[1712] miR-17-5b Inhibition Decreases Rhabdomyosarcoma Cell Growth In Vitro.

Chunyan Liu, Susana Galli, Maria Tsokos. NCI/NIH, Bethesda, MD

Background: microRNAs have been recognized as important players in tumorigenesis. One of these microRNAs, the cluster miR-17-92, exhibits potent oncogenic activity in many tumors. A recent study demonstrated overexpression of miR-17-92 in rhabdomyosarcoma, but its exact role in this tumor has not been clarified. Rhabdomyosarcoma is the most common soft tissue sarcoma in children and young adults. Even with aggressive therapy, many patients with rhabdomyosarcoma show poor clinical outcome. Investigation of tumorigenic pathways, such as that of the oncogenic miR-17-92 cluster, may lead to identification of novel therapeutic targets. Here we report that inhibition of miR-17-5b, one component of the oncogenic microRNA cluster, decreases rhabdomyosarcoma cell growth in vitro.
Design: Four rhabomyosarcoma cell lines were used: two (Rh4 and Rh28) alveolar and two (Rh36 and CTR) embryonal. The miR-17-5b miRIDIAN Hairpin Inhibitor was transfected with Lipofectamin 2000 to inhibit the function of endogenous miR-17-5b. Cell proliferation was measured using an assay that detected cellular dehydrogenase activity (CCK-8 kit) over a period of five days. Transfected cells were stained with propidium iodide and analyzed for cell cycle arrest using flow cytometry. Real-time RT-PCR and Western blotting were performed to evaluate the expression levels of genes that were involved in cell cycle arrest and apoptosis.
Results: Five days after transfection with the miR-17-5b inhibitor, all four rhabdomyosarcoma cell lines showed decreased cell proliferation. Flow cytometry demonstrated moderate increase in the percentage of G0/G1 arrested cells. p21, a key regulator of G0/G1 cell cycle arrest, was upregulated. BIM, an important proapoptotic protein in mitochondria-mediated apoptosis, was upregulated as well. These findings suggest that miR-17-5b promotes a malignant phenotype in rhabdomyosarcoma through p21-mediated cell cycle deregulation and BIM-mediated inhibition of apoptosis.
Conclusions: Our data show that miR-17-5b inhibition in rhabdomyosarcoma cells causes significant decrease in cell proliferation, promotes moderate cell cycle arrest and induces apoptotic pathways. Therefore, miR-17-5b may play an important role in the tumorigenesis of rhabdomyosarcoma and may serve as a potential therapeutic target for this tumor.
Category: Pediatrics

Monday, February 28, 2011 11:15 AM

Platform Session: Section H 2, Monday Morning

 

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