[Construction of shRNA eukaryotic expression vectors of pkm2 gene and their effect on drug resistant cell line of acute promyelocytic leukemia].
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 Western 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 pkm2 targeting shRNA remarkably promotes the differentiation of NB4R2 cells, showing the prospects of developing the gene target drug.
Sun
Ming-Li
ML
Cancer Center, The First Hospital, Jilin University, Changchun 130021, Jilin Province, China.
Wang
Guan-Jun
GJ
Li
Jiang
J
Cui
Jiu-Wei
JW
Zhang
Ai-Li
AL
Wang
Zhong-Nan
ZN
Li
Xiao-Meng
XM
Li
Wei
W
D001426:Bacterial Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics*; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
D001426:Bacterial Proteins; D034741:RNA, Small Interfering; C093969:Pkn2 kinase; D017346:Protein Serine-Threonine Kinases