Animal Expermental Study On The Relationship Between The Radiosensitivity Of Nasophatyngeal Carcinoma And Targeted Modulation Of RAC1

Objective: Construction of the stable poorly differentiated nasopharyngeal carcinoma CNE-2 cell lines with silent RAC1 and overexpression RAC1 gene.Establishment of nasopharyngeal carcinoma transplanted tumor in nude mice.In vivo studies on the close relationship between regulated RAC1 and radiosensitivity in nasopharyngeal carcinoma cells and further confirm the possibility of RAC1 as a radiosensitization target for nasopharyngeal carcinoma radiation therapy.Methods:1.Silencing and overexpressing of RAC1 gene lentiviral vector were constructed by using gene recombination technology which including RNA interference and overexpression and transfected nasopharyngeal carcinoma CNE-2 cells,then the best infected condition and interference vectors were selected by fluorescence microscopy and Western blotting.The stable silencing and overexpression of RAC1 gene in nasopharyngeal carcinoma CNE-2 cell line were established.2.The cell proliferation was measured by MTT assay after silencing and overexpressing of RAC1 gene in nasopharyngeal carcinoma CNE-2 cell line.The migration ability of cells was investigated by wound-healing.The cell cycle was analysis by flow cytometry.The expressions of P67,P47 and RAC1 were investigated by Western blotting.3.Human nasopharyngeal carcinoma xenografts were established by injecting CNE-2 cells with silencing and overexpressing of RAC1 gene into BALB/c athymic nude mice.Nude mice were given a single dose of 4Gy irradiation after inoculation 14 days and sacrificed after 10 days of irradiation.The changes of activities,weight and volume of nude mice were recorded.The pathological changes was observed by HE staining.The expressions of P67,P47 and RAC1 were detected by immunohistochemistry and Western blotting.The ultrastructural of cells was observed by transmission electron microscopy.Results:1.The sequencing results showed that silencing and overexpressing RAC1 gene lentivirus vectors were constructed successfully.The cells with silencing RAC1 gene were RAC1(-),which overexpressing RAC1 gene were RAC1(+)cells after CNE-2 was infected with virus particle.Fluorescence microscopy observed that the infection efficiency was higher than 95%.Western blotting analyses showed the expression of RAC1 protein in RAC1(+)cells was significantly higher than the control group,but RAC1(-)cells just the opposite.2.In vitro experiments showed that proliferation rate of RAC1(-)cells was increased,the migration rate was slowed down,what's more,P67,P47 and RAC1 protein expression were decreased significantly comparing with the control group(P<0.05).The rate of proliferation did not change in RAC1(+)cells,but the migration rate was increased,the P67,P47 and RAC1 protein expression were also increased significantly(P<0.05).In addition,silencing and overexpressing RAC1 gene,the cell cycle distribution in CNE-2 cells didn't change.3.The nasopharyngeal carcinoma transplanted tumor model in nude mice was established and of 100% tumor formation.Before irradiation,the weight of nude mice increased steadily,and there was no significant difference among different trial groups.Transplanted tumor of nude mice was rapid growth,tumor growth rate of RAC1(+)group and RAC1(-)group was slightly faster than the control group.However,the weight of nude mice decreased after treatment with 4Gy irradiation,especially RAC1(-)group was significantly decreased.Moreover,the tumor the volume of each group was significantly different from that untreated group,and the tumors of RAC1(+)and RAC1(-)were significantly reduced compared with those before irradiation.After nude mice were sacrificed,the weight of the transplanted tumor with irradiation was lighter than that of the untreated group,and the rate declining of tumor weight decreased of RAC1(+)was the most significant,high to 68%.HE staining showed significantly fibrosis and nuclear condensation in RAC1(-)group while significantly cell necrosis and nuclear fragmentation in RAC1(+)group.Western blot and immunohistochemistry showed that the expressions of P67,P47 and RAC1 in RAC1(-)group was down-regulated,and the down-regulation of RAC1(-)group was more obvious after radiation.The ultrastructural changes of tumor cells was observed under transmission electron microscope,compared with the control group,the structure of cells in RAC1(-)combined with radiation group was intact and clear.But the inner menbrances and out membrances of mitochondrial were interrupted.some mitochondrial was swelling,vacuolar obviously,ridge disappearance,partial ridge fusion.While the structure of cells in RAC1(+)combined with radiation group was completely destroyed.Cells were fused and necrosis,all organelle disappeared,karyorrhexis,a large number of cell debris can be seen.Conclusions:1.Silencing and overexpressing of RAC1 gene lentiviral vector were constructed and the CNE-2 cell lines with overexpression RAC1 and Sh-RNA RAC1 were established successfully.2.Silencing or overexpression of the RAC1 can positively inhibit or upregulate the expression of P67,P47 and RAC1 which are the key subunits of NADPH oxidas.3.Lentivirus-mediated silencing and overexpression of RAC1 xenograft model of nasopharyngeal carcinoma in nude mice was successfully established.The results in vivo comfirmed that overexpression of RAC1 can enhance the radiosensitivity of transplanted tumor of nasopharyngeal carcinoma after 4Gy irradiation.RAC1 may be as a new radiosensitization target for nasopharyngeal carcinoma.