UGCG通过调控MDR1/P-gp参与人结肠癌奥沙利铂耐药的初步研究

张秀芬; 刘贝贝; 郭子健; 李莉华

doi:10.3971/j.issn.1000-8578.2016.09.003

UGCG通过调控MDR1/P-gp参与人结肠癌奥沙利铂耐药的初步研究

UGCG is Involved in Oxaliplatin Resistance Mechanism of Human Colon Cancer Through Regulating MDR1/P-gp Expression

摘要

摘要:
目的建立人结肠癌耐奥沙利铂（L-OHP）细胞株，检测该细胞株的多药耐药性并初步探讨其可能的耐药机制。
方法以人结肠癌细胞HCT116为对象，采用药物浓度梯度递增诱导法建立人结肠癌耐奥沙利铂细胞株HCT-116/L-OHP。CCK-8法检测L-OHP、顺铂（DDP）、5-氟尿嘧啶（5-Fu）对亲本细胞和耐药细胞株的半数抑制浓度（IC50）。使用UDP-葡萄糖神经酰胺糖基转移酶（UGCG）siRNA转染HCT-116/L-OHP细胞，实时荧光定量 PCR和Western blot检测干扰前后UGCG基因和多药耐药基因1（MDR1）mRNA及其编码的蛋白表达水平。
结果 HCT-116/L-OHP对L-OHP的耐药指数为10.5，与DDP有一定程度的交叉耐药，耐药指数为4.61，但对5-Fu无交叉耐药。耐药细胞HCT-116/L-OHP中UGCG、MDR1 mRNA和UGCG、P-糖蛋白（P-gp, MDR1编码的蛋白）表达均增加，相比HCT-116细胞，差异具有统计学意义（P<0.05）。UGCG siRNA成功抑制HCT-116/L-OHP细胞中UGCG的表达，各干扰组MDR1 mRNA、P-gp表达减少，与对照组相比差异具有统计学意义（P<0.05）。
结论成功构建了人结肠癌耐药细胞株；UGCG基因通过调控MDR1/P-gp的表达参与人结肠癌奥沙利铂的耐药机制的形成。

Abstract:
Objective To establish and authenticate an oxaliplatin (L-OHP) resistant human colon cancer cell line, and to preliminarily explore the multidrug resistance mechanism.
Methods An L-OHP-resistant human colon cancer cell line, HCT116/L-OHP, was established by gradually increasing the dose of L-OHP in the culture. The half inhibition concentration (IC50) of L-OHP, cisplatin (DDP) and 5-fluorouracil (5-Fu) in HCT116 and HCT116/L-OHP cell lines were evaluated by CCK8 assay. The siRNAs of UDP-glucose ceramide glucosyltransferase (UGCG) were used to transfect HCT-116/L-OHP cells. The gene and protein expression levels of UGCG and multidrug resistance gene1 (MDR1) were examined by real-time reverse transcription-polymerase chain reaction(PCR) and Western blot.
Results The resistance index to L-OHP in HCT116/L-OHP cell line was 10.5. In addition, HCT116/L-OHP cell line had cross resistance with DDP by 4.61 of the resistance index, but not with 5-Fu. The expression levels of UGCG mRNA, MDR1 mRNA, UGCG protein and P-glycoprotein (P-gp, encoded by MDR1 gene) were significantly higher in HCT116/L-OHP cells than those in HCT116 cells (P<0.05). After transfected with UGCG siRNA, both UGCG gene and protein expression were inhibited in HCT116/L-OHP cells. Compared with the negative control, the expression of MDR1 mRNA and P-gp were also significantly decreased (P<0.05).
Conclusion L-OHP resistant human colorectal cancer cell line HCT116/L-OHP are successfully established, and UGCG is involved in L-OHP resistance mechanism of human colon cancer through regulating MDR1/P-gp expression.

HTML全文

参考文献(34)

施引文献

资源附件(0)