MiR-30A Inhibits Cell Migration, Invasion And Proliferation By Modulating Runx2 In Human Osteosarcoma Cells

Objective:In this study, we aimed to explore the expression level of miR-30a in different degree of malignancy human osteosarcoma cell lines and normal human marrow stromal cells. To investigate the role of miR-30a on human osteosarcoma cells in migration, invasion and proliferation and explore its molecular mechanisms.Methods:using Real-time quantitative PCR to detect the expression level of miR-30a in different degree of malignancy human osteosarcoma cell lines (143B, MG-63, U2OS, Saos2) and normal human marrow stromal cells (HS-5). The cell lines were treated with Ad-miR-30a or In-miR-30a. The cell migration, invasion were determined by Transwell assay. Wound healing assay was performed to detect the cell healing ability. The cell viability was analyzed by MTT assay. The target gene of miR-30a was identified by qRT-PCR and Western blot at mRNA level and protein level respectively. Luciferase report plasmids of miR-30a targeting gene were generated by pMIR-REPORT vector. Dual luciferase reporter gene experiment was used to investigate whether miR-30a bound to target gene 3-UTR or not.Results:The expression level of miR-30a in a series of human osteosarcoma cell lines were found to be markedly down-regulated in 143 B cell lines which is highly malignant osteosarcoma cell line when compared with low malignant cells (Saos2) and normal human marrow stromal cells (HS-5). Overexpression of miR-30a, migration and invasion ability of 143B cells were inhibited (P< 0.05). MTT assay results showed that 143B cell viability decreased by overexpression of miR-30a. miR-30a overexpression induced G1 cell cycle arrest in 143B cellse by FCM. When the endogenous miR-30a was inhibited in Saos2 cells, migration, invasion and proliferation ability of Saos2 cells increased. Ovexpression of miR-30a significantly repressed Runx2 protein expression, on the other hand, miR-30a inhibiting obviously promoted Runx2 protein expression. However, there was no significant difference in Runx2 expression after miR-30a overexpression or inhibiting. The 3’-UTR regions of target gene were insert into luciferase vector and the luciferase activity of Runx2 decreased by adding miR-30a. In rescue assay,143B were coinfected with Ad-miR-30a and Ad-Runx2, the vitality and scratch healing rate of 143B cells were recovered. On the other hand, miR-30a and Runx2 were inhibited simultaneously in Saos2 cells. Inhibiting Runx2 rescued miR-30a mediated promotion of cell vitality and scratch healing rate.Conclusion:miR-30a down-regulate in highly malignant 143B cell lines compared with low malignant cells (Saos2) and normal human marrow stromal cells (HS5). The migration, invasion and proliferation of osteosarcoma cells were regulated by miR-30a. This process is potentially achieved via suppressing Runx2 protein expression.