| ?Objective?In order to clarify the relationship between CD166 gene and radiosensitivity of nasopharyngeal carcinoma?NPC?,the CD166 gene in nasopharyngeal carcinoma CNE-2R cells was silenced by RNA interference technique.Then the effect and the possible mechanism of CD166 on radiosensitivity of CNE-2R cells were investigated by cell functional experiment.?Methods?1.We designed and synthesized a set of specific siRNA for the CD166/ALCAM gene,constructed lentiviral vector shRNA,transfected CNE-2R cells,and observed the transfection rate by inverted fluorescent microscope.2.RT-qPCR was used to detect the inhibitory effect of lentiviral shRNA vector on the target gene,and the perfect target of silencing was screened out.The inhibitory effect was confirmed by Western blot.3.The radiosensitivity of CNE-2R cells before and after CD166/ALCAM gene silencing was detected by cloning assay.4.CCK-8 assay was used to detect the proliferation rate of CNE-2R cells before and after the CD166/ALCAM gene silencing.5.Flow cytometry was used to detect the apoptosis rate of CNE-2R cells before and after the CD166/ALCAM gene silencing.6.Flow cytometry was used to detect the changes of CNE-2R cell cycle before and after the CD166/ALCAM gene silencing.?Results?1.Lentiviral vector shRNA was successfully constructed?titer of 5*108TU/mL?.After transfection of CNE-2R cells with lentiviral vector,the results showed that the infection efficiency was more than 90%,which indicated that transfection was successful.2.The best interference target CD166-RNAi-LV3 was screened by qRT-PCR.Western blot was used to verify the inhibitory effect of the best target.3.The results of clone formation experiment showed that the values of D0,Dq and SF2 were decreased in CD166-RNAi-LV3 group compared with non-transfected CNE-2R group and negative control?NC?group.The radiosensitization ratio was 1.477.4.The results of CCK-8 showed that the proliferation rate of CD166-RNAi-LV3 group was significantly lower than that of non-transfected CNE-2R group and NC group?F=16.716-57.675,p<0.05?after 2,4,6 and 8Gy irradiation,respectively.5.Apoptosis results show that before 10Gy irradiation,the apoptotic rates of CD166-RNAi-LV3 group,NC group and non-transfected CNE-2R group were?15.60±1.57?%,?8.60±1.80?%and?7.47±0.58?%,respectively.After10Gy irradiation,the apoptotic rates of CD166-RNAi-LV3 group,NC group and non-transfected CNE-2R group were?39.83±3.43?%,?20.53±1.81?%and?19.43±2.97?%,respectively.Before and after irradiation,the apoptosis rate of CD166-RNAi-LV3 group were significantly higher than that of non-transfected CNE-2R group and NC group?F=28.90,49.635,p<0.05?.6.The cell cycle results showed that the percentage of G2/M phase in CD166-RNAi-LV3 group,NC group and non-transfected CNE-2R group were?26.40±1.76?%,?13.30±3.24?%and?13.50±1.31?%,respectively.The proportion of G2/M in CD166-RNAi-LV3 group was significantly higher than that in NC group and non transfected CNE-2R group?F=33.208,0<0.05?.The percentage of S phase in CD166-RNAi-LV3 group,NC group and non-transfected CNE-2R group were?3.30±0.10?%,?12.23±1.67?%and?10.27±0.64?%,respectively.The proportion of S in CD166-RNAi-LV3 group was significantly lower than that in NC group and non transfected CNE-2R group?F=62.036,0<0.05?.The percentage of G0/G1 phase in CD166-RNAi-LV3 group,NC group and non-transfected CNE-2R group were?67.37±2.06?%,?67.37±3.78?%and?70.4±1.87?%,respectively,and there was no significant difference between the three groups?F=1.253,0>0.05?.?Conclusion?The RNA interference technique can effectively inhibit the CD166/ALCAM gene of CNE-2R cells in nasopharyngeal carcinoma by PCR and WB.The results showed that the silencing CD166/ALCAM gene may enhance the sensitivity of CNE-2R cells to radiation by inhibiting cell proliferation,inducing cell apoptosis and changing cell cycle distribution,suggesting that silencing of CD166/ALCAM gene is likely to be one of the effective treatment to improve the sensitivity of radiotherapy in patients with nasopharyngeal carcinoma. |
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