The Mechanism Research Of Proteins That Interact With The Novel Gastric Cancer Related Protein RKIP

Backgroud:At the present, the incidence of the gastric cancer ranks the fourth of tumors in the world, and its incidence and mortality rate is the first of malignant tumors of digestive tract.RKIP is a kind of small molecules cytoplasm, which is highly conservative, has a widely expression and many physiological and pathological functions. RKIP alias PEBP1(phosphatidyl diethanola-mine binding proteins1). Our previous study found that the expression of Raf kinase inhibitor protein (RKIP) in gastric cancer was reduced or absent, such changes may be connected with the occurrence, differen-tiation, invasion and metastasis of the gastric cancer, but those specific effects and mechanism of RKIP in the occurrence and metastasis of the gastric cancer have not been reported at home and abroad. Current studies suggested that the function and regulation of the most of enzymes were realized by a protein complex or the synergy of protein networks. Currently, researches on protein interactions are hot spots. Therefore, based on previous studies, we intend to offer the application of targeted proteomic techniques, which take affinity purification coupling mass spectrometry techniques as the principal thing, to isolate and identify the RKIP protein interactions in SGC7901gastric cancer cells. Thus do preliminary studies on the molecular mechanism that how RKIP inhibit the occurrence and development of GC, which provide the basis and the new clues for the early diagnosis, prognosis monitoring and targeted gene therapy of GC.Chapter one The construction of pCDNA3.1-PEBP1-3xFLAG expression plasmidObject:Constructing the3xFLAG marked PEBP1(namely RKIP) fusion protein expression vectorMethods:(1) combining3xFLAG according to the corresponding sequence of p3xFLAG-CMV-14carrier from the Sigma company;(2) amplify PEBP1CDS area with PCR, while introducing3xFLAG tags;(3)use Hindâ…¢ and Xhoâ…  to do double enzyme cut on the PCR clips and with purpose carrier pCDNA3.1(+) of PEBP1-3xFLAG,and then connect them.(use enzyme cut and sequencing to appraisal restructuring plasmid;(4) sequence the right plasmid conversion to competence escherichia coli, and amplify and extract lots of plasmids.Results:(1)conbined3xFLAG sequence successfully;(2) intruduced3xFLAG tags to the c-terminal of PEBP1gene sequences, and the double enzyme cut identified the validity of cloning; conne-cted joint gene fragments to carrier pCDNA3.1,the genetic sequences, and confirmed the vatality of sequence of genetic fragments.Conclusion:successfully constructe pCDNA3.1-PEBP1 3xFLAG fusion gene carrier. chapter two screening RKIP interaction proteinsObject:to isolate and identify the RKIP protein interactions in SGC7901gastric cancer cells. Thus do preliminary studies on the molecular mechanism that how RKIP inhibit the occurrence and development of GC, which provide the basis and the new clues for the early diagnosis, prognosis monitoring and targeted gene therapy of GC.Methods:(1)use liposome mediate to transiently transfect fusion expression plasmid pcDNA3.1-RKIP-3xFLAG into SGC7901cells, and use RT-PCR and Western blotting to respectively detect RKIP mRNA in transfected cells and protein expression levels;(2) use the3xFLAG label affinity chromatography technology to purify RKIP protein complex; use1D-sds-page protein complex technology to pre separateprotein complex;(3)use ESI-Q-TOF tandem mass spectrometry toanalysis and appraisal RKIP interaction protein;(4) do database query and protein GO function clustering analysis.(5) collecte a large amount of SGC7901cells, and extract the total protein;[6] RKIP antibodies and the immune IgY antibody coprecipitated the related proteins.[7] use HSP90, keratin or14-3-3application protein antibody to test the related protein expression.Results:(1)transfected and fuged the expression vector to SGC7901cells successfully, in RKIP carrying cell, wedetected obviously up-regulated RKIP by the RT-PCR and WB detection;(2) purificated and separated RKIP-3xFLAG fusion protein complex successfully;(3)In all,72RKIP related proteins were identified,whose functional classification includes protein metabolization, molecular partner, biological oxidation relevant enzymes, cytoskeleton protein, signal transduction related proteins, zymolysing related proteins and some proteins with unknown function, etc.(4)according to biological process classification by human GO slim vl.8analysis, in the72RKIP related proteins,the proportion of protein participating in the proliferation and growth of cells is30%, and the others are participating in the process of apoptosis in physical adjustment and organization of the interaction of protein; According to the comments, identified that most of proteins cells are inside proteins(90%),27%are the cytoplasm protein,16%belongs to the nucleus protein,12%belongs to membrane protein; According to the molecular function, it can be seen49%of proteins belongs to binding protein, including3%in signal transduction and1%in transcriptional regulation,25%of the proteins has catalytic activity, including10%has molecular structure adjustment activity,7%has enzyme activity(5)Detected HSP90,14-3-3, Keratin protein expression in RKIP immune complex.Conclusion:the72proteins of RKIP interaction proteins were identified successfully, including HSP90,keratin,14-3-3,GSTP1, Filamin,tubulin,Vimentin),Annexin A1,etc.And firstly identified the interaction among HSP90,14-3-3,Keratin protein and RKIP.it is said these proteins could play a major role in the occurrence and development of GC, or in regulating stomach cancer cells or invading or transfering the process, presumably, RKIP may through their interactions to the effect.