The Expression Characteristics Of TIPE3and Its Effects On Proliferation And Migration Of Tumor Cells

PurposeOncogenes and tumor suppressor genes play important roles in occurrence and development of tumor. Therefore discovering new oncogenes or tumor suppressor genes and their potential value in the diagnosis and treatment of tumor is just around the corner. TIPE (tumor necrosis factor-α-induced protein8) family is a newly discovered protein family in recent years, which is composed of TIPE, TIPE1, TIPE2and TIPE3. They are highly homologous with all containing seven conservative aspiral (a0-a6) structures. The existing research results suggest TIPE, TIPE1and TIPE2all play roles in a wide variety of tumors, while as new member of the TIPE family, effects of TIPE3in tumor formation and development have not yet been reported.In order to study the expression of TIPE3characteristics and its possible roles in cancer, in our research TIPE3polyclonal antibody was prepared and its specificity is identified; by means of immunohistochemical method, we use common tumor and normal tissue micro-array chip to analyze TIPE3expression in normal tissues and tumor tissues. As the expression of TIPE3 has obvious difference in two kinds of tumor-non-small cell lung cancer and esophageal cancer, we analyze the relationship between TIPE3expression and clinico-pathological parameters.Finally, by transfecting reconstructed TIPE3vectors we detected role of TIPE3in malignant behavior of tumor cell (including cell proliferation, cell cycle and cell migration], which suggests that TIPE3may serve as an oncogene.Method:1. Samples:(1) normal tissue chip:contains the18kinds of individual normal tissues such as liver, lung, and kidney;(2) tumor tissue chip:including totally10kinds of tumor tissues and corresponding tissue adjacent to carcinoma, each3cases;(3) non-small cell lung cancer (58cases) and esophageal cancer (29cases) chip.2. Construction of TIPE3eukaryotic expression vectors:use TIPE3gene cloning vector (pFL-B02cl) as the template, obtain different PCR fragmentsby designing different primers and insert them into the pflag-CMV-2carrier and get long-TIPE3, short-TIPE3and truncated-TIPE3plasmids respectively.3. TIPE3polyclonal antibody preparation:design and synthesize TIPE3antigen peptide mixed with carrier, inject the complex into rabbits to get polyclonal antibody of human TIPE3.4. Western Blotting test after cell protein lysis, PVDF membrane imprinting, TIPE2and TIPE3antibody incubation and ECL reaction, protein expression is detected 5. Immunohistochemical:using polyclonal antibody of TIPE3incubation and DAB, TIPE3protein expression in different tissue chips is detected.6. Cell vitality detection method (CCK8):after TIPE3vector transfection into tumor cells, confirm cell vitality by detecting OD value in0h,24h,48h and72h respectively by CCK8method7. Flow cytometry:cell suspension is made by trypsin digestion, by using PI staining, analyze cell cycle or apoptosis respectively by flow cytometry.8. Cell migration test (Transwell method):After transfection of different TIPE3vectors, cells were suspended in serum free medium in upper closet while the lower contains medium with10%serum.9. Statistics methods:application of nonparametric rank and inspection (Wilcoxon test) and rank correlation Pearson respectively.Results1. Reconstruction of vectors which contains two isoforms of human TIPE3and N-terminal truncated human TIPE3:There’re two isoforms of human TIPE3including long-TIPE3(292aa) and short-TIPE3(204aa), and there is a specific domain in short-TIPE3compared to human TIPE2(which here we call N-terminal domain). We reconstructed three kinds of expression vectors here including the long-TIPE3, short-TIPE3and truncated-TIPE3and identify their sequences to be specific.2. Preparation and identification of TIPE3polyclonal antibody A. We prepared polyclonal antibody of human TIPE3. We designed and synthesized TIPE3peptide, combined peptide with vectors and injected them into rabbits to get specific polyclonal antibody of human TIPE3with titer of1:400.B. Identification of the prepared TIPE3antibody:to clear the feasibility and specificity of the antibody preparation, we transfected human TIPE2and TIPE3(here using the long-TIPE3vector] vectors into HEK293cell respectively and detected protein expression by using the TIPE2、TIPE3antibody through western blotting method. Results showed that when using TIPE2antibody staining, TIPE2band could be only seen in group transfected withTIPE2vector, group transfected with TIPE3or control group showed no TIPE2band; while using TIPE3antibody staining, a clear band can be seen in the TIPE3group with the band on behalf of protein weight equal to the predicted molecular weight, which indicated that band is the TIPE3protein band and the human TIPE3antibody we prepared can specifically detected TIPE3protein with no cross-reaction with human TIPE2protein.3. TIPE3expression in human normal tissueA. Expression position:TIPE3mainly distributes in cell membrane and cytoplasm, with no expression in cell nucleus.B. Expression characteristics:a. TIPE3expression in covering epithelum:according to TIPE3expression result in chip, we seen mainly TIPE3expression in simple cuboidal epithelium cells including epithelial cells in the pancreas, sweat glands and prostate gland which possess secretion function, and also the kidney tubules and cholangiole tubules. TIPE3is also highly expressed in simple columnal epithelium cells of stomach, small intestine and large intestine etc.b. TIPE3expression in organon parenchymatosum:TIPE3is highly expressed in muscle tissue (skeletal muscle tissue and cardiac muscle tissue)and brain tissue(cerebrum and cerebellum) while that in liver is tow.4. TIPE3expression patterns in tumors and related clinical signifecanceA. TIPE3expression profiling in tumors:in order to analyze the possible relationship between TIPE3and tumor, we used immunohistochemical method to analyze the TIPE3protein expression on tumor tissue microarray and found that TIPE3expression is higher than its adjacent tissues in many kinds of tumor tissues, suggesting that it is closely related to the growth and metabolism of tumor.B. TIPE3expression in esophagus cancer and its related clinical significance:a. TIPE3expression in tumor and adjacent tissue:TIPE3protein expression in esophagus cancer tissue was significantly higher than that in adjacent tissue, the difference was statistically significant. b. Relationship between TIPE3expression intensity and the clinic-pathological parameter:By classification of the tumor clinical pathological data of patients in chip, we found that TIPE3expression degree increases with the increase of pathological classification of esophageal cancer tissue.C. TIPE3expression in lung non-small cell carcinoma tissue and its clinical significancea. TIPE3expression in tumor and adjacent tissue:TIPE3protein expression in lung non-small cell carcinoma tissue was significantly higher than that in adjacent tissue, the difference was statistically significant.b. Relationship between TIPE3expression intensity and the clinic-pathological parameter:By classification of the tumor clinical pathological data of patients in chip, we found that there was no significant correlation between TIPE3expression strength and ages、 gender and TNM staging of patients who provide tissues in the chip.c. Correlation analysis between TIPE3expression pattern and clinical data:Depending on TIPE3expression site in lung cancer cells we divide TIPE3expression in tumor tissue into patterns---membrane expression and cytoplasm expression. Statistical analysis showed that TIPE3membrane expression tissue accounted for a greater proportion in T2-T3stage patients than that in T1stage patients. 5. TIPE3effects on tumor cell malignant behaviorA. Detection of TIPE3transfection efficiency in tumor cells:For subsequent research of TIPE3effect on tumor cell malignant, we transfected these three kinds of vectors into tumor cells and detected TIPE3expression by western blotting. Results showed that all three kinds of TIPE3expression can be well detected, which indicates that the transfected efficiency is high.B. TIPE3influence on cell proliferation:In order to research TIPE3effects on tumor cell growth, we transfected the long-, short-and truncated-TIPE3vector into tumor cells and detected cell vitality by CCK8method after24h,48h and72h respectively. Results showed that tang TIPE3had no obvious effects on tumor cell growing, short TIPE3significantly increases it and truncated TIPE3inhibits that, which indicates that short TIPE3maybe an oncogene and N terminal domain play an important role in TIPE3function.C. TIPE3effects on tumor cell cycle:In order to study TIPE3effects on tumor cell cycle, we transfected tumor cells with TIPE3vectors and detected cell cycle changes by PI staining. Results showed that cell cycles of cells transfected with TIPE3vectors has no difference compared to that of cells transfected with empty vector.D. TIPE3effects on cell migration:In order to detect TIPE3effects on tumor cell migration, we transfected the long-, short-and truncated-TIPE3 into tumor cells respectively.24hours later we transferred cell suspension to transwell room to detected cell migration. Results show that long-TIPE3had no significant effects on tumor cell migration while short-TIPE3promotes it. Truncated TIPE3which lack N-terminal domain the promoting effects of TIPE3disappeared, which indicates that short-TIPE3may promote cell migration and N-terminal domain play an important role in its function.Conclusion1. We successfully prepared polyclonal antibody of human TIPE3which has no cross-effects with TIPE2.2. TIPE3protein has a wide distribution in human body, which has a high expression in epithelial cells etc. TIPE3expression in tumor tissues varies in different kinds of tissues and is especially high in lung non-small cell carcinoma and esophageal carcinoma, which man indicate important role of TIPE3in these two kinds of tumors.3. Short-TIPE3has a role of oncogene as its overexpression could promote both cell proliferation and migration of tumor cells, and N-terminal domain is necessary in short-TIPE3function. Long-TIPE3shows no effects.Innovation and significance1. We prepared polyclonal antibody for the first time and confirmed TIPE3expression characteristics in human normal and tumor tissues(especially in lung non-small cell carcinoma and esophageal carcinoma], which laid foundation for further research of TIPE3.2. We primarily confirm that short-TIPE3has the kind of oncogene effect and N-terminal domain plays an important role in its function, which provides clues for further research of effect and mechanism of TIPE3.