| Objective: It has been confirmed that the expression of FANCD2 gene in the cervical lymph node metastasis cell HSC-4 of head and neck squamous cell carcinoma(HNSCC) could be silenced efficiently and stably for a long time by short hairpin RNA(sh RNA) interference in vitro experiment of our previous study. After the expression of FANCD2 gene of HSC-4 cell was silenced by sh RNA interference, the radiosensitivity was improved by adjusting the proliferation, apoptosis and cell cycle of HSC-4 cell after radiotherapy, and the mechanism of radiotherapy sensitization may be associated with activating the p38 Mitogen-activated protein kinase(MAPK) signaling pathway and regulating the expression of apoptosis-associated proteins Bax/Bcl2 preliminarily. The present study is on these basis for further explore the effects of FANCD2 sh RNA interference on the HSC-4 cell xenograft growth and sensitivity to radiotherapy in nude mice through animal experiment, and discuss the possible mechanism of it, in order to provide the experimental foundation and theoretic basis for FANCD2 as a novel target in the treatment of HNSCC. Methods: With the cervical lymph node metastasis cell line HSC-4 as the research object which the FANCD2 gene was silenced stably in our previous study, the subcutaneous tumor model in nude mouse was established. 15 female BALB/c-nu nude mice were numbered and divided into 3 groups according to random number table method, 5 in each group. Experimental group: FANCD2-sh RNA(inoculated with HSC-4 cells transfected effective interference sequence and the silencing effect of FANCD2 is stable), negative control group: FANCD2-sh RNA-C(inoculated with HSC-4 cells transfected invalid FANCD2 interference sequence), blank control group: HSC-4(inoculated with wild type HSC-4 cells without any processing). The tumorigenicity, tumor-forming time and the growth of tumors were observed in each group by animal experiments; the local tumors were exposed to ionizing radiation with conventional fractionated radiation therapy after the HSC-4 cells were inoculated 28 days, 2Gy for each time, daily 1 time, for five consecutive days, the volume change of tumors in each group were observed after radiotherapy, tumors volume growth curve was figured; the mice were sacrificed by cervical vertebra dislocation at the 10 th day after radiotherapy, the tumors were stripped quickly and weighed, the tumor inhibition rate was calculated; all specimens were routine stained with HE, the pathological characteristics of tumors were observed; the cell apoptosis in tumor was detected by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL); the changes of the expression of Bax、Bcl2、p38 and p-p38 proteins after radiotherapy were detected by the Immunohistochemistry(IHC) Envision method and Western Blot(WB), explored the relationship between FANCD2 sh RNA interference on the influence of radiosensitivity of HSC-4 cell and the expression of Bax 、 Bcl2 、 p38 and p-p38 proteins, the possible mechanism was investigated. Results:(1) The results of nude mice transplantation tumor experiment displayed that the tumors were visible in 15 mice of 3 groups, the formation rate of transplanted tumor was 100%; the formation time of subcutaneous tumor nodule was prolonged, growth rate was slowed down, significant decline in tumor volume and weight in the experimental group, compared with the controls. The time of formating subcutaneous tumor nodule was 9~10 days in the experimental group, the median was 10 days; the time of formating subcutaneous tumor nodule in the negative control group and blank control group was 5~7 days, the median was 6 days. The tumor volume of experimental group before radiotherapy was 57.88±5.12mm~3, significantly lower than negative control group 89.95±7.44mm~3 and blank control group 98.83±6.31mm~3(F=56.474, P<0.05); the tumor volume of experimental group after radiotherapy was 11.11±5.25mm~3, significantly lower than negative control group 57.96±11.36mm~3 and blank control group 67.42±4.40mm~3(F=77.466, P<0.05), and the tumor volume of experimental group after radiotherapy was decreased by 46.77±2.76mm~3, significantly higher than negative control group 31.41±3.52mm~3 and blank control group 31.09±7.69mm~3(F=15.206, P<0.05); the tumor weight of experimental group after radiotherapy was 0.034±0.015 g, significantly lower than negative control group 0.092±0.023 g and blank control group 0.100±0.045g(F=6.950, P<0.05); the tumor weight inhibition rate of experimental group was 66.00±15.17%, the tumor volume inhibition rate was 83.52±7.78%; the HE staining of tumors showed that the carcinoma cells with nest-like distribution, the annular and eosinophilic keratin pearls and intercellular bridges were visible, marked pleomorphism, mitotic figures were readily identified, conform to the pathological characteristics of squamous cell caicinoma.(2) TUNEL assays revealed that there were different degrees of apoptosis cells in the tumors of three groups after radiotherapy, but the amount of apoptosis cells in experimental group were significantly more than the negative control group and blank control group; the apoptotic index(AI) of experimental group was 56.02±6.51%, significantly higher than negative control group 30.80±4.47% and blank control group 26.22±5.82%(F=80.216, P<0.05).(3) The results of IHC indicated that the expression of Bax and p-p38 proteins in experimental group were increased significantly after radiotherapy, the expression of Bcl2 and p38 protein were decreased significantly, compared with the controls; the mean optical density(MOD) value of Bax protein in experimental group was 0.190±0.022, significantly higher than negative control group 0.168±0.015 and blank control group 0.163±0.012(F=6.594, P<0.05); the MOD value of p-p38 protein in experimental group was 0.224±0.015, significantly higher than negative control group 0.173±0.016 and blank control group 0.165±0.015(F=20.778, P<0.05); the MOD value of Bcl2 protein in experimental group was 0.147±0.013, significantly lower than negative control group 0.176±0.012 and blank control group 0.168±0.008(F=16.137, P<0.05); the MOD value of p38 protein in experimental group was 0.161±0.016, significantly lower than negative control group 0.191±0.010 and blank control group 0.200±0.029(F=15.330, P<0.05).(4) The results of WB showed that the expression of Bax and p-p38 protein in experimental group after radiotherapy were significantly increased, the expression of Bcl2 and p38 protein were significantly decreased, compared with the controls; the grey scale value of Bax protein in experimental group was 0.219±0.008, significantly higher than negative control group 0.089±0.005 and blank control group 0.091±0.005(F=6.594, P<0.05); the grey scale value of p-p38 protein in experimental group was 0.129±0.005, significantly higher than negative control group 0.020±0.004 and blank control group 0.024±0.004(F=652.917, P<0.05); the grey scale value of Bcl2 protein in experimental group was 0.053±0.005, significantly lower than negative control group 0.097±0.013 and blank control group 0.105±0.008(F=25.200, P<0.05); the grey scale value of p38 protein in experimental group was 0.021±0.002, significantly lower than negative control group 0.215±0.007 and blank control group 0.233±0.006(F=1314.201, P<0.05). Conclusions:(1) FANCD2 sh RNA interference prolongs the tumor- forming time of HSC-4 cells in nude mice, and inhibits the growth of HSC-4 cells in nude mice;(2) FANCD2 sh RNA interference reduces the volume and weight of HSC-4 cells transplanted tumor in nude mice after radiotherapy, increases the tumor inhibition rate after radiotherapy, promotes the apoptosis of HSC-4 cells caused by radiotherapy, indicating that FANCD2 sh RNA interference can increase the radiosensitivity of HSC-4 cells in vivo;(3) The radiosensitization mechanism of FANCD2 sh RNA interference on HSC-4 cells transplanted tumor in nude mice is related to activating the p38 MAPK signaling pathway,and regulating the expression of apoptosis-associated proteins Bax/Bcl2;(4) FANCD2 is expected to be a new therapeutic target of HNSCC, and FANCD2 sh RNA interference will provide a new concept for clinical targeted therapy of HNSCC. |
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