Pkm2-shRNA真核表达载体的构建和对急性早幼粒细胞白血病耐药细胞株的作用研究

Construction of shRNA Eukaryotic Expression Vectors of pkm2 Gene and Their Effect on Drug Resistant Cell Line of Acute Promyelocytic Leukemia

本研究旨在构建M2型丙酮酸激酶(M2-pyruvate kinase;PKM2)的小发夹结构RNA(shRNA)的真核表达载体,研究特异性沉默pkm2基因对急性早幼粒细胞白血病(APL)耐药性的影响。设计pkm2基因的3个特异shRNA序列,利用基因重组技术将其克隆到含有U6启动子的PBSU6载体上,通过酶切和测序等方法鉴定重组质粒的正确性。利用Western blot方法检测了pkm2-shRNA对急性早幼粒细胞白血病耐药细胞株NB4R2细胞内源性M2-PK蛋白表达的影响,通过NBT还原实验检测了pkm2基因沉默后NB4R2细胞分化情况。结果表明:成功构建了3个有效特异的pkm2-shRNA,在蛋白水平上证明了其对NB4R2细胞M2-PK基因沉默的有效性。发现干涉M2-PK基因后,可显著地促进耐药细胞NB4R2细胞的分化。结论:DNA载体途径的pkm2-shRNA能促进早幼粒细胞白血病细胞的分化,具有基因靶向药物开发前景。

This study was aimed to construct the shRNA eukaryotic expression vectors of M2-pyruvate kinase gene （pkm2） and to investigate the effects of pkm2 gene interference on the drug resistance of acute promyelocytic leukemia （APL） cells in vitro. Three specific shRNAs of pkm2 gene were designed and cloned into PBSU6 vector containing a U6 promotor. The constructed plasmids were identified and proved by the restriction sequence analysis. Then the effect of pkm2-shRNA on the protein expression of endogenous PKM2 was detected in NB4R2 cells, a drug resistant cell line of APL by Westem blot. The alteration of NB4R2 cell differentiation with the interference of pkm2 gene was also validated by nitroblue tetrazolium （NBT） reduction test. The results showed that three specific shRNA eukaryotic expression vectors targeting pkm2 were successfully constructed. The efficiency of pkm2 gene silence was proved at protein level. The interference of pkm2 gene could significantly enhance the cell differentiation in the drug resistant NB4R2 cell line. It is concluded that the DNA vector containing pkrn2 targeting shRNA remarkably promotes the differentiation of NB4R2 cells, showing the prospects of developing the gene target drug.