[HOXA9 is direct target of miR-196a]

To analyze the possible epigenetic regulation mechanism of HOXA9 gene expression and find the possible microRNA regulating HOXA9 gene expression.

Targetscan software was used to analyze potential microRNA target sites in 3'-UTR of human HOXA9. 3'-UTR fragment of HOXA9 was amplified by PCR. PCR products were cloned into Xba I/Pst I-digested pGL3-M reporter vector, placing the 3'-UTR with potential microRNA binding sites downstream of coding sequence of luciferase. Mutant 3'UTRs were generated by overlap extension PCR method. The construct was cotransfected in 293T cells with control plasmid or plasmids expressing microRNAs regulating HOXA9 potentially. Western blot and RT-PCR were used to detect the expression level of HOXA9 protein and mRNA in MV4-11 cells after transfection of miR196a.

The results of luciferase assays revealed that overexpression of miR-196a could reduce the luciferase activity from the reporter construct containing the HOXA9 3'-UTR significantly. The activity of the reporter construct mutated at the specific miR-196a target site was unaffected. Protein and mRNA of HOXA9 was found to be downregulated by miR-196a.

Theses results suggested that miR-196a regulates the expression of HOXA9 by targeting the complementary sites.