Research On The Function Of Tubulin Polymerization Promoting Protein 3 (TPPP3) And Prokineticin 2 (PK2)

Part 1TPPP3 plays an important role in cell proliferation and tumor growth Objective:To observe the role of TPPP3 in cell proliferation and tumor growth. Methods:Full-length open reading frame of TPPP3 gene was cloned using polymerase chain reaction (PCR), and comfirmed by sequencing analysis. Northern blotting was used to establish the gene expression profile of TPPP3. The prokaryotic expression vector PET28a-TPPP3 was constructed and the His-TPPP3 fusion protein was expressed in E.coli. The fusion protein was purified to immunize the Newzealand rabbits to generate polyclonal antibody against TPPP3. To construct recombinant eukaryotic expression vector of TPPP3 gene, and transfect cell. Using MicroRNA based RNA inference (RNAi) technology to delete the TPPP3 in HeLa cell, and then observe the proliferation of the cell in vitro. Transfect the RNAi plasmid into Lewis Lung Carcinoma (LLC) cell, select cell using blasticidin, and pick up stably tranfected LLC cell colonies (TPPP3-knockdown LLC or control LLC). Transplant TPPP3-knockdown LLC or control LLC into C57B1/6 mice subcutaneously, and observe the tumor growth. Using transwell assay to analysis the ability of migration and invasion in TPPP3-knockdown LLC cell and control cell. Inject the TPPP3-knockdown LLC cell or control LLC into mice by tail vein, and observe the metastasis in lungs.Results:Northern Blotting analysis demonstrated that TPPP3 mRNA was abundantly expressed in lung and aorta. Depleting TPPP3 by RNAi, it was indicated that the proliferation of HeLa cells was retarded, cell cycle pregression was arrest, abnormalities in mitosis were happened and apoptosis was increased. As we transplanted the TPPP3-knockdown LLC and control LLC cells to C57B1/6 mice subcutaneously, we found that the TPPP3-knockdown LLC tumors were much smaller. The transwell assay showed that the migration and invasion ability of TPPP3-knockdown LLC cells were decreased compared to the control. As we injected the LLC cell into the mice, we found that the metastasis was much lower in TPPP3-knockdown LLC bearing mice, which only had fewer and smaller nodes on the lungs.Conclusion:1) TPPP3 mRNA was widely expressed in many tissues and was abundantly in lung and aorta.2) Depleted TPPP3 in HeLa, the cell proliferation was suppressed.3) Knockdown of TPPP3 affected tumor growth.4) Knockdown of TPPP3 affected the ability of migration and invasion in LLC.5) Knockdown of TPPP3 affected the metastasis of tumor cell in mice.Part 2Prokineticin 2 is involved in the thermoregulation and energy expenditureObjective:To study the thermoregulation and energy expenditure in PK2 null mice during the food limitation.Methods:We used the situ hybridization to explore the PK2 mRNA expression in the paraventricular hypothalamic nucleus (PVN) and analyzed using a video-based computer image analysis system. C57BL/6 mice were injected with 2DG (250mg/kg) or saline to assay for PK2 mRNA expression in PVN. Use the locomotor boxes to monite the mice locomotor activites. A radio transmitter device was implanted in the abdominal cavity to measure body temperature of mice. The mice were implanted with cortical electroencephalogram (EEG) electrodes, placed over the frontal and parietal cortexes and electromyogram (EMG) electrodes in the dorsal neck muscles. The signal of EEG and EMG was recorded and analyzed. We used a Comprehensive Lab Animal Monitoring System (CLAMs) to asscess the food intake, oxygen and carbon dioxide gas fraction of mice daily.Results:The PK2 expression was rapidly induced in the PVN after fasting, which can be mimicked by 2-DG injection. During the food deprivation, PK2-/- mice exhibited lower body temperature and less induced locomotor activity compared the wild-type mice. The fasting-induced arousal was absent in PK2-/- mice. Furthermore, PK2-/-mice showed less energy expenditure and body weight loss than wild-type control upon fasting. Supply of limited food (equal to 5% of body weight) daily rescued the body weight loss and hypothermal phenotype in WT mice, but not in PK2-/- mice, which likely due to the lower efficiency of food utilization in PK2-/- mice.Conclusion:Our study demonstrated that PK2 was a regulator in the thermoregulation and energy expenditure. Losing PK2 signal, mice were more vulnerable to enter torpor during the fast. Meanwhile, PK2 could elevate the food efficiency in mice.