Ras基因与鼻咽癌西妥昔单抗耐药的相关性研究

Correlation between ras gene and the resistance of nasopharyngeal carcinoma to cetuximab

目的探讨ras基因与鼻咽癌西妥昔单抗耐药的相关性。方法以人鼻咽癌细胞株5-8F细胞为研究对象,采用逐步增加剂量法诱导建立西妥昔单抗耐药细胞5-8F/Erbitux;MTT法检测西妥昔单抗对细胞的半数抑制浓度IC50,计算耐药指数(RI);Western Blot法检测细胞H-ras、K-ras的蛋白表达;实时荧光定量PCR检测细胞ras-p21、H-ras以及K-ras基因的表达量;测序方法检测H-ras和K-ras基因第12、13、59、61密码子是否突变。结果建立了西妥昔单抗耐药的人鼻咽癌细胞系5-8F/Erbitux,西妥昔单抗作用3天和5天的RI分别为1.2和1.1;5-8F/Erbitux细胞H-ras和K-ras蛋白表达水平显著升高(P<0.001),H-ras基因表达量显著升高(P=0.016),K-ras基因表达量显著下降(P=0.000);ras-p21基因表达量有所升高,但差异不显著(P=0.113);通过比对,5-8F/Erbitux细胞H-ras基因、K-ras基因第12、13、59、61密码子均无突变。结论 H-ras基因扩增与过表达是导致5-8F/Erbitux细胞对西妥昔单抗耐药的主要机制之一。

Objective To explore the correlation of ras gene to the drug resistance of nasopharyngeal carcinoma to cetuximab.Methods Cultured 5-8F/Erbitux cells were induced by stepwise exposure to increasing doses of cetuximab.MTT assay was used to determine the IC50（half inhibitory concentration） of cetuximab and the drug resistance index（RI）.Western blotting was employed to detect the protein levels of H-ras and K-ras.Real-time PCR was used to detect the expression of H-ras and K-ras.Gene sequencing was performed to identify potential mutations in H-ras and K-ras genes.Results We successfully induced cetuximab-resistant 5-8F/Erbitux hNPC cells by stepwise exposure to increasing doses of cetuximab.After treatment with cetuximab for 3 and 5 days,the RI of 5-8F/Erbitux cells was 1.2 and 1.1,respectively.The 5-8F/Erbitux cells had increased levels of H-ras and K-ras protein expressions（P0.001） and enhanced gene expressions of H-ras（P=0.016） and ras-p21（P=0.113） with decreased K-ras gene expression（P=0.000）.Sequence analysis identified no mutations in the H-ras and K-ras genes in codons 12,13,59,and 61.Conclusion Gene amplification and overexpression of H-ras is the major mechanism that causes the drug resistance of 5-8F/Erbitux cells to cetuximab.