Plac8 Gene Knockout Enhances The Radiosensitivity Of Nasopharyngeal Carcinoma Xenograft Tumors In Nude Mice By Promoting Apoptosis

Background and objectiveOur previous in vitro cell experiments found that knocking out the Plac8 gene significantly enhanced the sensitivity of nasopharyngeal carcinoma cells to radiation.In this research,we studied the relationship between Plac8 expression and prognosis of nasopharyngeal carcinoma by tissue chip.Then,we used the xenograft model of nude mice to investigate the role of Plac8 gene knockout in radiotherapy sensitization of in vivo nasopharyngeal carcinoma and its molecular mechanism.MethodsTo detecte the expression of Plac8 in 120 NPC tissues and 30 NPG(nasopharyngitis)tissues by immunohistochemistry(IHC).To analyze the relationship between the expression of Plac8 gene and survival prognosis in patients with nasopharyngeal carcinoma.2.We cultured immoralized nasopharyngeal epithelial cell lines NP69 and 5 human nasopharyngeal carcinoma cell lines(CNE-2、HK1、SUNE1、5-8F、6-10B)in vitro.To detecte the transcriptional level of Plac8m RNA in different nasopharyngeal carcinoma cell lines by RT-PCR.3.We cultured CNE-2 cells and Plac8 knocked CNE-2 cells in vitro and they were inoculated subcutaneous in nude mice to form tumors.The two groups of nude mice were exposed to a dose of 8GY for once respectively to establish the radiotherapy model of nude mice with tumor.The experiment was divided into IR+CNE-2 group(control group)and IR+ko PLAC8 CNE-2 group(experimental group).4.We regularly observed,measured and recorded the volume changes of tumor bearing tumor in nude mice,and analyzed the effect of Plac8 gene knockout on the radiotherapy sensitivity of nasopharyngeal carcinoma cells in vivo.5.We used immunofluorescence to detect the expression differences ofγH2AX,Bax and Bcl-2 in the tumor.6.We used immunohistochemistry to detect the expression differences ofγH2AX,Bax and Bcl-2in the tumor.7.We used western blotting to detect the expression differences of Plac8,pathogenicγH2AX,Bax,Bcl-2 and Caspase-3 in tumor tissues.Results1.Tissue microarray results showed that the positive expression rate of Plac8 in NPC samples was significantly higher(84.2%,101/120)than that in NPG samples(46.7%,14/30)(P<0.01).Patients with nasopharyngeal carcinoma with Plac8upregulation had worse prognosis(P<0.01).2.Compared with NP69,Plac8 of human nasopharyngeal carcinoma cell line had higher transcriptional level(P<0.05).3.We successfully established the radiotherapy model of subcutaneous tumor transplantation in nude mice.4.Before radiotherapy,the tumor bearing volume of IR+CNE-2 group and IR+ko PLAC8 CNE-2 group gradually increased,but the tumor growth of IR+ko PLAC8 CNE-2 group was slightly slower than that of IR+CNE-2group.When the nude mice were sacrificed at the 10th day after radiotherapy,it was found that the average tumor volume of the two groups were(1.571±0.055)cm~3and(0.533±0.132)cm~3,respectively(P<0.05).The mean weight of transplanted tumor in the two groups was(0.82±0.08)g and(0.21±0.04)g,respectively(P<0.01),the tumor inhibition rate of IR+ko PLAC8 CNE-2 group was 75.0%.5.Immunofluorescence(IF)staining results showed that the fluorescence intensity ofγH2AX and Bax in IR+ko PLAC8 CNE-2 group was higher than that in IR+CNE-2 group,while the fluorescence intensity of Bcl-2 was lower than that in IR+CNE-2 group.6.Immunohistochemical staining results showed that the mean optical density values ofγH2AX and Bax in IR+ko PLAC8 CNE-2 group were significantly higher than those in IR+CNE-2 group(P<0.01,P<0.05),while the mean optical density values of Bcl-2 were just the opposite(P<0.01).7.Western blotting results showed that Plac8was hardly expressed in IR+ko PLAC8 CNE-2 group(P<0.01).The expression ofγH2AX,Bax,Caspase-3 and other proteins increased significantly(P<0.01),and the expression of anti-apoptotic protein Bcl-2 decreased significantly(P<0.05).Conclusion1.Knockout of Plac8 gene can increase the radiosensitivity of nasopharyngeal carcinoma cells in vivo by promoting cell apoptosis,which is an effective target for radiotherapy sensitization of nasopharyngeal carcinoma.2.Plac8’s high expression was associated with a shorter survival time for patients with nasopharyngeal cancer and a lower sensitivity to radiation therapy.