| Aims:1. To confirm the drug resistance of HNE1/DDP.2. To investigate the expression of miR-181 c in cisplatin-sensitivity and cisplatin-resistant nasopharyngeal carcinoma cells and explore the effect of mi RNA onapoptosis in nasopharyngeal carcinoma cells.3. To investigate the effect on the apoptosis of nasopharyngeal carcinoma cells by regulating of mi R-181 c.Methods:1. We used cisplatin(DDP) with different concentrations in HNE1 and HNE1/DDP cells,respectively, the OD number of 24、48、72h was detected to calculate the IC50 and resistance index(RI) by MTT assay; The expression of Anti-apoptotic protein MDR、Bcl-2 and pro-apoptotic protein Bax and Bim were detected by Western blot to verify the drug resistance of HNE1/DDP.2. MiR-181 c was selected because of the different expression in HNE1 and HNE1/DDP used the mi RNA microarray assay and verify the selected mi RNA used Quantitative real-time RT-PCR.3. The mi R-181 c inhibitors were transfected into HNE1 with a high expression of mi R-181 c and the mi R-181 c mimics were transfected into HNE1/DDP with a low expression of mi R-181 c. The expression of mi R-181 c after transfection in the two cells was detected by RT-PCR; We used MTT assay to detect the effect on the proliferation of HNE1 transfected with mi R-181 c inhibitors and HNE1/DDP transfected with mi R-181 c mimics. The change of apoptosis of the two cells was detected by colony formation assay、PI and Annexin V/PI assay.4. The expression of Mcl-1 in HNE1 and HNE1/DDP after regulation of mi R-181cwas deteced by Western blot; colony formation assay、PI and Annexin V/PI assay were used to detected the effect on apoptosis after transfected with Mcl-1 si RNA.Results:1.Compared with HNE1 cell, HNE1/DDP cell exhibit chemoresistance to DDP(1) The proliferation of HNE1 and HNE1/DDP cells were inhibited by differentconcentrations of DDP(2, 4, 8, 16, 32μmol·L-1) for 24, 48, 72 h by MTT array, in concentration-and-time-dependent manners. The IC50 values of HNE1/DDP and HNE1 cells were 32.15±0.37、21.88±0.49、14.76±0.56 μmol·L-1 and 7.87±0.33、5.25±0.15、3.45±0.35 μmol·L-1 for 24, 48, and 72 h, and RI were 4.08、4.16、4.27. From the above result, we can know that compared with HNE1 cells, HNE1/DDP cells were not sensitive to DDP.(2) In HNE1/DDP, the expression of anti-apoptotic protein and anti-apoptotic m RNA MDR、Bcl-2 increased, at the same time, the expression of pro-apoptotic protein and m RNA Bax、Bim decreased.2.The expression of mi R-181 c was different in HNE1 and HNE1/DDPThe expression of mi R-181 c was different in the two cells by a mi RNA microarray of our group. We verified the difference by RT-PCR and found that the expression of mi R-181 c was up-regulation in HNE1.3. The effect on apoptosis of nasopharyngeal carcinoma cells by regulation of miR-181c(1) The mi R-181 c inhibitors were transfected into HNE1 with a high expression of mi R-181 c and the mi R-181 c mimics were transfected into HNE1/DDP with a low expression of mi R-181 c. We found that the mi R-181 c inhibitors could increase the expression of mi R-181 c in HNE1 and the mi R-181 c mimics could decrease the expression of mi R-181 c in HNE1/DDP by the RT-PCR assay.(2) The mi R-181 c inhibiors could enhance the drug-resistance to DDP in HNE1, On the contrary, the mi R-181 c mimics reduced the drug-resistance to DDP in HNE1/DDP in MTT assay. In addition, in colony formation assay、PI and Annexin V/PI assay, the results suggested that mi R-181 c inhibitors could reduce the apoptosis rate of DDP-induced in HNE1 and the mi R-181 c mimics could increased the the apoptosis rate of DDP-induced in HNE1/DDP.4. The mi R-181 c has an effect on apoptosis in nasopharyngeal carcinoma cells through regulation of Mcl-1.(1) The expression of Mcl-1 in HNE1/DDP was higher than HNE1 by Western blot.(2) The result of Western blot suggested that mi R-181 c inhibitors could increase the expression of Mcl-1 in HNE1 and mi R-181 c mimics could reduce the expression of Mcl-1 in HNE1/DDP.(3) We found that the expression of Mcl-1 was reduced after transfecting with Mcl-1 si RNA in western blot and RT-PCR assay. The colony formation assay result showed that transfected with Mcl-1 si RNA could inhibit the colony formation. In addition, transfected with Mcl-1 si RNA were markedly increased the apoptosis rate of DDP-induced in HNE1/DDP by PI and Annexin V/PI assay.Conclusion:1. HNE1/DDP has drug-resistance to DDP compare to HNE1.2. The expression of mi R-181 c was different in HNE1 and HNE1/DDP. Inhibition of mi R-181 c could inhibit the apoptosis in HNE1, On the contrary, overexpression of mi R-181 c could increase the apoptosis in HNE1/DDP.3. The change of Mcl-1 was consistent with mi R-181 c and Mcl-1 si RNA were markedly increased the apoptosis rate of DDP-induced in HNE1/DDP... |
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