[Targeted regulation of mll-af4 fusion gene by miR-142-3p].

In order to analyze the possible epigenetic regulation mechanism of mll-af4 gene expression and find the possible microRNA regulating mll-af4 gene expression, targetscan software was used to analyze potential microRNA target sites in 3'-UTR of mll-af4. 3'-UTR fragment of mll-af4 was amplified by PCR. PCR products were cloned into EcoR I/Pst I-digested pGL3-M reporter vector, placing the 3'-UTR with potential microRNA binding site downstream of coding sequence of luciferase. The construct was cotransfected in 293T cells with control plasmid or plasmids expressing microRNAs regulating mll-af4 potentially. Western blot and RT-PCR were used to detect the expression level of mll-af4 protein and mRNA in RS4; 11 cells after transfection of miR-142-3p, respectively. The results showed that the pGL3-AF4-3'UTR of luciferase reporter recombinant plasmid contain the 3'UTR sequence of mll-af4 gene with 1935 bp, 2104 bp and 1371 bp was constructed successfully and was confirmed by enzyme digestion and gene sequencing. The luciferase assay revealed that overexpression of miR-142 could reduce the luciferase activity from the reporter construct containing the mll-af4 3'-UTR significantly. Protein and mRNA expressions of mll-af4 were found to be downregulated by miR-142-3p. It is concluded that miR-142-3p regulates the expression of mll-af4 through target binding the mll-af4 gene 3'UTR site.