| Objective: Nasopharyngeal carcinoma is a kind of malignant tumor derived from the mucosal epithelium of nasopharynx.According to the WHO classification criteria,it can be divided into keratinising and non-keratotising squamous cell carcinomas,differentiated non-keratotising carcinomas,undifferentiated carcinomas.China is one of the high incidence country of nasopharyngeal carcinoma,the incidence of the Southern China is higher than that of the northern China,the undifferentiated carcinomas counted for the majority.Raidotherapy is the main treatment of nasopharyngeal carcinoma.Although clinical efficacy is satisfactory,there are still some radioresistant patients,the effectiveness of re-radiotherapy is not very well.FANCD2,a member of the fanconi anemia family involved in DNA damage repair and forms the fanconi anemia pathway,plays a central role in fanconi anemia pathway.In our previous study,the expressions of FANCD2 protein in patients with recurrent nasopharyngeal carcinoma were detected and suggested that FANCD2 was involved in radioresistance of nasopharyngeal carcinoma.On the basis of this,the aim of this study was to investigate the effect of silencing the expression of FANCD2 on cell clony formation,cell proliferation,cell cycle and cell apoptosis before and after radiotherapy,and to explore the possibility of improving the radiosensitivity of nasopharyngeal carcinoma CNE-2 cell by silencing the expression of FANCD2.Methods:CNE-2 was used as the research object,it was cultured in RPMI-1640 medium+ 10% fetal bovine serum,37 ?,5% CO2,saturated humidity incubator.The experimental cells were divided into three groups: wild-type CNE-2 cells without any intervention;CNE-2NC containing the scrambled sequence of the control group;CNE-2sh containing the shRNA-FANCD2 interference sequence of the experimental group.(1)To construct a stable cell line,CNE-2 cells wereinfected with lentivirus,infection efficiency was observed under a fluorescence microscope.Puromycin was used to screen for more than one week until no new dead cells occure,then the table cell line CNE-2sh and CNE-2NC were obtained.The total RNA was extracted from three groups of cells and reverse transcribed into cDNA.The expression of FANCD2 mRNA was detected by fluorescence quantitative real time PCR.The total protein was extracted from three groups,the expression of FANCD2 protein was detected by Western Blot.(2)To conduct plate clony assays,the three groups of cells CNE-2,CNE-2NC,CNE-2sh were inoculated in 6-well plates.After 0Gy,2Gy,4Gy,6Gy,8Gy,10 Gy dose of radiotherapy,cells were continued to culture for another 7-14 days until cells grow to the visible clone which was about 0.1-0.3cm in diameter.Cells were fixed by paraformaldehyde,then dyed and rinsed.After drying in the air,taking pictures,observing and counting,each clone including more than 50 cells was counted under an inverted microscope.To obtain survival fraction and the radiotherapy biological parameters,the clony formation rate was fitted by using a multi-target single hit model and a linear quadratic model.(3)To conduct cell proliferation experiments,the three groups of cells were inoculated in 96-well plates.After 0Gy and 6Gy dose of radiotherapy,cells were continued to culture for antother 24,48,72,96 hours.The optical density of each well was detected by microplate reader.(4)To complete cell apoptotic rate detection,the three groups of cells were inoculated in 6-well plates.After 0Gy and 6Gy dose of radiotherapy,cells were continued to culture for another 72 hours.Cells were stained with Annexin V-PE and7 AAD according to the manufacture's instruction,and detected by flow cytometry.(5)To complete cell cycle detection,the three groups of cells were inoculated in 6cm Petri dishes.After 0Gy and 6Gy dose of radiotherapy,cells were continued to culture for another 24 hours.The cell cycle was detected by flow cytometer after being stained with PI according to the protocol of the cell cycle detection kit.Results:(1)The results of silencing efficiency showed thatthe relative expression of FANCD2 mRNA in the three groups was statistically different(F=75.035,P<0.05).The expression of FANCD2 mRNA in CNE-2sh group(0.254±0.072)was significantly lower than that in CNE-2 group(1.000±0.000)and CNE-2NC group(0.696±0.078).Western Blot showed that the expression of FANCD2 protein in three groups was statistically different(F=2450.370,P<0.05).The expression of FANCD2 protein in CNE-2sh group(0.130±0.025)was significantly lower than that in CNE-2 group(1.672±0.028)and CNE-2NC group(1.336±0.013),the protein silencing efficiency was92.20%.(2)The results of plate clony formation assay showed that there was no significant difference of clony formation rate between the three groups without radiotherapy(0Gy)(F=0.600,P>0.05),the difference was statistically different between the three groups with radiotherapy(2Gy,4Gy,6Gy,8Gy,10Gy),statistical values were(F=49.132,P < 0.05),(F=590.113,P < 0.05),(F=286.798,P<0.05),(F=77.167,P<0.05),(F=27.000,P<0.05)respectively.Cell clony formation rate in CNE-2sh groups were significantly smaller than that in CNE-2 groups and CNE-2NC groups.Cell survival curve showed that the survival curve of CNE-2sh group was lower than that of CNE-2 group and CNE-2NC group,the sensitization enhancement ratio fiited by muti-target single hit modle was 1.44,and by linear quadratic model was 1.18,both of them were greater than 1.(3)The results of cell proliferation assays showed that there was no significant difference between CNE-2sh,CNE-2 and CNE-2NC groups after 0,24 and 48 hours without radiotherapy(0Gy),(F=0.303,P>0.05),(F=0.193,P>0.05),(F=0.067,P>0.05).After 72 and 96 hours,the difference of optical density between the three groups was statistically different(F=24.148,P<0.05),(F=15.029,P<0.05).The optical density of CNE-2sh groups(0.342±0.018,0.671±0.045)was significantly lower than that in CNE-2 groups(0.453±0.019,0.900±0.017)and CNE-2NC groups(0.411±0.022,0.825±0.075).There was no significant difference betweenCNE-2sh,CNE-2 and CNE-2NC group after 0,24 and 48 hours with radiotherapy(6Gy),(F=0.303,P>0.05),(F=0.193,P>0.05),(F=0.067,P>0.05)(F=0.172,P>0.05),(F=1.224,P>0.05),(F=0.444,P>0.05).After 72 and 96 hours,the difference of optical density between the three groups were statistically different(F=88.598,P<0.05),(F=77.371,P<0.05).The optical density of CNE-2sh group(0.300±0.009,0.411±0.020)was significantly lower than that of CNE-2 groups(0.443±0.018,0.634±0.027)and CNE-2NC groups(0.402±0.012,0.574±0.021).(4)The apoptotic rate results showed that the cell aopoptotic rates were significantly different between the three groups without radiotherapy(0Gy)(F=7.218,P<0.05).The apoptotic rate of CNE-2sh group(4.90±0.21)was significantly higher than that of CNE-2 group(3.36±0.89)and CNE-2NC group(3.09±0.59).After radiotherapy(6Gy),the apoptotic rate of three groups were significantly different between three groups(F=30.872,P<0.05),the cell apoptotic rate in CNE-2sh group(9.05±1.99)was significantly higher than that in CNE-2 group(11.26±0.85)and CNE-2NC group(11.08±1.17).(5)Cell cycle test results showed that the cell cycle in three groups without radiotherapy(0Gy)is mainly distributed in G1 and S phase,less in G2 phase.The distribution of three groups in the G1 phase,S phase,G2 phase were statistically different(F=7.555,P<0.05),(F=19.382,P<0.05),(F=27.031,P < 0.05).The proportion of CNE-2sh group in G1 phase(38.54±1.29)and S phase(41.06±1.24)were significantly higher than those in CNE-2 group(33.78±2.03),(37.70±0.75)and CNE-2NC group(34.24±1.55),(36.48±0.72).The proportion of G2 phase in CNE-2sh group(20.07±1.52)was significantly lower than that in CNE-2 group(28.28±1.58)and CNE-2NC group(29.94±2.12).The cell cycle distribution of three groups changed after radiotherapy(6Gy),the proportion of G1 phase and S phase decreased,G2 phase increased.The distribution proportion of G1,S and G2 between three groups were statistically different(F=26.785,P<0.05),(F=10.571,P<0.05),(F=39.523,P<0.05),G1 phase and S phase in CNE-2sh group(13.64±1.93),(16.56±3.39)were significantly smaller than that in CNE-2 group(21.37±1.19),(24.72±1.53)and CNE-2NC group(21.66±1.35),(22.86±1.32),the proportion of G2 phase in CNE-2sh group(66.60±0.74)was significantly higher than that in CNE-2 group(53.91±2.70)and CNE-2NC group(55.47±1.75).Conclusions:(1)CNE-2sh cell,whose FANCD2 was scliecned,can be obtained from shRNA interference technique.Silencing efficiency was achieved as expected.(2)Silencing FANCD2 expression can inhibit the cell clony formation of nasopharyngeal carcinoma CNE-2 cell after radiotherapy.(3)Sensitization enhancement ratios,calculated by the multi-target single hit model and the linear quadratic model,were both greater than 1,and suggested that silencing the expression of FANCD2 could improve the radiosensitivity of nasopharyngeal carcinoma CNE-2 cell.(4)Silencing the expression of FANCD2 can significantly inhibit the proliferation of nasopharyngeal carcinoma CNE-2 cell before and after radiotherapy.(5)Silencing the expression of FANCD2 can significantly induce cell apoptosis of nasopharyngeal carcinoma CNE-2 cell before and after radiotherapy.(6)Silencing the expression of FANCD2 can significantly change the cell cycle distribution of nasopharyngeal carcinoma CNE-2 cell.After radiotherapy,the cells in G2 phase significantly increased and the radiotherapy sensitivity improved.(7)In this study,silencing the expression of FANCD2 through shRNA interference can improve the radiosensitivity of nasopharyngeal carcinoma CNE-2 cell by in vitro study.On this basis,we will continue to do in vivo study on improving the radiosensitivity and reveal its possible mechanisms,aim to provide new targets and theoretical basis on improving the nasopharyngeal carcinoma radiosensitivity. |
PDF Full Text Request