Preliminary Research Of Function And Differentiation Regulating Mechanism Of Rai 16 Protein Interacting With Tec Tyrosine Kinase

Background and objectiveTumor is a diseas with polygene, multistep, multifactor, many networks and complex pathomechanism. Cell dysdifferentiation is the nature of tumorigeness. The induced differentiation therapy for tumor, which can make tumor cells become mormal cells or similar to the normal cells by using some compounds, is a new therapy pattern following the surgery therapy, radiotherapy and chemotherapy and become the focus of research.Tec is a kind of non-receptor tyrosine kinase which is a crucial joint molecule in signal transduction pathway of liver stem cells and liver cell proliferation, differentiation and apoptosis. We use Tec kinase domain as the bait protein in our preliminary work and take advantage of the technique of yeast two-hybrid system to filter the human foetus liver cDNA library that was built from transcription activation domain vector, and through auxotrophic selecting , inducing filtering and preliminary identification, we found and confirmed a new positive clone. After sequencing and making BLAST comparison analysis on the gene fragment got from the filtering result of gene library in this study, we were sure that it was the RAI16 (Homo sapiens retinoic acid induced 16 gene), which has no homology sequence and has been embodied by Genebank and the number is BC052237, and the source of tissue is Brain astrocytoma (grade IV). Its coding protein is the Homo sapiens Retinoic Acid induced protein 16. Afterwards we looked on RAI16 as the cut through point. We mainly used related computer softwares and some bioinformatics websites to analyze RAI16,from which we obtained the significant information of RAI16 protein structure and thus we could predict its unknown new functions according to its structure. The analyses of bioinformatics shows that RAI16 protein contains six protein kinase C phosphorylation sites, nine casein kinase II phosphorylation sites, three tyrosine kinase phosphorylation sites, six N-myristoylation sites, two amidation sites, three tyrosine sulfation sites and one ATP/GTP-binding site. Furthermore, this protein has no transmembrane domain, which contains a section of signal peptide consisted of 31 amino acid and the cleavage site is located between 30-31aa. So we predict it to be a kind of important new decapentaplegic molecule of RA signal path in intracytoplasm.At present there is a few of documents reported about RAI16 and its function study is vacuity. While learning from its name it is a kind of inducing protein of RA. Because signal transduction process is the key to reveal its mechanism of action and thinking of its unknowning function and the outstanding effection of RA for cancer cells in induced differentiation, we choose HCC cells which were induced to differentiate by RA or RAI16 to act as our study object and we looked on'Preliminary Research of function and differentiation regulating mechanism of RAI16 Protein Interacting withTec Tyrosine Kinase'as the breakthrough point. Herein, from the induced differentiation of HCC cells and intracellular signal transduction path, we looked on phenonmenon that RAI16 inacted with Tec in yeast two-hybrid system as clue to identify expression spectrum and subcellular localization of RAI16, to analyze its function in the process of liver regeneration, hepatocarcinogenesis and HCC differentiation induced by ATRA, to investigate RAI16 effect for many kind of biological behavior of HCC cells, such as cell differentiation, cell cycle, cell proliferation or apoptosis , cell migration or invasiveness and etc, and explored the new function by antibody preparation, the construction of eukaryotic expression vector and adenovirus vector, immunohistochemisty, laser confocal microscop, immunoblotting, MTT, FCM, and so forth, which can make us to learn and interpret its crucial role in signal transduction pathway that RA induced HCC cells to differentiate. This study will provide new idea and theoretical evidences for the molecule mechanism of tumorous cell induced differentiation and signal transduction, and provide a new target for molecule targeted therapy of HCC, which can make the found base for the next step of the function mechanism study of RAI16.Materials and Methods1. Rabbit anti-human RAI16 polyclonal antibody was prepared by using its protein synthetic peptide and a modified rapid immune procedure followed by purification of specific avidity column. The efficacy of this polyclonal antibody was certificated by ELISA, Western blot and immunohistochemistry. The expression of RAI16 was preliminarily detected in regenerating liver tissue and cancer tissue of liver. This can provide antibody for the next step of the function study of RAI16.2. To detect the influence of RAI16 for cell cycle, cell proliferation and apoptosis by flow cytometer after HepG2 cells were induced by ATRA, then extracted total RNA and total protein to detect the expression of RAI16 by RT-PCR and Western blot and analyze the relation of ATRA with RAI16 in inducing differentiation, which can provide the theoretical base for the next step of the function mechanism study of RAI16 during the induced differentiation process.3. First,the RAI16 genes was amplified by PCR from plasmid cloning templates which contains total length gene fragment of RAI16 then inserted into pEGFP-C1 between EcoRI and XhoI sites. Then, the plasmid vector was sequenced and named as pEGFP-C1-RAI16. Laser confocal microscopy was used to observe the subcellular localization of recombinant RAI16. Western blot was applied to measure the increase level of RAI16 protein and induced differentiation function. Subsequently, cellular survival and proliferation was assayed by MTT.4. First,the RAI16 genes was amplified by PCR from plasmid cloning templates which contains total length gene fragment of RAI16 then inserted into pDC315-EGFP at the side of EcoRI sites. The plasmid vector was sequenced and named as pDC315-EGFP-RAI16. Second, AdMax system was used to construct the Ad-RAI16 and to enclose, amplify, depurate and detect titer.Then laser confocal microscopy was used to observe the subcellular localization of recombinant RAI16. Western blot was applied to measure the increase level of RAI16 protein and induced differentiation function. Subsequently, cellular survival and proliferation was assayed by MTT, cellular cycle and apoptosis was analyzed by FCM and the effect for cell biological behaviour was investigated by Transwell cell migration experiment and Metrigel invasion experiment.Results1. Rabbit anti-human RAI16 polyclonal antibody has been preparated successfully: The titer of the antibody obtained in this experiment determined by ELISA was up to 1∶125 000.The kaff value of the antibody was 10-5～10-6 M-1. Western blot results showed the antibody has high specificity to RAI16 protein. RAI16 can express in many tissues, such as liver, heart, esophagus, stomach, brain, pancreatic and so forth, and its expression was up-regulated during the liver regeneration and hepatocarcinogenesis process. 2. The induced effect of ATRA: That the expression of RAI16 was up-regulated after induced by ATRA was showed by Western blot. MTT results show that ATRA can inhibition HepG2 cells to proliferate in vitro, cell growth in induced group begin to be restrained obviously after two days comparing with control(P<0.05)and there are significant difference(P<0.05)in different phase after treating. FCM shows that ATRA can promote HCC cells to differentiate and apoptosis, suppress DNA synthesis and make them stagnate in G1 period.3. Construction and biological effects of plasmid vector overexpressing RAI16 specifically in eukaryotic: Sequencing analysis show that pEGFP-C1-RAI16 was constructed successfully. After transfected into HepG2 RAI16 was found to mainly localizate in endoplasmic reticulum and intracytoplasm and to significantly suppress cell proliferation and the expression of AFP andγ-GT, which are differentiation molecules.4. Construction and biological effects of adenovirus plasmid vector overexpressing RAI16: Sequencing analysis show that Ad-RAI16 was constructed successfully. After infected HepG2 RAI16 was found to mainly localizate in endoplasmic reticulum and intracytoplasm and to significantly suppress cell proliferation and the expression of AFP andγ-GT, which are differentiation molecules, to inhibite cell migration and to weaken invasing ability of tumorous cells.Conclusion1. Rabbit anti-human RAI16 polyclonal antibody, with high titer and specificity, was successfully produced by a modified rapid immune procedure. This polyclonal antibody can be further applied in ELISA, Western blot and immunohistochemistry to elucidate the roles of RAI16 protein played in many important cellular procedures. The result of initiatory experiment shows that RAI16 genes express in many tissues and regulate liver regeneration and hepatocarcinogenesis.2. The proliferation of HepG2 was significantly inhibited,the differentiation and apoptosis was induced and the expression of RAI16 was obviously up-regulated by ATRA. RAI16 may play important role in the process of ATRA induced differentiation.3. The eukaryotic overexpressing vector pEGFP-C1-RAI16 was successfully constructed, which may satisfy experiment demand. RAI16 proteins localizate in endoplasmic reticulum and intracytoplasm,which can inhibit the proliferation of HCC cells and make them differentiate.4. The recombinant adenovirus overexpressing vectorwas successfully constructed. It was proved that RAI16 can inhibit proliferation, invasion and migration of HCC cells, promote cancer cells to differentiate and apoptosis and play induced differentiating function in HCC through regulating Wnt signal transduction path.