| Objective:Nasopharyngeal carcinoma is a common malignant tumor in China,especially in Guangdong and Hong Kong,and non-keratinized carcinoma is the most common pathological type.Due to its high sensitivity to ionizing radiation and the limited scope of surgical resection by special anatomical location,radiotherapy is still the primary method in nasopharyngeal carcinoma treatment up to now.However,development of radioresistance makes some patients unable to benefit from this treatment.FA(Fanconi Anemia)pathway is an important signal pathway for DNA damage repairing.As a member of FA family,FANCD2(Fanconi Anemia Complementation Group D2)plays a crucial role in process of FA pathway completing the biological function of DNA damage repairing.This research group has successfully obtained nasopharyngeal carcinoma CNE-2 cells with stable FANCD2-silencing effect in previous studies,and cell function experiments have proved that silencing FANCD2 expression improves radiosensitivity of nasopharyngeal carcinoma CNE-2 cells in vitro by inhibiting cell proliferation,promoting apoptosis and changing cell cycle distribution.This study intends to further explore the effect of shRNA silencing FANCD2 expression on radiosensitivity of nasopharyngeal carcinoma in vivo through subcutaneously implanted tumor model in nude mouse based on previous experiments,and preliminarily explore its mechanism in regulating radiosensitivity,so as to provide theoretical basis for radiosensitizing therapy by targeting FANCD2 in nasopharyngeal carcinoma.Methods:This study was carried out with two parts:The first part was to explore effect of shRNA silencing FANCD2expression on radiosensitivity of nasopharyngeal carcinoma CNE-2 cells in vivo through nude mouse transplanted tumor model.The main methods were as follows:(1)Taking nasopharyngeal carcinoma CNE-2 cells as the research object,a nude mouse subcutaneous transplantation tumor model was established.Nude mice used in the experiment were randomly divided into two groups:group A with non-radiotherapy and group B with radiotherapy.Then,nude mice of group A and group B were further divided into the following three groups:(1)Experimental group:inoculated wih CNE-2 cells stably transfected with shRNA-FANCD2 interference sequence(CNE-2SH).Group A and group B were labeled with N-CNE-2SH and R-CNE-2SH respectively.(2)Negative control group:inoculated wih CNE-2 cells transfected with ineffective FANCD2-interfering sequence(CNE-2NC).Group A and group B were labeled with N-CNE-2NC and R-CNE-2NCrespectively.(3)Blank control group:inoculated wih wild-type CNE-2 cells without any treatment(CNE-2).Group A and group B were labeled with N-CNE-2 and R-CNE-2 respectively.The effects of silencing FANCD2 expression on growth of CNE-2 cells transplanted tumors in nude mice and sensitivity of radiotherapy in vivo were compared in group A and group B.(2)Transplanted tumors were stained with HE,and histopathological features of tumors in each group were observed under microscope.(3)Apoptotic cells in tumors were detected in situ by TUNEL[Terminal Deoxynucleotidyl Transferase(TdT)-Mediated dUTP Nick-End Labeling]and compared in each group.The second part was to primarily explore mechanism of radiosensitization mediated by silencing FANCD2 in nasopharyngeal carcinoma CNE-2 cells.The main methods wre as follows:(1)CNE-2 and CNE-2SH cells were taken as research objects,and genes with differential expression after silencing FANCD2 were screened based on gene chips.(2)DAVID(The Database for Annotation,Visualization and Integrated Discovery)and NCBI(National Center of Biotechnology Information)were conducive to further understand the function of differential genes,and the possible downstream genes of silencing FANCD2 enhancing radiosensitivity in nasopharyngeal carcinoma CNE-2 cells were preliminarily determined subsequently.(3)RT-qPCR(Quantitative Real-Time Polymerase Chain Reaction)was used to detect the mRNA expressions of the differential genes—NUPR1(Nuclear Protein 1),FLI1(Friend Leukemia Integration 1)and FGF21(Fibroblast Growth Factor 21)in nasopharyngeal carcinoma CNE-2 cells and corresponding transplanted tumor tissues of nude mice in each group.(4)IHC(Immunohistochemistry)was used to detect the expression of NUPR1,FLI1 and FGF21 proteins in transplanted tumor tissues of each group.The experimental results were mainly analyzed by SPSS 23.0 statistical software,supplemented by GpraphPad Prism 8 statistical mapping and Adobe Photoshop CC 2018 picture layout.Gene expression difference and P value in gene chip were analyzed by GeneSpring GX software,assisted by R language statistical mapping.The experimental data were expressed with x±s.The comparison among three groups of samples was analyzed by variance,and that between two groups of samples was analyzed by t test.The test level is?=0.05;When p<0.05,the difference is considered statistically significant.Results:The first part:(1)All groups of cells form tumor bodies after inoculated in nude mice.Tumor formation rate is 100%.The volume changes of tumors before and after radiotherapy were compared with self-control method in radiotherapy group(group B).The results showed that,before radiotherapy,the transplanted tumor volume of group R-CNE-2SH(453.300±27.312mm~3)was significantly smaller than that of group R-CNE-2 and group R-CNE-2NC(737.746±23.027mm~3,710.358±33.234mm~3)(F=124.115,P=0.000),and the tumor volume inhibition rate was 38.600±1.839%.After radiotherapy,the volume of transplanted tumor in group R-CNE-2SH(216.350±27.759mm~3)was also significantly smaller than that in group R-CNE-2 and group R-CNE-2NC(619.681±37.900mm~3,577.652±27.484mm~3)(F=199.175,P=0.000),and the tumor volume inhibition rate was 65.200±2.302%.Further comparing the volume changes of transplanted tumors before and after radiotherapy,the results demonstrated that the volume of group R-CNE-2SH decreased by236.950±3.602mm~3 after radiotherapy,which was significantly larger than that of group R-CNE-2 and group R-CNE-2NC(118.065±17.510mm~3,132.707±35.332mm~3)(F=32.164,P=0.000).A parallel control method was used to compare the weight measurement of tumors in non-radiotherapy(group A)and radiotherapy(group B)groups.The results revealed that,in group A(non-radiotherapy group),the average tumor weight in group N-CNE-2SH(0.893±0.199g)was significantly lower than that in group N-CNE-2 and group N-CNE-2NC(1.826±0.438g,1.708±0.606g)(F=5.180,P=0.032),and the weight inhibition rate was 50.700±5.115%.In group B(radiotherapy group),the average tumor weight in group R-CNE-2SH(0.338±0.140g)was also significantly lower than that in group R-CNE-2 and group R-CNE-2NC(1.200±0.561g,1.103±0.398g)(F=5.429,P=0.028),and the weight inhibition rate was 70.650±9.576%.Further comparison of tumor weight between group R-CNE-2SH and group N-CNE-2SH showed that the tumor weight of group R-CNE-2SH was significantly smaller than that of group N-CNE-2SH(t=4.558,P=0.004).(2)HE staining results of transplanted tumor tissues indicated that the cancer cells were distributed in nests,stripes or disordered arrangement in sheets,with obvious cell atypia and no obvious cell keratosis and keratotic bead formation,which were consistent with the histopathological characteristics of nasopharyngeal carcinoma.In group A(non-radiotherapy group),the cancer cells of group N-CNE-2 and group N-CNE-2NC were arranged compactly,with large and deep nuclei and no obvious necrotic areas,while in group N-CNE-2SH focal necrotic areas could be seen in tumor tissues,and nuclear condensation and fragmentation could be seen in necrotic areas.In group B(radiotherapy group),focal or patchy necrotic areas were seen in the tumor bodies of group R-CNE-2 and group R-CNE-2NC,with localized nuclear consolidation and fragmentation,while large areas of liquefied necrosis were seen in the tumor bodies of group R-CNE-2SH,with nuclear consolidation,fragmentation and even dissolution in the necrotic areas,presenting large areas of cytoplasmic red staining.(3)TUNEL apoptosis test results showed that without radiotherapy(in group A),the average number of apoptotic cells in group N-CNE-2SH(46.800±20.327)was significantly higher than that in group N-CNE-2 and group N-CNE-2NC(10.000±3.873,7.200±2.490)(F=16.864,P=0.000).After radiotherapy(in group B),the average number of apoptotic cells in group R-CNE-2SH(110.000±16.912)was also significantly higher than that in group R-CNE-2 and group R-CNE-2NC(45.200±12.153,56.000±8.860)(F=35.297,P=0.000).Further comparison of apoptosis cells in tumors between group R-CNE-2SH and group N-CNE-2SH showed that the average number of apoptotic cells in group R-CNE-2SH was significantly higher than that in group N-CNE-2SH(t=-5.344,P=0.001).The second part:(1)A total of 313 genes were screened by gene chip technology,showing differential expression in CNE-2 cells and CNE-2SH cells,of which 193 genes were up-regulated and 120 genes were down-regulated.(2)According to the retrieval results in DAVID and NCBI databases,the differential genes NUPR1,FLI1 and FGF21 were preliminarily determined as candidate downstream genes for shRNA silencing FANCD2 expression to improve radiotherapy sensitivity of nasopharyngeal carcinoma CNE-2 cells.The original signal values of the three genes in CNE-2SH cells(3933.004±509.674,117.751±9.070,143.325±18.535)were significantly lower than that in CNE-2cells(13907.892±3520.280,278.602±35.726,663.739±78.371).The differences were statistically significant(t=4.857,P=0.037),(t=7.558,P=0.002),(t=11.193,P=0.005).(3)RT-qPCR results demonstrated that,without radiotherapy(in group A),the relative expressions of the three differential genes NUPR1,FLI1and FGF 21 in nasopharyngeal carcinoma CNE-2 cells of group N-CNE-2SH(0.148±0.005,0.434±0.017,0.236±0.016)were significantly lower than those of group N-CNE-2(1.000±0.000,1.000±0.000,1.000±0.000),and the differences were statistically significant(t=322.026,P=0.000),(t=57.568,P=0.000),(t=84.715,P=0.000).After radiotherapy(in group B),the relative mRNA expressions of the above three genes in nasopharyngeal carcinoma CNE-2 cells of group R-CNE-2SH(0.067±0.014,0.236±0.044,0.078±0.026)were also significantly lower than those of group R-CNE-2(0.363±0.029,0.504±0.031,0.483±0.030),and the differences were statistically significant(t=15.983,P=0.000),(t=8.637,P=0.001),(t=17.790,P=0.000).Moreover,the relative mRNA expressions of the three genes in nasopharyngeal carcinoma CNE-2 cells of group R-CNE-2SH were significantly lower than those of group N-CNE-2SH(t=9.645,P=0.001),(t=7.251,P=0.002),(t=8.977,P=0.001).In addition,the change trends of relative mRNA expression of the above three genes were detected in each group of transplanted tumor tissues without radiotherapy and after radiotherapy,which were consistent with the change trends in cells.(4)IHC results showed that,without radiotherapy(in group A),the MOD(Mean Optical Density)values of NUPR1,FLI1 and FGF21 in nude mouse transplanted tumor of group N-CNE-2SH(0.019±0.003,0.016±0.002,0.021±0.003)were significantly lower than those of group N-CNE-2(0.038±0.002,0.021±0.001,0.029±0.003),and the differences were statistically significant(t=9.171,P=0.001),(t=4.438,P=0.011),(t=3.893,P=0.018).After radiotherapy(in group B),the MOD values of NUPR1,FLI1,and FGF21 in group R-CNE-2SH(0.012±0.002,0.006±0.002,0.009±0.002)were significantly lower than those in group R-CNE-2(0.022±0.001,0.010±0.000,0.019±0.003),and the differences were statistically significant(t=8.552,P=0.001),(t=3.536,P=0.024),(t=4.733,P=0.009).And the MOD values of the three genes in group R-CNE-2SH were significantly lower than those in group N-CNE-2SH(t=3.550,P=0.024),(t=6.653,P=0.003),(t=7.060,P=0.002).Conclusion:(1)Silencing FANCD2 expression can inhibit the growth of nasopharyngeal carcinoma CNE-2 cells transplanted tumors in nude mice and promote cells apoptosis in tumor tissues;(2)Compared with silencing FANCD2 expression or radiotherapy alone,the silencing of FANCD2 expression combined with radiotherapy has a more significant effect on inhibiting tumors'growth and promoting cells apoptosis,which indicates that silencing FANCD2 expression can enhance radiotherapy sensitivity of nasopharyngeal carcinoma CNE-2 cells in vivo;(3)Silencing of FANCD2 gene expression in nasopharyngeal carcinoma CNE-2 cells causes down-regulation expression of gene NUPR1,FLI1 and FGF21;(4)The molecular mechanism of silencing FANCD2 expression mediated radiosensitization in nasopharyngeal carcinoma CNE-2 cells may be related to the down-regulation expression of gene NUPR1,FLI1 and FGF21 after silencing FANCD2 expression. |
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