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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 investigate the effect of RNA interference on the expression of B cell-specific murine leukemia virus integration site-1 (BMI-1) gene in doxorubicin (Dox) ) Chemoresistance and its related molecular mechanisms. Methods: The Dox-resistant human hepatoma cell line MHCC-97H / Dox (97H / Dox) was established and its parent cell line MHCC-97H (97H) was used as control. MTT, cell clone formation and Transwell invasion assay The drug resistance, clonality and invasion ability of the cells were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. Transient transfection of small interfering RNA (si RNA) was used to inhibit the expression of BMI-1 in both cell lines, and then the drug resistance of 97H / Dox and 97H to Dox were detected by MTT assay The changes of tumor-related proteins in parental cells 97H were detected by Western blotting. Results: The drug resistance of 97H / Dox was stable and multidrug resistant, and the number of clone formation was also significantly higher than that of 97H (P <0.05). Compared with 97H parental cells, 97H / Dox The expression of BMI-1, ABCG2 and MMP-2 in drug-resistant cells was up-regulated (P All <0.05). siRNA-mediated BMI-1 gene silencing significantly increased the sensitivity of chemosensitive Dox to 97H / 97H / Dox resistant cells (P <0.05). In 97H parental cells, silencing of BMI-1 significantly decreased phospho-Akt (p-Akt), phospho-c-Jun N-terminal kinase (p- And ABCG2 (all P <0.05). CONCLUSION: Up-regulation of BMI-1 gene may be involved in the drug resistance of 97H cells. Inhibition of BMI-1 expression may enhance the sensitivity of this cell to chemotherapeutic drug Dox. Therefore, intervention of BMI-1 expression may be a candidate strategy to improve the chemotherapeutic response of hepatocellular carcinoma.