-19b-3p mimics respectively,the mRNAs level of FXR1?Dusp6?Rhob were down-regulated(P<0.05),the mRNAs level of RAB18?USP32 were up-regulated(P<0.01),while no significantly difference in the mRNA level of CD47 and Cdkn1a;(3)Western Blot indicated that the protein level of FXR1?Dusp6 and Rhob were down-regulated(P<0.01),while the protein level of RAB18 and USP32 were up-regulated(P<0.05)in SH-SY5 Y cells when transfected with hsa-miR-19a-3p mimics and hsa-miR-19b-3p mimics respectively.(4)MTT showed that when transfected with hsa-miR-19a/b-3p mimics respectively,both the proliferation of SH-SY5 Y cells was inhibited,while hsa-miR-19a/b-3p inhibitor mimics could promote the proliferation of SH-SY5 Y cells.(5)Apoptosis test results showed that compared with miRNA NC mimics,when transfected with hsa-miR-19a/b-3p mimics the apoptosis of SH-SY5 Y cells was promoted by FACS and Hoechst 33342;compared with miRNA inhibitor NC mimics,the apoptosis of SH-SY5 Y cells was inhibited by hsa-miR-19a/b-3p inhibitor mimics.(6)Cell cycle test showed that compared with miRNA NC mimics,when transfected with hsa-miR-19a/b-3p mimics,the proportion of cells was increased 6.92% and 3.98% in G1 stage,reduced 4.02% and 2.28% in S stage as well as 2.90% and 1.71% in G2 stage respectively by FACS;compared with miRNA inhibitor NC mimics,the proportion of cells was reduced 2.58% and 2.68% in G1 stage,increased 5.11% and 3.78% in S stage,2.54% and 2.27% in G2 stage respectively.Part 2:Study on the Biological Function of FXR1 in SH-SY5 Y Cells:(1)QRT-PCR indicated that when FXR1 was over-expressed in SH-SY5 Y cells,the expression level of hsa-miR-19a/b-3p was up-regulated(P<0.05),as well as the mRNA level of USP32(P<0.01),and the mRNA levels of Cdkn1 a ? Dusp6 ? Rhob were down-regulated(P<0.05),while there was no significant change in RAB18(P>0.05);(2)Western Blot demonstrated that compared with control group,when FXR1 over-expressed,the protein level of USP32 was up-regulated(P<0.01),while the protein levels of Cdkn1a?Dusp6 and Rhob were down-regulated(P<0.05);(3)MTT indicated that FXR1 could inhibit the proliferation of SH-SY5 Y cells;(4)By FACS and Hoechst33342 we found that FXR1 could promote the apoptosis of SH-SY5 Y cells;(5)Cell cycle test showed that FXR1 could decrease the proportion of cells in G1 stage and G2 stage 9.0% and 13.83% respectively,and increase the proportion of cells in S stage 14.57%.Part 3:Study on the interaction between FXR1 P and DUSP6:(1)PCR amplified got Dusp6 product about 1.1Kb,which was cloned into pDsRed2-N1 empty vector,the results of double enzyme digestion,sequencing and Blast analysis showed that DNA sequence is correct;(2)When transfected with pEGFP-N1-FXR1 and pDsRed2-N1-Dusp6 respectively,the fluorescence microscopy showed that the cytoplasm was visible in green and red fluorescence respectively,indicating that both FXR1 P and DUSP6 are located in the cytoplasm.When transfected with them together,the results showed that FXR1 P and DUSP6 could be expressed in the same position of cytoplasm,and the co-localization occurred in cytoplasm.Conclusions:FXR1 could interact with hsa-miR-19a/b-3p,FXR1?RAB18?USP32?Dusp6 and Rhob are targeted genes of hsa-miR-19a/b-3p,and USP32?Dusp6?Rhob and Cdkn1 a are targeted mRNA of FXR1;Both FXR1 and hsa-miR-19a/b-3p could inhibit the proliferation of SH-SY5 Y cells,promote the apoptosis,and influence the process of cell cycle;(3)Construct recombinant vector p DsRed2-N1-Dusp6 successfully;and FXR1 P and Dusp6 have the co-localization in cytoplasm.