Construction And Application Of Artificial MicroRNA Targeting EZH2 Gene

Histone methyltransferase EZH2 is an anti-tumor target in the field of epigenetics.Many inhibitors have developed to the clinical stage.Therefore,the research on the inhibitor of EZH2 is very important for biomedical application.In this study,10 interference vectors were constructed by using NF-κB exclusive promoter DMP and miRNA targeting EZH2 to regulate the activity of EZH2 in cells.The main research contents are as follows:The 10 pairs of artificial miRNA single strand oligos were designed and synthesized with EZH2 selected as a target.These annealed miRNA single strand oligos were then ligated into the universal interference vector pEASY-Blunt-DMP-miR-lac that contained murine miR-155 backbone to construct 10 interference vectors targeting EZH2 coding sequences,while pEASYBlunt-DMP-miR-Neg was a negative control.TALENs and recombinant homology arms were used to evaluate the effect of subsequent interference.The results of cell transfection experiments demonstrated that the 10 interfering vectors constructed in this study all promoted the apoptosis of cancer cells in varying degrees.Each group of interference vectors was transfected into HepG2 cells,respectively.Then total RNA was extracted and cDNA was obtained from it by reverse transcription Kit.The expression level of EZH2 in treatment cells was detected by qPCR assay.The results showed that all of these constructed vectors could up-regulate the mRNA level of target genes.Based on the apoptosis rate detected by flow cytometry,pDMP-EZH2-miR1741 was selected as the most effective interference vector.To further explore the effect of pDMP-EZH2-miR1741 on protein levels of target genes,pDMP-EZH2-miR1741 was transfected into PANC-1 and HepG2 cells,respectively.The total proteins were extracted after 48 h.The results of western blot analysis showed that pDMPEZH2-miR1741 significantly reduced the protein expression of target genes.At the same time,the green fluorescent protein ZsGreen was fused with EZH2 by gene editing technology TALEN.The TALENs were co-transfected with pDMP-EZH2-miR1741 into HepG2 cells.Then the fluorescence intensity of cells was measured by fluorescence microscopy and flow cytometry.The fluorescence intensity of cells decreased with the interference of pDMP-EZH2-miR1741,which was consistent with the results of western blot,indicating that pDMP-EZH2-miR1741 down-regulated the protein level of target genes.The pDMP-EZH2-miR1741 was transfected into HepG2,PANC-1,A549 and HL7702 cells,respectively.The cell growth was measured through microplate reader and the growth curve was drawn with CCK-8 reagent treatment.The results displayed that pDMP-EZH2-miR1741 inhibited the growth of cancer cells and had few effects on viability of normal cells.Therefore,the selected pDMP-EZH2-miR1741 vector in this research is hopeful to be a novel anti-tumor agent targeting EZH2 gene for gene therapy.