MiR-144 Inhibits Tumor Growth and Metastasis in Osteosarcoma via dual-suppressing RhoA/ROCK1 Signaling Pathway.

Background Several miRNAs have been found expressed differentially in osteosarcoma (OS), thus they may function in the onset and progression of osteosarcoma. Method The osteosarcoma cell proliferation ability was evaluated by CCK8 assay, cell cycle and colony formation assay in vitro and xenograft NOD mice model in vivo. The osteosarcoma cell mortality was analyzed by wound healing assay and transwell invasion assay in vitro and by zebrafish cancer metastasis model in vivo. The level of miR-144 were detected by real-time PCR and ISH. The level of ROCK1 and RhoA1 were detected by real-time PCR, Western Blot and IHC. RNA pull down, RNA immunoprecipitation and dual-luciferase assay were used to determine the direct binding between miR-144 and ROCK1/RhoA. Results We found that miR-144 significantly suppresses osteosarcoma cell proliferation, migration and invasion ability in vitro, and inhibited tumor growth and metastasis in vivo. Mechanically, we demonstrated that Ras homolog family member A (RhoA) and its pivotal downstream effector Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) were both identified as direct targets of miR-144. Moreover, the negative co-relation between downregulated miR-144 and upregulated ROCK1/RhoA was verified both in the osteosarcoma cell lines and clinical patients'specimens. Functionally, RhoA with or without ROCK1 co-overexpression resulted a rescue phenotype on the miR-144 inhibited cell growth, migration and invasion abilities, while individual overexpression of ROCK1 had no statistical significance compared with control in miR-144 transfected SAOS2 and U2-OS cells. Conclusion This study demonstrates that miR-144 inhibited tumor growth and metastasis in osteosarcoma via dual-suppressing of RhoA and ROCK1, which could be a new therapeutic approach for the treatment of osteosarcoma.