Study On Anti-leukemia Mechanism Of Silencing Of MIR-17and MIR-20a Mediated By Mirna Sponge

Objective:This study was aimed to construct miRNA Sponge targeting miR-20aand to establish a stable cell line Jurkat-S, paving the way for further research onfunction of miR-20a and application of RNAi in gene therapy.This study was aimed toquantitatively detect the expression levels of pre-miR-17and pre-miR-20a in acuteleukemia patients and cell lines, and to investigate the anti-leukemia mechanism ofsilence of miR-17and miR-20a mediated by miRNA Sponge.Methods:One pair of two-repeated oligonucleotide sequences containing bulgedsites that are mispaired opposite miR-20a positions9-12were designed andsynthesized with enzyme cutting sites. The annealed oligonucleotide fragments weresubcloned into pCDNA3.0-L expressing vector. After double-enzyme cutting, thevector was ligated to the annealed oligonucleotide fragments again. Enzyme cuttingand luciferase activity assay were used to identify after four repeats. Then the ligatedfragment was subcloned to lentivirus expressing vector. Virus particles were collectedafter the control or Sponge vectors were co-transfected with the psPAX2packagingplasmid and the envelope plasmid pMD2.G into HEK-293T cells usingLipofectamine2000. The Jurkat cells were transdused with recombinantlentivirus-transdusing units plus6μg/ml of Polybrene. Real-time PCR and Westernblot were used to detect the Mrna and protein expression of P21and E2F1afterlentivirus transdusion respectively. Then the proliferation ability and cell cycle ofJurkat cells was evaluated by CCK-8and flow cytometry respectively. Quantitativereal-time PCR was used to detect the mRNA expression levels of pre-miR-17andpre-miR-20a in various types of leukemia and cell lines. The Jurkat cellsover-expressing miR-17and miR-20a were transdused with recombinantlentivirus-transdusing units targeted at miR-17and miR-20a plus6μg/ml ofPolybrene. Results:Luciferase activity assay demonstrated that the sponge targeting miR-20awas constructed successfully and the virus was packaged in293T. The titer of viruswas5×107TU/ml. Stable transfected Jurkat-S cell line was established. As wasexpected, the mRNA and protein level of P21and E2F1was upregulated significantlyin Jurkat-S cells. The results showed that the pre-miR-17and pre-miR-20a expressionlevel in all leukemia patients was significantly higher than that in normalgroup(p<0.05), the expression of pre-miR-17and pre-miR-20a in acute lymphoidleukemia was significantly higher than that in acute myeloid leukemia(p<0.05), andthat the pre-miR-17and pre-miR-20a expression level was not significant with highwhite blood cell count of>20.0*109/L(p>0.05). Finally, silencing of miR-17andmiR-20a mediated by miRNA Sponge led to a significant decrease of cell growth andrestored G1accumulation.Conclusion:The miR-20a Sponge was constructed successfully, and Jurkat-Sstable cell line was established, in which the expression of miR-20a was inhibitedstably.It is concluded that miR-17and miR-20a expression is upregulated in leukemiapatients and may contribute to leukemogenesis. Over-expressed miR-17and miR-20apromote cell growth and cell cycle progression maybe by negatively-regulating P21and E2F1post-transcriptionally.