耐药肝癌细胞p62表达与顺铂敏感性的关系

Relationship between p62 expression in cisplatin-resistant liver cancer cells and sensitivity to cisplatin

目的观察顺铂耐药的肝癌细胞中p62的表达情况,及p62基因沉默后耐药肝癌细胞对顺铂敏感性的变化。方法培养肝癌亲本细胞Hep G2、Huh7,通过浓度梯度法建立顺铂耐药细胞模型,采用Western blotting法检测亲本Hep G2、Huh7细胞和耐药Hep G2、Huh7细胞中的p62蛋白。将耐药Hep G2、Huh7细胞分为沉默组和耐药组,分别转染p62 siRNA和siRNA Negative Control。在转染48 h后的0、1、2、3、4、5 d测算沉默组和耐药组细胞存活率。转染后48 h进行Hochest 33258染色观察沉默组和耐药组细胞凋亡核形态、计数凋亡细胞,采用Annexin VFITC/PI流式细胞术测算细胞凋亡率。转染后48 h采用Western blotting法检测沉默组与耐药组细胞中的核因子红细胞前体2相关因子2(Nrf2),RT-PCR法检测Nrf2蛋白下游的谷氨酸半胱氨酸合成酶(GCL)、谷胱甘肽S转移酶(GST)、NADP(H)醌氧化还原酶(NQO1)、血红素氧化酶1(HO-1)、多药耐药蛋白(Mrp)基因。结果耐药的Hep G2、Huh7细胞中p62蛋白相对表达量分别高于亲本Hep G2、Huh7细胞(P均<0.05)。转染48 h后的0、1、2、3、4、5 d沉默组Hep G2、Huh7细胞存活率低于耐药组(P均<0.05)。沉默组凋亡细胞数及凋亡率均高于耐药组(P均<0.05)。沉默组细胞中Nrf2蛋白及NQO1、GCL、GST、HO-1、Mrp mRNA相对表达量均低于耐药组(P均<0.05)。结论顺铂耐药的肝癌细胞中p62蛋白表达上调;p62基因沉默后耐药肝癌细胞对顺铂的敏感性增强,其机制可能与Nrf2及其下游基因表达下调有关。

Objective To investigate the expression of p62 in cisplatin-resistant liver cancer cells and the chemotherapeutic sensitivity of p62-silenced cisplatin-resistant liver cancer cells to cisplatin. Methods Human liver cancer cell lines Hep G2 and Huh7 were used to induce cisplatin-resistant cell lines by slowly increasing cisplatin concentrations. The protein expression of p62 was detected in parental cells and cisplatin-resistant cells by Western blotting. The liver cancer cisplatin-resistant cell lines（ Hep G2/CDDP and Huh7/CDDP） were divided into the p62-silenced group and cisplatin-resistant group,and were treated by p62 siRNA and siRNA Negative Controls,respectively. After 48-hour transfection,MTT assay was applied to detect cell viability of the two groups at 0,1,2,3,4,and 5 d. Apoptotic nuclear changes were evaluated with Hochest 33258 staining,and apoptotic cells were quantified. Meanwhile,the apoptosis rate was calculated by Annexin V-FITC/PI flow cytometry at 48 h after transfection. The protein expression of NF-E2-related factor 2（ Nrf2） and the mRNA expression of its target genes glutamate cysteine ligase（ GCL）,glutathione S transferase（ GST）,quinone oxidoreductase 1（ NQO1）,glutathione S transferase（ GST）,heme oxygenase-1（ HQ-1） and multidrug resistance-associated protein（ Mrp） was evaluated by Western blotting and RT-PCR,respectively. Results The cisplatin-resistant cells（ Hep G2 and Huh7） expressed much higher protein levels of p62 than their parental cells（ all P〈0. 05）. The cell viability of the two groups at 0,1,2,3,4,and 5 d of the p62-silenced group was lower than that of the cisplatin-resistant cells（ all P〈0. 05）,and the apoptosis of cisplatin-resistant group increased as compared with that of the p62-silenced group（ all P〈0. 05）. The expression of Nrf2 protein and its target genes NQO1,GCL,GST,HO-1,and Mrp mRNA of the p62-silenced group was lower than that of the cisplatin-resistant group（ all P〈0. 05）. Conclusions The protein level of p62 is up-regulated in cisplatin-resistant liver cancer cells. Silencing p62 increases the sensitivity of liver cancer cells to cisplatin by down-regulating the expression of Nrf2 and its target genes.