沉默HOXA10基因表达联合川芎嗪可逆转K562/ADR细胞的多药耐药性  

HOXA10 gene-silencing combined with tetramethylpyrazine reverses multidrug resistance of K562/ADR cells

作　　者：贾秀红[1] 伊英杰[1] 任秀敏[1] 王建勇[1] 李有杰[2] 

机构地区：[1]滨州医学院附属医院儿科,山东滨州256603 [2]滨州医学院医学分子遗传研究所,山东烟台264003

出　　处：《肿瘤》2016年第6期635-643,共9页Tumor

基　　金：山东省自然科学基金(编号:ZR2014HL032);山东省医药卫生科技发展计划(编号:2014WS0183)~~

摘　　要：目的 :研究靶向沉默人慢性髓系白血病耐多柔比星(adriamycin,ADR)细胞株K562/ADR中同源盒A10(homebox A10,HOXA10)基因表达后联合川芎嗪(tetramethylpyrazine,TMP)对逆转K562/ADR细胞多药耐药性的影响,并探讨其可能的作用机制。方法 :采用脂质体法将构建有靶向HOXA10基因的sh RNA重组表达载体p GPHI/GFP/Neo-HOXA10-sh RNA稳定转入K562/ADR细胞中,分别采用实时荧光定量PCR和蛋白质印迹法检测对HOXA10 m RNA和蛋白表达的影响。实验分为6组:空白对照组、阴性对照-sh RNA(negative controlsh RNA,NC-sh RNA)组、HOXA10-sh RNA组、TMP单药组、NCsh RNA+TMP(2μg/m L)组和HOXA10-sh RNA+TMP(2μg/m L)组。采用MTT法检测各组细胞的逆转耐药倍数。FCM法检测细胞内ADR平均荧光强度的变化;蛋白质印迹法检测细胞中P-糖蛋白(P-glycoprotein,P-gp)和多药耐药相关蛋白1(multidrug resistance-associated protein 1,MRP1)的表达。结果:p GPHI/GFP/Neo-HOXA10-sh RNA表达载体能有效抑制K562/ADR细胞中HOXA10 m RNA及蛋白的表达。HOXA10-sh RNA+TMP(2μg/m L)组K562/ADR细胞对ADR的逆转耐药倍数为5.08倍,均较HOXA10-sh RNA组和TMP单药组提高(P值均<0.05);细胞内ADR的平均荧光强度均较HOXA10-shR NA组和TMP单药组提高(P值均<0.05);P-gp和MRP1的表达水平均较HOXA10-shR NA组和TMP单药组明显下调(P值均<0.05)。结论 :靶向沉默HOXA10基因表达联合TMP能逆转K562/ADR细胞的多药耐药,两者间有协同作用,可为临床逆转白血病多药耐药提供重要的实验依据。Objective： To investigate the reversal effect of homebox A10 （HOXA10） gene-silencing combined with tetramethylpyrazine （TMP） on multi-drug resistance of adriamycin （ADR）-resistant chronic myeloid leukemia K562/ADR cells, and to explore its possible molecular mechanism. Methods： The recombinant pGPHI/GFP/Neo-HOXA10-shRNA vector targeting HOXAIO gene was transfected into K562/ADR cells by using liposome. The expression levels of HOXA10 mRNA and protein were measured by real-time fluorescent quantitative PCR and Western blotting, respectively. Then the cells were divided into six groups： blank control group, negative control （NC）-shRNA group, HOXA10-shRNA group, TMP （2 I^g/mL） group, NC- shRNA＋TMP （2 ~g/mL） group, and HOXA10-shRNA＋TMP （2 ~g/mL） group. The reversal effect of HOXAIO gene-silencing combined with TMP （2 i^g/mL） on drug-resistance of K562/ADR cells to ADR was detected by M＇IF method. The change of mean fluorescence intensity of ADR in K562/ADR cells was detected by FCM. The expression levels of P-glycoprotein （P-gp） and multidrug resistance-associated protein 1 （MRP1） were detected by Western blotting. Results： Transfection of pGPHI/GFP/Neo-HOXA10-shRNA vector effectively inhibited the expression levels of HOXA10 mRNA and protein in K562/ADR cells. In HOXA10-shRNA＋TMP （2 μg/mL） group, the reversal fold of drug-resistance of K562/ADR cells to ADR （5.08）, and the mean fluorescence intensity of ADR in K562/ADR cells were significantly higher than those in HOXA10-shRNA group and TMP group （all P 〈 0.05）. Compared with HOXA10-shRNA group and TMP group, the expression levels of P-gp and MRP1 in HOXA10-shRNA＋TMP group were significantly decreased （all P 〈 0.05）. Conclusion： HOXAIO gene-silencing combined with TMP can reverse multidrug resistance of K562/ADR cells, and there is a synergistic effect between them. It provides an important experimental basis for clinical reversing the multidrug resistance of leukemia.

关 键 词：白血病 抗药性 多药 RNA 小分子干扰 川芎嗪 HOXA10基因 

分 类 号：R733.7[医药卫生—肿瘤]