靶向沉默HuR基因增强人肝癌细胞Hep3B对多柔比星的化疗敏感性
Gene silencing of HuR enhances the chemosensitivity of human liver cancer cells Hep3B to doxorubicin
摘要 目的:探讨沉默人抗原R(HuR)基因对人肝癌细胞Hep3B多柔比星化疗敏感性的影响及相关机制。方法脂质体包裹靶向HuR基因的短发夹RNA(shRNA)转染Hep3B细胞,real-time PCR和Western blot分别检测沉默HuR基因后HuR、MDR1 mRNA和蛋白的表达,MTT法检测沉默HuR基因后Hep3B细胞对多柔比星化疗的敏感性,流式细胞术(FCM)检测沉默HuR基因后对Hep3B细胞凋亡的影响。结果 HuR shRNA质粒转染Hep3B细胞能有效沉默HuR基因,mRNA和蛋白水平的抑制率分别为82%和75%。与未处理Hep3B细胞(Mock组)比较,沉默HuR基因的Hep3B细胞(siHuR组)对多柔比星的半数抑制浓度(IC50)显著降低[(86.36±6.54)μg/L vs.(318.56±9.89)μg/L, P<0.05],细胞凋亡率明显升高[(30.48±5.12)%vs.(8.86±1.35)%,P<0.05],MDR1 mRNA和P-gp蛋白表达显著降低,比值分别为0.36±0.08和0.28±0.11,差异均有统计学意义(P<0.05)。Mock组与转染对照shRNA质粒Hep3B(Control组)的各检测指标均无统计学差异(P>0.05)。结论沉默HuR基因可能通过抑制耐药蛋白P-gp的表达增强其对多柔比星的化疗敏感性。
Abstract:
Objective To investigate the effect and underlying mechanisms of silencing human antigen R (human antigen R, HuR) gene to the chemotherapy sensitivity of human liver cancer cells Hep3B to doxorubicin. Methods Plasmids of short hairpin RNA (short hairpin RNA, shRNA) targeting HuR gene were transfected into Hep3B cells by Lipofectimine 2000. The mRNA and protein levels of HuR and MDR1 in Hep3B cells after transfection of these plasmids were detected by real-time PCR and Western blot analysis, respectively. The chemosensitivity to doxorubicin and apoptosis rates of Hep3B cells were measured by MTT assay and flow cytometry (FCM). Results Transfection of HuR shRNA plasmids effectively inhibited the mRNA and protein levels of HuR in Hep3B cells by 82%and 75%, respectively. In comparison with untransfected Hep3B cells (Mock group), the half inhibitory concentration (IC50) of Hep3B cells transfected with HuR shRNA plasmids (siHuR group) was significantly lower [(86.36±6.54)μg/L vs. (318.56±9.89)μg/L, P<0.05)]. Meanwhile, the apoptosis rates of siHuR group were increased significantly [(30.48±5.12)%vs. (8.86±1.35)%, P<0.05], and the MDR1 mRNA and P-gp protein expression was significantly lower, with ratio was 0.36±0.08 and 0.28±0.11 (P<0.05). Conclusion Gene silencing of HuR may increase the chemosensitivity of Hep3B to doxorubicin through inhibiting the expression of MDR1 gene and P-gp.
Author: Liu Liping Bao Shiyun Zhang Yusen He Wan
作者单位: 暨南大学第二临床医学院 深圳市人民医院肝胆外科, 广东深圳,518020 暨南大学第二临床医学院 深圳市人民医院肿瘤科, 广东深圳,518020
年,卷(期): 2015, (9)
在线出版日期: 2015年5月25日
基金项目: 国家自然科学基金