抑制BMI-1表达可增强人肝癌细胞对多柔比星的敏感性

Inhibition of BMI-1 expression increases the sensitivity to doxorubicin in human hepatocellular carcinoma cells

目的 :研究RNA干扰技术抑制B细胞特异性小鼠白血病病毒插入位点1(B cell-specii c murine leukemia virus integration site-1,BMI-1)基因表达对肝癌细胞多柔比星(doxorubicin,Dox)化疗耐药性的影响及其相关分子机制。方法 :建立Dox耐药的人肝癌细胞系MHCC-97H/Dox(简称97H/Dox),同时以其亲本细胞系MHCC-97H(简称97H)为对照;采用MTT、细胞克隆形成和Transwell侵袭实验分别检测细胞的耐药性、克隆形成能力和侵袭能力;实时荧光定量PCR和蛋白质印迹法检测细胞中肿瘤相关分子的m RNA和蛋白表达水平。采用小干扰RNA(small interference RNA,si RNA)瞬时转染的方法抑制2种细胞中BMI-1基因的表达,然后再用MTT法检测耐药细胞97H/Dox和亲本细胞97H对Dox的耐药性变化,以及采用蛋白质印迹法检测亲本细胞97H中肿瘤相关蛋白的表达变化。结果 :肝癌耐药细胞系97H/Dox的耐药性稳定,并呈多药耐药特点,其克隆形成数也明显多于亲本细胞97H(P<0.05);相对于97H亲本细胞,97H/Dox耐药细胞中BMI-1、ATP结合转运蛋白G超家族成员2(ATPbinding cassette superfamily G member 2,ABCG2)和金属基质蛋白酶-2(matrix metalloproteinase-2,MMP-2)分子的表达水平上调(P值均<0.05)。si RNA介导的BMI-1基因沉默可以显著提高97H亲本细胞及97H/Dox耐药细胞对化疗药物Dox的敏感性(P值均<0.05)。在97H亲本细胞中,BMI-1基因沉默可显著降低磷酸化Akt(phospho-Akt,p-Akt)、磷酸化c-Jun氨基末端激酶(phospho-c-Jun N-terminal kinase,p-JNK)和ABCG2的表达水平(P值均<0.05)。结论 :BMI-1基因表达上调可能参与97H细胞耐药性的获得,抑制BMI-1表达可增强该细胞对化疗药物Dox的敏感性。因此,干预BMI-1表达可能是提高肝癌化疗反应性的一个候选策略。

Objective： To study the effect of B cell-specific murine leukemia virus integration site-1 （BMI-1） gene expression inhibited by RNA interference on doxorubicin （Dox） -resistance of hepatocellular carcinoma cells, and to explore its related molecular mechanism. Methods： The Dox-resistant MHCC-97H cell line was established and named as 97H/Dox, while the parental MHCC-97H cell line （named as 97H） was used as the control. The drug-resistance, cell colony-forming capacity and invasive ability were measured by MTT method, cell colony formation assay and in vitro Transwell invasive experiment, respectively. The mRNA and protein levels of tumor-related genes were detected by real-time fluorescent quantitative-PCR （RFQ- PCR） and Western blotting. Then the small interference RNA （siRNA） targeting BMI-1 gene （BMI- -siRNA） was transfected into 97H/Dox and 97H cells. After BMI-1 gene silencing, the changes of Dox-resistance of 97H/Dox and 97H cells expressions of tumor-related proteins in 97H were detected by MTT method, and the change of cells was detected by Western blotting. Results： The 97H/Dox cell line had a stable multiple-drug resistance, and its colony formation capacity was higher than that of the control cell line 97H （P 〈 0.05）. The levels of BMI-1, ATP-binding cassette superfamily G member 2 （ABCG2） and matrix metalloproteinase-2 （MMP-2） were significantly increased in 97H/Dox cells compared with those in the control 97H cells （all P 〈 0.05）. Knockdown of BMI-1 gene by siRNA significantly increased the sensitivity to Dox in 97H/Dox and 97H cells （both P 〈 0.05）, and resulted in a significant reduction of the expression levels of phospho-Akt （p-Akt）, phospho-c-Jun N-terminal kinase （p-JNK） and ABCG2 in 97H cells （all P 〈 0.05）. Conclusion： The upregulation of BMI-1 gene expression may be involved in the acquisition of Dox-resistance in hepatocellular carcinoma 97H cells. Inhibition of BMI-1 expression can enhance the sensitivity to Dox in hepatocellular carcinoma cells. Therefore, interfering the expression ofBM/-1 gene may be a potential strategy for enhancing the response to chemotherapy in treatment of hepatocellular carcinoma.