Expression inhibition of multidrug resistance-associated protein (MRP1) by multi-ribozyme expression system in HEK293 cells.
To study application of multi-ribozyme expression system on expression inhibition of multidrug resistance-associated protein (MRP1) in HEK293 cells, the multi-ribozyme expression system containing 20 cis-acting ribozymes for self-cleavage and 10 trans-acting ribozymes for targeting to MRP1 gene specific site were constructed. HEK293 cells cotransfected multi-ribozyme expression system with MRP1 target gene or full length of MRP1 gene respectively were analyzed by RT-PCR, Western blot analysis and MTT assay. The results showed that multi-ribozyme systems were able to dramatically decrease fluorescent fusion protein expression in HEK293 cells. RT-PCR analysis indicated that the extent of MRP1 target mRNA decrease was correlated with the number of trans-acting ribozyme contained in multi-ribozyme expression system. Similar changes have been observed from Western blot. MTT assay showed that multi-ribozyme systems were able to reverse MDR generated by MRP1 gene in HEK cells. These results suggested that inhibitory effects of multiple copies of ribozymes contained in the system were better than that of single ribozyme contained. Therefore, this strategy could be used in treatment of tumor or other diseases via gene therapy.
Tian
Sheng Li
SL
College of Life Sciences, Shenzhen Key Laboratory of Microbiology and Gene Engineering, Shenzhen University, Shenzhen 518060, China. sltian@szu.edu.cn
Liu
Gui Yun
GY
Zheng
Shuo
S
Liang
Hui Qing
HQ
Liu
Shi De
SD
Zhang
Jian Hua
JH
D015153:Blotting, Western; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism* / Q000502:physiology*; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction
D027425:ATP-Binding Cassette, Sub-Family C Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger