NRAGE Inhibits Autophagy And Its Molecular Mechanism

Background:NRAGE(neurotrophin receptor p75-interacting MAGE homologue)gene was found to interact with the p75 neurotrophin receptor(p75NTR)by Salehi in 2000 with Yeast Two-Hybrid System.It is the member of the MAGE(melanoma associated antigen)protein family.NRAGE belongs to the MAGE-D protein subfamily,and it interacts with the transcription factors Dlx5/Dlx7 and Msx2,so it was also named MAGE-D1 or Dlxin-1(Dlx-interacting protein-1).Studies have shown that NRAGE plays an important role in regulating cell apoptosis,transcriptional regulation,cell cycle and proliferation and tumorigenesis process.Objective:The expression of NRAGE gene declins in many tumor tissues,which belongs to the potential tumor suppressor genes.However,according to our previous studies,we have found that the expression of NRAGE gene was unusually high in liver cancer cell lines and tissues compared with normal liver cells and tissues adjacent to carcinoma,suggesting that NRAGE may exist an special regulation mechanism in the liver cancer which is different from other types of cancer cells.It was reported that autophagy served as a tumor suppressor in the process of the occurrence and development of liver cancer,autophagy level was generally lower in liver cancer cells than normal liver cells.Our previous study also found that,the level of autophagy has improved significantly in MEF cells of mouse where NRAGE was knockout.Therefore,we presume that the high expression of NRAGE gene may be related to the low level of autophagy in liver cancer cell lines.Beclinl combines with the class ? phosphatidylinositol 3-kinase(PI3K)to formate PI3KC3 complexs.The class ? PI3K product,phosphatidylinositol 3-phosphate(PI(3)P)recruits some proteins to the pre-autophagosomal structure(PAS);in addition,it activates two ubiquitin-like protein conjugation systems,which promote the elongation and closure of the phagophore membrance.The class ? PI3K and it's product PI(3)P are important regulatory factors for the initiation of autophagy.Our cooperation team found that,NRAGE can combine with Beclinl,so we presume that NRAGE inhibits autophagy through inhibiting the activity of the class ? PI3K.The activity of the class ? PI3K is reflected indirectly by detecting the content of PI(3)P in the cells.The recombinant plasmid pGEX-6p-1-p40phox-PX needs to be constructed firstly and the recombinant protein GST-p40phox-PX also needs to be expressed and purified,because the PX domain of p40phox can specifically combine with PI(3)P.After making preparation,then we will study the key problems in the subject:the effects on autophgy in hunman HepG2 cell and the activity of the class? PI3K when the expression of NRAGE gene is interfered;whether the role and molecular mechanism of NRAGE in HepG2 cell fits normal cells,such as 293 cell.Methods:1?To construct the recombinant plasmid pGEX-6p-1-p40phox-PX:The human p40phox-PX gene was amplified by PCR(polymerase chain reaction)with the template of thp-1 cells' cDNA;after the target gene and expression vector pGEX-6p-1 were cut by EcoR I and Xho I,they were ligased with T4 DNA ligase overnight;the ligation product was transformed into competent cell E.coli DH5a,then the positive clones were picked to identify by the clony PCR and electrophoresis;the recombinant plasmid was sequenced in the company and was alignmented in the NCBI.2?To express and purify the recombinant protein GST-p40Phox-PX:The recombinant plasmid was transformed into competent cell BL21or Rosetta(DE3),it was coated on the LB solid culture medium;then the positive clones were cultured in the LB liquid medium for 14 h;the bacterium solution was inoculated according to the proportion of 1%,the E.Ecoli containing recombinant vector expressed fusion protein after induction by IPTG.Under optimum conditions,a large number of E.coli containing recombinant vector was induced,and the fusion protein was purified by GST system.3?Two oligonucleotides of NRAGE siRNA were designed and synthesized,which were transfected into HepG2 cell to interfere the expression of NRAGE gene.The protein levels of NRAGE and LC3B ?/? were measured by Western blot.The activity of the class ? PI3K was detected by protein-lipid overlay assay by indirectly detecting the content of PI(3)P.That is to say:Lipids extracted from a 35mm plate was spotted onto PVDF membrane and allowed to dry overnight.The membrane was incubated with lipid blocking buffer for 1 h,and incubated with GST-p40phox-PX overnight,then the membrane was incubated with anti-GST.4?100nM siRNA were transfected into 293 cell,the protein levels of NRAGE and LC3B ?/? were measured by Western blot;the content of PI(3)P was detected by protein-lipid overlay assay.Results:1?The bactirum solution of positive clone was sent to the company for sequencing,the result was alignmented in the NCBI,similarity was 100%,which showed that the recombinant plasmid pGEX-6p-1-p40phox-PX was successfully constructed.2?The fusion protein was expressed only in the E.coli containing recombinant vector by inducted with IPTG except the control group without IPTG and empty vector,and the molecular weight was consistent with the theoretical value,which showed that it was the target protein.The recombinant protein mainly existed in the soluble form under the condition of 0.1 mM IPTG?18??18h.Purified protein products had a specific band,which showed that the recombinant protein GST-p40phox-PX was successfully purified.3?After transfected into HepG2 cell with siRNA for 48 hours,the NRAGE proteins decreased,indicating that the expression of NRAGE gene was interfered;the ratio of LC3B ?/? increased,indicating that autophagy was enhanced;the content of PI(3)P increased,indicating that the activity of class ? PI3K was enhanced.4?siRNA was transfected into 293 cell for 48 hours,after the expression of NRAGE gene was interfered,autophagy was enhanced and the activity of class ? PI3K was also enhanced.Conclusion:NRAGE inhibits autophagy through inhibiting the activity of the class ? PI3K,related molecular mechanism needs further research.