Hsa-miR-181a Post-transcriptionally Down-regulates RALA And Contributes To Cell Growth And Apoptosis In Leukemia K562 Cells

Objective:Our goal is to identify target genes of hsa-miR-181a and investigate their potential roles in leukemia K562 cells. This study is also expected to provide new experimental evidence for miRNA therapeutics in leukemia.Methods:In previous study, hsa-miR-181a dsRNA duplexes(hsa-miR-181a mimics) were designed and synthesized according to the mature sequence of hsa-miR-181a. Agilent Human 1A Oligo microarray was employed to generate data of expression profiles through overexpression of hsa-miR-181a mimics in K562 cells. In this study, we analyze the hsa-miR-181a targets by combining bioinformatic software prediction and expression profiling in leukemic K562 cells. Three overlapping genes were found to be the potential targets of hsa-miR-181a. RALA, one of the three, was observed to play important roles in tumorigenesis. For this reason, we detected RALA expression level by real-time PCR and Western blot at 48 hours post-transfection, and constructed RALA 3'UTR and RALA mut-3'UTR dual lueiferase report vector (psi-CHECK-2). Meanwhile, K562 cells were transfected with hsa-miR-181a and RALA siRNA respectively. Cell growth was detected by MTT. Cell apoptosis was detected by flow cytometry using double-staining with Annexin V-FITC and propidium iodide (PI), and further morphologically confirmed by hoechst 33258 staining. Cell cycle progression was determined by flow cytometry using PI staining.Results:Three overlapping genes (RALA, PLCL2 and SEMA4C) were found to be the potential targets of hsa-miR-181a. hsa-miR-181a significantly downregulated the expression level of RALA mRNA and protein. Further research showed that hsa-miR-181a significantly decreased the activity of RALA-3'UTR reporter without affecting the activity of RALA-mut-3'UTR reporter in the K562 cells. Both hsa-miR-181a and RALA siRNA effectively suppressed cell growth, induced apoptosis and G2-phase arrest.Conclusion:1. The identification of miRNA target using a combination of computational prediction and expression profiling analysis might be a good choice.2. RALA is one of the direct target genes of hsa-miR-181a.3. hsa-miR-181 a post-transcriptional effectively inhibits cell growth, induces cell apoptosis and G2-phase arrest partially by targeting RALA in leukemic K562 cells.