Exploration Of The Role Of Tumor Suppressor Genes Foxo1、PTEN、SHP-1 And Chromosome 13q14 In Tumorigenesis Of Mouse Natural Killer-Cell Lymphoma

［Objective］Extranodal NK/T-cell lymphoma is one of most common aggressive malignancies,accounting for almost 6.02% of lymphoma in China,but the causes of extranodal NK/T-cell lymphoma are largely unknown.The results of chromosome analysis,gene copy number analysis and expression profiling studies of tumors tissues have highlighted that some tumor suppressor genes were involved in the occurrence and development of Extranodal NK/T-cell lymphoma.This study is based on the NKp46-iCre transgenic mice constructed earlier,to investigate whether tumor suppressor genes Foxo1,PTEN,SHP-1 and chromosome 13q14 played a key role in tumorigenesis of mouse natural killer-cell lymphoma.This study can be divided into three parts:PartⅠ:Exploration of the Role of Tumor Suppressor Genes Foxo1 and PTEN in Tumorigenesis of Mouse Natural Killer-Cell Lymphoma［Methods］1.Crossed NKp46-iCre mice and ROSA26-YFP^{loxP/stop/loxP}[ROSA26-YFP（+/+）] mice to generate mice with genotype NKp46-iCre;ROSA26-YFP（+/-）.Flow cytometry was used to detect the percentages of YFP positive cell in various organs and various lymphatic cell of spleen.2.NKp46-iCre mice were crossed with mice carrying floxed Foxo1 alleles (Foxo1^fl/fl)as well as floxed PTEN alleles(PTEN^fl/fl)to generate mice in which Foxo1 and PTEN were disrupted in NK cells,referred as to Foxo1^△NKPTEN^△NK.Observed the growth and development of the mice and identified whether the mice were tumorigenic.Flow cytometry was used to detect the percentages of NK cell in various lymphatic organs.3.B16F10 melanoma model of tumor metastasis was utilized to investigate NK cell-mediated tumor surveillance.［Result］1.Compared control group[(NKp46-iCre;ROSA26-YFP（-/-）],the percentages of YFP positive cell in blood,periphery lymph node,bone marrow and spleen from mice with genotype NKp46-iCre;ROSA26-YFP（+/-）were very significantly higher [（BL:8.03%±1.64%vs 0.01%±0.008%）;（LN:0.26%±0.03%vs 0.01%±0.001%）;（BM:1.37%±0.003%vs 0.003%±0.001%）;（SP:2.59%±0.67%vs 0.029%±0.013%）],（n=5,P<0.05）.YFP was barely expressed in heart cell,and no statistically differences in percentages of YFP positive cell in heart between this two types of mice[（Heart:0.009%±0.007%vs 0.023%±0.01%）（n=5,P<0.05）].YFP positive cell percentages in NK cell,T cell and B cell from spleen lymphocyte were 81.33%±1.43%,0.19%±0.08%,0.11%±0.009%.YFP positive cell percentages in NK cell were higher than in T cell and B cell from spleenlymphocyte.2.The mouse model with NK cell-special Foxo1 and PTEN double knockout was established.Compared with control group(Foxo1^fl/flPTEN^fl/fl),Foxo1^△NKPTEN^△NK mice were born alive and appeared healthy over a period of 46 weeks.No spontaneous tumor formation was observed at this stage.There were no statistically differences in NK cell percentages of gated lymphocyte from various organs include blood,bone marrow cell,periphery lymph node and spleen between Foxo1^△NKPTEN^△NK mice and Foxo1^fl/flPTEN^fl/fl mice[（BL:4.76%±1.13%vs4.17%±2.20%）（P>0.05,n=8）,（BM:1.13%±0.57%vs 1.31%±0.24%）（P>0.05,n=8）,（LN:0.50%±0.25%vs 0.85%±0.52%）（P>0.05,n=8）,（SP:4.41%±1.72%vs 3.5%±0.63%）（P>0.05,n=8）]3.B16F10 melanoma model of tumor metastasis were established,no differences in median survival time were observed in this two types of mice（P>0.05,n=13）.［Summary］The simultaneous deletion of the Foxo1 and PTEN genes play less important role in the tumorigenesis of mouse natural killer-cell lymphoma and NK cell-mediated tumor surveillance in vivo.PartⅡ:Exploration of the Role of Tumor Suppressor Genes Foxo1 and SHP-1 in Tumorigenesis of Mouse Natural Killer-Cell Lymphoma［Methods］1.NKp46-iCre mice were crossed with mice carrying floxed Foxo1 alleles(Foxo1^fl/fl) as well as floxed SHP-1 alleles(SHP-1^fl/fl)to generate mice in which Foxo1 and SHP-1 were disrupted in NK cells,referred as to Foxo1^△NKSHP-1^△NK.Observed the growth and development of the mice and identified whether the mice were tumorigenic.2.Flow cytometry was used to detect the percentages of NK cell in various organs.［Result］1.The mouse model with NK cell-special Foxo1 and SHP-1 double knockout was established.Compared with control group(Foxo1^fl/flSHP-1^fl/fl),Foxo1^△NKSHP-1^△NK mice were born alive and appeared healthy over a period of 42 weeks.No spontaneous tumor formation was observed at this stage.2.Compared with control group,NK cell percentages of gated lymphocytes from blood,and periphery lymph node decreased very significantly[（BL:6.26%±0.478%vs 4.39%±0.23%）（P<0.01,n=3）,（LN:0.431%±0.08%vs 0.17%±0.04%）（P<0.05,n=3）].NK cell percentages of gated lymphocytes from bone marrow cell increased（BM:1%±0.09%vs 2.81%±0.76%）（P<0.05,n=3）.However,the percentages of NK cell gated lymphocytes in spleen and liver were not statistically significantly different between this two types of mice[（SP:1.59%±0.07%vs 2.27%±0.76%）（P>0.05,n=3）,（8.11%±2.99%vs 8.17±0.73）（P>0.05,n=3）.［Summary］The simultaneous deletion of the Foxo1 and SHP-1 genes play less important role in the tumorigenesis of mouse natural killer-cell lymphoma,which can significantly influence the NK cell distribution.PartⅢ:Exploration of the Role of tumor suppressor genes Foxo1,PTEN and chromosome 13q14 in the Tumorigenesis of Mouse Natural Killer-Cell Lymphoma［Methods］1.NK cell special Foxo1 and 13q14 defiency mice(NKp46-iCre;Foxo1^fl/fl13q14^fl/fl) were crossed with mice carrying floxed PTEN alleles(PTEN^fl/fl)to generate mice in which Foxo1,13q14 and PTEN were disrupted in NK cells,referred as to Foxo1^△NK13q14^△NKPTEN^△NK.We observed the growth and development of the mice and investigated whether the mice were tumorigenic in NK cell special Foxo1,13q14 and PTEN defiency mice.2.Flow cytometry was used to detect the percentages of NK cells in various lymphatic organs.［Result］1.The mouse model with NK cell-special Foxo1,13q14 and PTEN knockout was established.Compared with control group(Foxo1^fl/fl13q14^fl/flPTEN^fl/fl mice),Foxo1^△NK13q14^△NKPTEN^△NK mice were born alive and appeared healthy over a period of 42 weeks.No spontaneous tumor formation was observed at this stage.2.No significant differences in NK cell percentages of gated lymphocytes from various organs include blood,bone marrow cell,periphery lymph node and spleen between this two types of mice[（BL:5.23%±0.87%vs 6.98%±2.04%）（P>0.05,n=5）,（BM:1.21%±0.24%vs 1.49%±0.19%）（P>0.05,n=5）,（LN:0.51%±0.11%vs 0.66%±0.04%）（P>0.05,n=5）,（SP:2.51%±0.61%vs 2.76%±0.48%）（P>0.05,n=5）].［Summary］Tumor suppressor genes Foxo1,PTEN and chromosome 13q14 play less important role in the tumorigenesis of mouse natural killer-cell lymphoma.［Conclusion］The simultaneous deletion of the Foxo1 and PTEN genes may play not significant role in NK cell-mediated tumor surveillance in vivo.Tumor suppressor genes Foxo1,PTEN,SHP-1 and chromosome 13q14 may play not significant role in the tumorigenesis of mouse natural killer-cell lymphoma.