Evaluation of the expression of microRNAs associated with ROCK kinases and their role in the invasion process in osteosarcoma

Osteosarcoma (OS) is a neoplasia that mainly occurs at the metaphyses of long bones, being the most common pediatric bone tumor. The treatment is based on surgical resection and the multimodal chemoterapy adjuvant and neoadjuvant. However, despite the treatment, around 80% of patients who evolve to metastais present a poor survival. Therefore, understanding the metastatic process is essencial, as well as the search for new therapy targets. The mainly pathway related to invasion and migration in neoplasic cells is regulated by the Rho GTPases, and their main effectors are the kinases ROCK1 and ROCK2, which are responsible for cytoskeleton control. The hyperexpression of these kinases has been described in different cancers and it has been associated to poor prognostic. In parallel, several studies have extensively demonstrated miRNA deregulation in tumorigenesis, and the hipoexpression of some miRNA are related to ROCK upregulation, consequently, involved with metastasis. Herein, we studied the expression profiles of ROCK1 and 2 and associated miRNAs in OS tumor samples by means of qRT-PCR. We found downregulation of ROCK1 in OS samples when compared to normal bone (control), while ROCK2 did not show differences. MiR-138 showed hiperexpression and was correlated with ROCK2, and an association with survival rates. MiR-139 and miR-708 were found downregulated in tumor samples, though miR- 196b and miR-584 did not show differences in expression. Afterwards, miR-708 expression was induced in three OS cell lines, aiming establish miR-708 role. Proliferation and clonogenic essays did not present any effects when miR-708 was induced. In the wound healing essay, miR-708 reduced the migration of SAOS-2 cells, and in invasion essay, miR-708 induced invasion of MG-63 cells in a matrigel matrix, while reduced the invasive potential of HOS and SAOS-2 cell lines in a gelatin matrix. An in silico analysis of miR-708 targets highlighted its association with WNT, MAPK and Adherent Junction pathways. Therefore, we suggest that miR-708 can be involved in process that leads to metastasis, mainly related to extracellular matrix interation. (AU)