Study On Transplanted Tumor In Nude Mice And Differential Gene Expression Of Thymoma Thy0517 Cells After Reduced Wnt4 And FoxN1

Objective: By observing down regulation the Wnt4 and Fox N1 of Thy0517 thymoma cells formed tumors in nude mice,further confirmed the role of Wnt signaling pathway.Using gene chip technology to research the differential gene expression of thymoma Thy0517 cells reduced Wnt4 and Fox N1 and find the upstream and downstream genes of Wnt4 and Fox N1,to provide the basis of the future experiment and find new methods for treatment of thymoma.Methods:1.Cell culture and transfection Using conventional methods recovery a strain of thymoma Thy0517 cell,complete medium for culture(containing 10% bovine serum)and passage to logarithmic growth phase.Lipo2000,Wnt986 and Fox1069 were transfected with liposome method,and then use semi quantitative RT-PCR to verify whether Wnt4 and Fox N1 were down regulated,and successful transfection could be used for the next experiment.2.Morphological observation of tumor formation in nude mice Dividing 24 BALB/c nude mice into four groups: blank group,Lipo2000 group,Wnt986 group,Fox1069 group.In the SPF class animal feeding room observation for 1 weeks,the nude mice were in good condition.The four groups of cells successfully transfected with logarithmic growth phase were inoculated in four groups of nude mice in the same position(right axillary subcutaneous)by the same person.3 days after inoculation,all nude mice grew tumors,every 3 days to measure the tumor volume.All the mice were sacrificed 4 weeks after inoculation,measure the tumor volume,remove the tumor,observe the tumor morphology,weigh the tumor,draw the growth curve,paraffin sections and immunohistochemistry.3.Screening of differentially expressed genes by gene chip Extraction of total RNA for quality inspection,the total RNA was extracted for quality control,then screening of differentially expressed genes before and after transfection by Agilent gene expression microarray.Analysis the differences of GO and pathway,and randomly selected part of differential genes,use semi quantitative RT-PCR method to test after down-regulation of Wnt4 and Fox N1,the level of m RNA expression in thymoma Thy0517 cells,verify that the results are consistent with the gene chip.Results:1.Three days after inoculation the mice in four groups all had tumor growth,the tumor growth of lobulated,cut off white,crisp.After tumor removal,the volume of the four groups was: blank(3.23±0.74)cm2,Lipo2000(2.86±0.35)cm2,Wnt986(1.89±0.30)cm2,Fox1069(1.82±0.40)cm2,there was no difference between blank and Lipo2000 group(P>0.05),the volume of Wnt986 and Fox1069 group was decreased compared with blank,the difference was statistically significant(P<0.05);the weight of tumor in the four groups was: blank(2.18±0.54)g,Lipo2000(1.68±0.18)g,Wnt986(1.53±0.30)g,Fox1069(1.36±0.28)g,there was no difference between blank and Lipo2000 group(P>0.05),the weight of Wnt986 and Fox1069 group was decreased compared with blank,the difference was statistically significant(P<0.05).2.Four groups of cells were successfully transfected.The gene chip results showed that genetic differences caused by thymoma Thy0517 cells after transfection of Wnt986 for a total of 1054,including 631 up-regulated and 423 down regulated genes,screened 7 differentially expressed genes,after semi quantitative PCR verification the results are basically consistent with the microarray results,and the concordance rate was 85.7%,the functions of these differentially expressed genes are mainly concentrated in biological regulation,cell regulation,cell membrane,binding,cell composition,and other functional clusters,the main signal pathway of differential genes is asthma,autoimmune thyroid disease,inflammatory bowel disease,axon guidance,ABC transporters,RAS,MAPK signaling pathway.After transfection with Fox1069 produced 1140 differential genes,up 575,down 565,creened 7 differentially expressed genes,after semi quantitative PCR verification the results are basically consistent with the microarray results,and the concordance rate was 85.7%,the functions of these differentially expressed genes are mainly concentrated in protein kinase B signal,multicellular biological process,protein binding,cell processes,biological processes,molecular functions and other functional clusters,the main signal pathway of differential genes is transcriptional dysregulation in cancer,physiological cycle,Hedgehog,FOX-O,Hippo and other signaling pathways.Conclusion:1.All nude mice were inoculated for three days to observe the tumor formation,and the growth rate was faster,indicating that the thymoma Thy0517 cells can be successfully established in nude mice transplantable tumor model for experimental study.2.The tumor growth rate of Wnt group and Fox group compared with blank group slow,indicated that after down-regulation of Wnt4,Fox N1 thymoma Thy0517 cell tumorigenicity decreased significantly,it was suggested that Wnt4 and Fox N1 could promote the occurrence of thymoma,and down-regulation of Wnt4 and Fox N1 could inhibit the invasion of Thy0517 cells in nude mice.3.The down-regulation of Wnt4 and Fox N1 can induce the expression of thymoma Thy0517 cells gene expression profile of widely spectrum changes,Wnt4,Fox N1 and its regulation on Upstream and downstream genes are not through a separate pathway play a role,but to participate in the related pathway of complex network system,resulting in the occurrence and development of thymoma,construct the regulatory network is more conducive to the exposure of key genes of thymoma and its pathogenesis.