MDR1、GSTπ特异性siRNA真核表达载体的构建及表达

Construction and examination of a specific siRNA expression vector inhibiting MDR1 and GST mRNA activity

目的构建多药耐药基因(MDR1)、谷胱甘肽S-转移酶(GSTπ)特异性小干扰RNA(siRNA)真核表达载体并检测其表达。方法参照siRNA模板设计原则,设计并化学合成2条siRNA模板序列,退火后将其插入质粒pSilencer2.1-U6,限制性酶切和基因测序进行鉴定。脂质体介导下转染K562/Adr细胞,实时荧光定量PCR分析MDR1、GSTπmRNA的表达,荧光免疫组化检测Pgp、GSTπ蛋白的表达。结果重组质粒pSilenc-er2.1-MDR1、GSTπ经酶切、测序分析表明siRNA模板序列成功插入预计位点,并且序列正确。用pSilencer-mdr1、pSilencer2.1-GSTπ分别转染K562/Adr细胞株,mdr1mRNA表达量下降了71.5%,GSTπmRNA表达量下降了39.8%,荧光免疫组化显示Pgp、GSTπ表达均显著减少(P<0.01)。结论成功构建了MDR1、GSTπ特异性siRNA真核表达载体,该载体可不同程度逆转K562/Adr细胞的多药耐药。

Purpose GSTπ mRNA activity To construct a specific siRNA expression vector which inhibits MDR1 and Methods Two templates sequence for siRNA were designed, synthesized, and inserted into pSilencer2.1-U6 neo expression vector. Restriction analysis and sequencing were performed to verify the pSilencer2.1U6 neo-MDR1 ,GSTTπ vector. Expression of mdrl and GSTTπ mRNA were assayed by SYBR Green Ⅰ real-time PCR. Immunofluorescence test was used to examine the expression of the multidrug gene （mdr-1） product P-glycoprotein （P-gp） and GSTTr protein. Results It was demonstrated that correct template sequence has been inserted in the designed location site. After transletted with pSilencer neo-mdrl ,the expression of mdrl mRNA in K562/Adr was reduced 71.5 % compared to the mock transfection; the expression of Pgp was greatly decreased by immunofluorescence test; while the expression of GSTTπ mRNA in K562/Adr cell transfected with pSilencer neo-GSTTπ was reduced 39.8% compared to the mock transfection, the expression of GSTTπ was greatly decreased by immunofluorescence test. Conclusions The siRNA expression vector against MDR1 and GSTTπ mRNA was constructed successfully. The hairpin siRNA could effectively mudolate the multidrug resistance of K562/Adr cell line.