Screening,identification And Its Preliminary Functional Studies Of Protein Interacting With FAP In OSCC

Background and ObjectiveOral squamous cell carcinoma(OSCC)is one of the most lethal malignancies worldwide and is also the most common oral cancer worldwide which represents 90%of all forms of oral cancer.Despite the great progress that has been made in diagnosis and combined treatments including surgical resection,chemotherapy,radiation therapy,and targeted biological agents,the 5-year overall survival rate is less than 50%.Thus,to find out the pathogenesis of OSCC and searching for new therapeutic targets are basic problems to be solved.The carcinogenisis and development of OSCC,like other tumors,are related to disorder regulation of genes,including the activation of oncogenic genes and the down-regulation of tumor suppressor genes,and abnormal regulation of cellular energy metabolism signaling pathways and cell differentiation signaling pathways.Proteins,as gene coding products and important carriers of biological signaling,play an important role in the process of carcinogenisis and cancer development.In recent years,the development of proteomics and mass spectrometry(MS)is helpful to study the role of proteins in tumors.Exploring the function of key proteins and their interaction proteins network in OSCC is helpful to elucidate the molecular mechanism of invasion and metastasis of OSCC,which is helpful for the diagnosis,treatment and prognosis of OSCC.Our previous study had confirmed that over-expression of FAP in OSCC was significantly related with the clinical stage and cervical lymph node metastas which indicating more poorly prognosis.Further experiments confirmed that FAP promoted OSCC cell growth,migration and invasion.Inhibition the expression of FAP in OSCC cells significantly reduced the proliferation,metastasis and invasion of OSCC cells through Akt/PI3K signaling pathway.However,the mechanism of FAP in OSCC is unclear and further research is needed.In this study,based on the previous researches,we use immunoprecipitation and mass spectrometry method to screen FAP interaction protein to explore the exact molecular mechanism of FAP in OSCC and provide new ideas for targeted treatment of OSCC.Methods1.Screening of FAP interaction proteinsThe full length of FAP was obtained by reverse transcription and then inserted into pCDH-CMV-MCS-EFl-copGFP vector using gene recombination techniques.The recombinant virus expression vectors(pCDH-FAP)and the auxiliary vectors(psPAX2,pMD2.G)cells were co-transfected to tool cells.The supernatant was collected and infected OSCC cells.The positive cells were screened by FACS to obtain cells stablely expression FAP.The total protein of OSCC cells stably over-expressing FAP was extracted.And with the co-immunoprecipitation technique and mass spectrometry,the proteins interacting with FAP were obtained.According to databases,the proteins interacting with FAP was further verified by bioinformatics analysis.2.Expression of DPP9 in OSCC and its effect on SCC9 cell biology functionThe expression of DPP9 in OSCC specimens and matched non-cancerous tissues was examined by qRT-PCR and immunohistochemistry.The expression of DPP9 in SCC9 was inhibited specifically by siRNA,and the interference efficiency was detected by qRT-PCR and Western Blot.And then we detected the cell proliferation,migration and invasion.Results1.The OSCC cell line that over-expresses FAP stably was constructed successfullyThe green fluorescence was observed with fluorescence microscope.The positive rate of green fluorescence was 91.44%.qRT-PCR and Western Blot showed that FAP was highly expressed at both mRNA and protein levels compared to the blank control group.2.Screening FAP-interacting proteins by Co-immunoprecipitation combined with mass spectrometryThe results of Coomassie bright blue showed that the endogenous FAP-interacting proteins were obtained by immunoprecipitation.The total protein was digested and the peptide was extracted and subjected to ESI mass spectrometry.The results of mass spectrometry were analyzed by software and the types of interaction proteins were identified.Two independent experiments were conducted.The results showed that there were 24 identical proteins,of which six proteins had the same GO function as FAP,13 proteins had common subcellular localization with FAP,and one protein had a similar domain.DPP9 was selected as the target protein for further study as a protein that has the same location,the same GO annotation,and similar domain with FAP.3.Immunoprecipitation confirmed that DPP9 was an interacting protein of FAP,the expression level of DPP9 in OSCC is down-regulated than paired normal tissues,inhibition the expression of DPP9 could enhance cell proliferation,migration and invasionImmunoprecipitation was carried out using tagged antibody and magnetic beads,and FAP and DPP9 were detected in both input and co-precipitated eluents,which confirmed that DPP9 is an interaction protein of FAP.Immunohistochemical and qRT-PCR showed that the expression of DPP9 in OSCC was lower than that in matched non-cancerous tissues.After the expression of DPP9 in SCC9 was down-regulated by siRNA,cell viability was enhanced,cell migration and invasion ability was decreased,and the apoptotic ratio was also lower than control group.Conclusions1.The recombinant eukaryotic expression vector pCDH-FAP and stable over-expressing FAP cell line were successfully constructed;2.24 identical proteins were obtained by two independent experiments,6 of 24 proteinshad the same GO function with FAP,13 proteins had common subcellular localization with FAP,and one protein had a similar domain with FAP;3.DPP9 interacts with FAP in OSCC cells.The expression level of DPP9 in OSCC tissues was significantly down-regulated than the matched normal tissues.Inhibition the expression of DPP9 could enhance the activity of SCC9 cells,promote migration and invasion,and inhibit apoptosis.