The Experimental Research Of Interaction Of ?-arrestin1 And STAT3 And Its Role Of Prolification In Glioblastoma Cells

Objective:According to the result of Preliminary test, we hypothesis:?-arrestinl can enhance the modification of STAT3,and play an important influence on the proliferation of glioma.This study confirmed the enhanced effect of ?-arrestinl.The interaction of?-arrestinl and STAT3 and its influence on the proliferation of glioma is discussed in this paper.Methods:1. To verify the interaction of ?-arrestinl and STAT3:? transfect 6myc-?arrestinl plasmid and flag-STAT3 plasmid together into 293T cells together,Using Co-Immunoprecipitation(COIP) to detect the interaction of ?arrestinl and STAT3? transfect pds-red-STAT3 plasmid and YFP-N1-?arrestinl plasmid together into 293T cells together,then detecting the interaction of them with fluorescence microscope2. The influence of overexpressing ?-arrestinl on expression and modification of STAT3 in glioma cells:? transfect 6myc-?arrestinl plasmid into C6 cells according to the gradient(0.5,1,2?g).Using immune precipitation (Immunoprecipitation, IP) to purificate endogenous STAT3 protein.To detect the change of acetylation, phosphorylation and the expression of STAT3 by Western blotting3. The influence of overexpression of ?-arrestinl on glioma cell growth? transfect 6myc-?arrestinl plasmid into C6 cells, and setting up blank control group.? To detect cell proliferation by MTT:Adding 20ul MTT solution to every hole of 96 96-well plates and cultivating for 4 hs, then terminate the reaction.Adding 150 ul dimethyl sulfoxide to every hole,then vibrating with low speed on shaking bed for 10mins.Finally, measuring OD490nm absorbance value of each hole in enzyme-linked immune detector4. The influence of glutamine on glioma cell growth:Cultivate rat glioma cells (C6 cells) with normal DMEM medium containing no Glutamine respectively for 24hs.Then Observed cell growth condition under inverted microscope.5.The influence on expression and modification of endogenous STAT3 by stimulating with glutamine in glioma cells:? Stimulate rat glioma cells (C6 cells) with Glntamine by time gradient (0,1,5,10,15,30,60,90 minutes).Using immune precipitation (Immunoprecipitation, IP) to purificate endogenous STAT3 protein.To detect the change of acetylation, phosphorylation and the expression of STAT3 by Western blotting6.To detect the impact of STAT3 and ?-arrestinl by stimulating with glutamine:Transfecting the 6myc-?arrestinl plasmid into C6 cells. To stimulathe the experimental group with 1 mm glutamine,then by observing the interactiong of endogenous STAT3 and 6 myc-?arrestinl by COIP.Results:1. Results of western blotting show that 6myc-?arrestinl can interact with flag-STAT3. Fluorescence microscope shows that pds-red-STAT3 can interacat with ?-arrestinl-GFP2. Results of western blotting show that the overexpression of ?arrestinl enhances the phosphorylation of STAT3 in C6 cells, but have no obvious effect on the expression of STAT3 protein.3. According to enzyme-linked immune detector absorbance value, the C6 cells which overexpressing ?-arrestinl have a higher rate of proliferation than the normal4.Inverted microscope showed that the use of excluding glutamine DMEM medium C6 cells, compared with glutamine DMEM medium, cell count decreased significantly, and cell morphology change5. Results of western blotting show that by stimulating with Glntamine in C6 cells, the phosphorylation of STAT3 had significant changes in 60 minutes, while the expression of STAT3 protein has no obvious change6.Results of western blotting show that the overexpression of glutamine can enhance the interaction of STAT3 and ?-arrestinlConclusion:1. ?-arrestinl can interact with STAT32. Overexpression of ?arrestinl enhances the phosphorylation of STAT3 in glioma cells, but have no obvious effect on the expression of STAT3 protein.3.Overexpressing ?arrestinl can enhance the proliferation of glioma cells4. Glutamine can promote the proliferation of glioma cells5. Glutamine can enhance the phosphorylation of endogenous STAT3, while the expression of STAT3 protein has no obvious change6.Glutamine can enhance the interaction of STAT3 and p-arrestinlInnovation and significance:Neuroglioma,hich is glioma short for,is the most common primary central nervous system tumor.It accounts for about half of all intracranial primary tumors.The damage of glioma is very serious.Signal Transducer and Activator of Transcription 3 (STAT3) is one of the cytoplasm of cytokines and growth factor receptor transcription factor.When are connected to the JAK cytokines receptors,JAK is activated by phosphorylation itself.After activation of JAK phosphorylation of one or more receptor sites and presented to the STAT3 combination,which leading to phosphorylation of STAT3 Tyr705 and Ser727 and the receptor turned away.,then transferred to the nucleus in the regulation of gene expression.A variety of STAT3 in tumor cells have abnormally high expression.Abnormal STAT3 activation cause cell aberrant proliferation and apoptosis,and promote the formation and development of tumor.STAT3 signal channel may be a new action of tumor gene therapy.The above results show that the p-arrestinl can enhance the modification of STAT3, and a certain impact on the proliferation of glioma. And the impact of Gln on the proliferation of glioma may enhance the interaction of ?-arrestinl and STAT3.This may be a potential treatment for glioma therapy.