Tongue Cancer Resistance-associated Protein1(TCRP1) Polyclonal Antibody Development And Identification Of Its Interacting Proteins

Objective:In our previous study, we screened a new multi-drug resistance gene TCRP1from Tca8113/PYM cell lines by gene chips. We also shown previously that transmition of TCRP1can promote the cisplatin resistance of tongue cancer cells, which indicated that TCRP1may have a close relationship with the cisplatin-resistant, but the endogenous TCRP1expression and distribution in tissue cells, the mechanism of cisplatin resistance is not clear. Therefore, in this study we firstly analyzed the physicochemical properties of TCRP1and predicated its function by bioinformatics tools. Then by producing the polyclonal antibody against TCRP1, we looked for proteins that interact with TCRP1and explored its function. The final goal of this study is to clarify the mechanism of TCRP1in tongue cancer resistance from molecular level and to provide a theoretical basis for the tongue cancer chemotherapy.Methods:1. Through bioinformatics approach and analysis of the full-length gene, we get information about the physical and chemical properties, epitope and the sub-cellular localization characteristics of TCRP1.2. Constructing pGEX-6P/TCRP1prokaryotic expression vector and expression biologically active TCRP1protein; Using purified TCRP1protein immunize mice to producing polyclonal antibody; Detection TCRP1endogenous expression and sub-cellular localization in a variety of tumor cell lines and tongue cancer samples by WB, IFA and IHC;3. Using Human Toxicology and Drug Resistance Microarray (OHS-401) to identify differentially expressed gene profile between Tca/PYM and TCRP1interference cell lines. Validation a partial of these genes by real-time PCR and Western blot. These genes were further grouped into the eight categories according to their functions as described by GO analysis;4. Co-immunoprecipitation experiments were performed to determine the presence of any protein that could bind to TCRP1directly. The Tca/PYM cell line was transfected with MT1X siRNA or scramble siRNA to silence corresponding gene. After detected MTIX expression by real-time PCR and Western blot analysis, we further characterize MTIX with DDP resistance by FCM, WB, MTS and soft agar colony formation.Results:1. The bioinformatics analysis indicate that TCRP1is located on human chromosome11q13.4, contains eight exons and PI is9.327, the theoretical molecular weight is25kDa, we postulate that the TCRP1-encoded protein should be located primarily in the cytoplasm and will not be cells secrete extracellular.2. Constructed pGEX-6P/TCRP1prokaryotic expression vector, orthogonal experiment optimized TCRP1induction conditions and successfully purified active recombinant TCRP1protein. The protein has DNA protective effect which can antagonize cisplatin's DNA damage and cause Tca8113cells'cisplatin resistance. Western blot analysis identified that prompted TCRP1expression is not tumor cell but cisplatin-resistant cells specific, that TCRP1expression in cisplatin-resistant cells was significantly higher than other cells; IFA and IHC detected TCRP1mainly expressed in the cytoplasm.3. The expression of30genes was changed in the TCRP1interfering cells compared with the original population. Among these genes, nine genes were upregulated whereas21genes were downregulated. Most of the upregulated genes are associated with the chaperones/heat shock proteins and the downregulated genes are associated with the transcription factors and regulators.4. Co-immunoprecipitation experiments found that MT1X and Akt can bind to TCRP1directly. The MTS and soft agar colony formation experiments indicated that silence of TCRP1and/or MT1X expression can reverse the tolerance of tongue cancer resistant cells to cisplatin. The FCM results indicated that apoptosis was significantly increased in cells with lower expression of TCRP1compared with the control, whereas apoptosis ration was significantly increased from8.04%to15.47%and20.20%when treated with siMT1X and siTCRP1RNA respectively. When we treated the Tca/PYM cells with combination of siMT1X and siTCRP1, the apoptosis ration increased to37.48%(P<0.05). Conclusion:1. Successfully constructed TCRP1prokaryotic expression vector. Purified recombinant TCPR1protein has high quality and immunized mice produced high titer polyclone antibody.2. TCRP1proteins mainly localized in the cytoplasm. It is expression in a variety of tumor cells, but only upregulated in cisplatin-resistant tumor cells;3. TCRP1associated with DDP resistance pathway in OSCC though inhibition of apoptosis-related genes, promotion the metabolism of cisplatin, acceleration transport of cisplatin to the outer membrane.4. MT1X and Akt could bind to TCRP1directly. TCRP1can upregulate MTX1expression and increase chelation of MTX1to cisplatin which is the mainly pathway of TCRPl mediated cisplatin-specific tolerance.5. Silence TCRP1and the MT1X gene expression by siRNA can reverse OSCC cells resistant to cisplatin by promotion of apoptosis.