| Objective:Nasopharyngeal carcinoma(NPC)is a head and neck malignant tumors.Radiotherapy-based comprehensive treatment is the main treatment of nasopharyngeal carcinoma in clinical,because the main pathological types of NPC are poorly differentiated squamous cell carcinoma and undifferentiated carcinoma,that are sensitive to radiation and have the unique anatomical features.As some patients have radioresistance,tumor residual,recurrence and distant metastasis are the main reasons for the failure of NPC treatment.Therefore,the application of gene therapy to improve radiosensitivity may increase the survival rate in patients with NPC.In recent years,the study was found and confirmed that Ig can promote the growth of tumor cells,inhibit the apoptosis of tumor and participate in the occurrence and development of tumors in many kinds of tumor cells.Previous experiments in our group have confirmed that there was the ectopic expression of IgM in NPC that anti-IgM antibody to inhibit its expression can significantly inhibit the proliferation and promote the apoptosis in NPC HNE-1 cell,and the higher the expression of IgM in NPC tissue,the lower the sensitivity of NPC to radiotherapy.In this study,human NPC CNE1 cells were transfected with the lentivirus of human IgM gene RNAi to observe its effect on cell function and radiosensitivity in order to explore the relationship between IgM gene and the biological function and radiosensitivity of NPC CNE1 cells.Methods:CNE1cells were cultured in vitro using 1640 medium+10%fetal bovine serum.(1)After the shRNA sequence was designed,the lentivirus was packaged,transfected and screened to obtain stable transfected CNE1 cells,which were CNE1-sh cell line stably interfered with IgM expression and CNE1-NC cell line negative control group with ineffective interference IgM sequence.CNE1cells without any treatment were used as blank control group.The expression of IgM mRNA and protein in three groups of cells was detected by qRT-PCR and Western blot respectively.(2)The effect of interfering IgM expression on the survival fraction of CNE1 cells and the effect on the survival fraction of cells in three groups after combining radiotherapy were detected by plate clone formation assay.(3)The influence of interfering IgM expression on the proliferation of CNE1 cells and the effect on the proliferation of cells in three groups after combining radiotherapy were detected by MTT assay.(4)The effect of interfering IgM expression on the apoptotic rate of CNE1 cells and the effect on apoptotic rate of cells in three groups after combining radiotherapy were detected by Flow cytometry(Annexin V/PI staining method).(5)The changes of the cell cycle of CNE1 cells with interfering IgM expression and the effect of cell cycle of cells in three groups after combining radiotherapy were detected by FCM(PI/RNase staining method).(6)Female BALB/c-nu nude mice were randomly divided into A group and B group.Each group was randomly divided into three groups in order to be inoculated cells in three groups respectively.Then the nude mice xenograft models were constructed.After radiotherapy in group B,compare the effects of lentivirus-mediated IgM-RNAi on tumorigenesis and radiotherapy sensitivity in the A and B groups.Results:(1)The results of qRT-PCR showed that the IgM was expressed in all the cells of three groups,and the relative expression level of IgM in CNE1-sh group(0.220±0.013)was significantly lower than that in CNE1 group(1.000±0.000)and CNE1-NC group(0.890±0.050)(F=615.069,P=0.000).The results of Western blot experiment results showed that the IgM protein was expressed in all the cells of three groups,and the expression of IgM protein in CNE1-sh group(0.320±0.007)was significantly lower than that in CNE1 group(1.053±0.001)and CNE1-NC group(1.037±0.012)(F=8228.347,P=0.000).(2)The results of plate clony formation assay showed that interfering IgM expression can significantly reduce cell colony formation rate of CNE1 cells.The cell colony formation rate in CNE1-sh group(0.385±0.005)was significantly lower than that in CNE1 group(0.658±0.015)and CNE1-NC group(0.648±0.010)(F=589.727,P=0.000).The cell clony formation rate of CNE1-sh groups were significantly lower than that of CNE1 groups and CNE1-NC groups after radiotherapy(0Gy,2Gy,4Gy,6Gy,8Gy,10Gy),and the difference was statistically significant,statistical values were respectively(F=589.727,P=0.000),(F=565.344,P=0.000),(F=365.548,P=0.000),(F=434.569,P=0.000),(F=112.600,P=0.000),(F=173.000,P=0.000).The survival curve of CNE1-sh group was lower than that of CNE1 group and CNE1-NC group which was fitted by the single-hit multitarget model and the linear quadratic model,and the sensitization enhancement ratios were respectively 1.12 and 1.42 that were all greater than 1.(3)The results of MTT assay showed that interfering IgM expression can significantly inhibit the proliferation of CNE1 cells,and the difference was not statistically significant in OD value between the cells of three groups after 0 day(F=0.241,P=0.793).The OD value of CNE1-sh group was significantly less than that of CNE1group and CNE1-NC group after 2 days(F=9.037,P=0.015).Then the OD value of CNE1-sh group was significantly less than that of CNE1 group and CNE1-NC group after 3 and 4 days,statistical values were respectively(F=94.462,P=0.000),(F=17.658,P=0.003).After 6 Gy radiotherapy,there was no significant difference in OD value between the cells of three groups on the first 0 day(F=2.521,P=0.160).On the first day after radiotherapy,the OD value of CNE1-sh group was significantly lower than that of CNE1 group and CNE1-NC group(F=11.156,P=0.010).On the second day,third and fourth days after radiotherapy,the OD of CNE1-sh group was significantly lower than that of CNE1 group and CNE1-NC group(F=275.152,P=0.000),(F=81.788,P=0.000),(F=203.941,P=0.000).(4)The results of flow cytometry(Annexin V/PI staining method)showed that interfering IgM expression can significantly improve the apoptotic rate of CNE1 cells.The apoptotic rate of CNE1-sh group(11.393±0.240%)was significantly higher than that of CNE1 group(4.043±0.683%)and in CNE1-NC group(2.847±0.842%)(F=165.351,P=0.000).After radiotherapy with the exposure dose of 6 Gy,the apoptotic rate of CNE1-sh group(35.023±2.742%)was significantly higher than that in CNE1group(12.433±1.391%)and in CNE1-NC group(12.987±0.804%)(F=147.942,P=0.000).(5)The results of flow cytometry(PI/RNase staining method)showed that interfering IgM expression can significantly change the cell cycle distribution that was mainly distributed in G1 and S phase.Compared with CNE1 group and CNE1-NC group,the proportion of G1 phase of CNE1-sh group was significantly increased(F=28.086,P=0.001),and the proportion of S phase of CNE1-sh group was significantly decreased(F=9.303,P=0.014).The proportion of G2 phase of three groups was no difference(F=1.424,P=0.321).After radiotherapy with the exposure dose of 6 Gy,compared with CNE1 group and CNE1-NC group,the proportion of G2 phase of CNE1-sh group was significantly increased(F=34.470,P=0.001),the proportion of G1 phase CNE1-sh group was significantly decreased(F=22.812,P=0.002),and the proportion of S phase of CNE1-sh group was decreased(F=9.088,P=0.015).(6)The NPC nude mice xenografts models of three groups were constructed successfully,and the formation rate of xenografts tumor was 88.10%.On the eighteenth day after radiotherapy,the results showed that the average volume of nude mice xenografts of CNE1-sh group(789.600±253.163 mm~3)was significantly lower than that of CNE1 group(1785.320±624.497 mm~3)and CNE1-NC group(2028.541±741.848 mm~3)in the A group(F=5.148,P=0.032).The average volume of nude mice xenografts of CNE1-sh group(272.587±84.779 mm~3)was significantly lower than that of CNE1 group(700.681±49.123 mm~3)and CNE1-NC group(631.691±63.519 mm~3)in the B group(F=46.494,P=0.000).The inhibition rates of tumor volume of CNE1-sh group in the A and B group respectively were 51.95%,61.10%.The average weight of nude mice xenografts of CNE1-sh group(0.833±0.215 g)was significantly lower than that of CNE1 group(1.670±0.491 g)and CNE1-NC group(1.733±0.600 g)in the A group(F=4.684,P=0.040).The average weight of nude mice xenografts of CNE1-sh group(0.273±0.104 g)was significantly lower than that of CNE1 group(0.788±0.098 g)and CNE1-NC group(0.750±0.067 g)in the B group(F=39.492,P=0.000).The inhibition rates of tumor weight of CNE1-sh group in the A and B group respectively were53.12%,63.67%.The HE staining of xenografts showed that the necrotic lesions and marked pleomorphism of CNE1-sh group were more than that of CNE1 group and CNE1-NC group in the A and B group.And it was more obvious in the B group.Conclusions:(1)CNE1 cell line stably interfering with IgM can be obtained by lentivirus-mediated RNAi.(2)The interfering IgM expression in CNE1 cells can inhibit its clone formation and proliferation,induce apoptosis and promote G1 arrest,and CNE1 cell growth is inhibited.(3)The interfering IgM expression combined with radiotherapy can significantly inhibit colony formation and proliferation of CNE1 cells,promote cell apoptosis and arrest cell cycle in G2 phase in order to improve the radiosensitivity of CNE1 cells.(4)That interfering IgM expression and radiotherapy can reduce the volume and weight of the tumors and promote the necrotic lesions of xenograft tumor,and it inhibit tumor growth more significantly with combining radiotherapy.It was confirmed that the interfering IgM expression can improve the radiosensitivity of CNE1 cells.(5)On the basis of our experiment,we will further explore its possible mechanisms that interfering with IgM expression enhances the radiosensitivity of CNE1 cells,that may have important clinical significance to improve the radiosensitivity of NPC. |
PDF Full Text Request