Mechanism Of RASSF1A Gene In Inhibiting Osteosarcoma And Its Relationship With Wnt Signaling Pathway

OBJECTIVE:Osteosarcoma is a kind of connective tissue tumor which is developed from the mesenchymal cells with osteogenic potential. It is one of the primary malignant bone tumors with highest incidence rate. Osteosarcoma is considered to be a very high degree of malignancy, which has a tendency of local invasion and distant metastasis. RASSF1A is a novel tumor suppressor gene, loss of its expression resulting from promoter hypermethylation is found in various cancers including osteosarcoma. Hovever, role and mechanism of RASSF1A on osteosarcoma is not clarified. In this study, the effects of RASSF1A on cell proliferation, apoptosis, invasion and metatasis of osteosarcoma cells, as well as the relationship between RASSF1A gene and Wnt/β-catenin pathway were studied, in order to explore the application of RASSF1A gene in gene therapy.METHODS:We constructed the adenovirus expressing RASSF1A gene and infected osteosarcoma cells to explore the relationship between RASSFIA gene with osteosarcoma at the cellular level. Firstly, we used Western blotting method to detect the expression of RASSF1A genes in osteosarcoma cell lines. Then, we transduced adenovirus expressing RASSFIA gene into osteosarcoma cell line MNNG/HOSC1without expression of RASSF1A. A series of experiments were performed to examine the effects of RASSF1A gene in cell proliferation (MTT test), cell cycle (flow cytometry), apoptosis (flow cytometry), migration (Transwell), invasion (Matragel assay). Realtime quantative PCR method was used for determine the expression of MMP7, C-myc, and Cyclin D1expression. Western blotting was conducted to examine the expression level and phosphorylation status of serveral key proteins involved in Wnt/β-catenin signaling pathway. At last, we used the xenograft tumor nude mice model to examine the tumor suppressive role of RASSF1A gene.RESULTS:We successfully constructed the adenovirus shuttle vector p-Ad4-shutter-RASSF1A and obtained recombinant adenovirus vector expressing RASSFIA gene. We confirmed the RASSF1A expression differences in different osteosarcoma cell lines and low RASSFIA expression in Saos-2and MNNG/HOSC1cell lines. We demonstrated that over-expression of RASSF1A gene in MNNG/HOSC1lead to G1/S phase arrest, reduced cell proliferation rate, enhanced apoptosis, as well as inhibited the abilities of invasion and migration. The key genes responsible for proliferation, migration, and invasion, such as MMP7, C-myc, Cyclin D are down-regulated by RASSFIA gene. We further analysis the cell signaling pathway and found that RASSF1A can affect the Wnt signaling pathway, such as promoting the degradation of β-catenin protein, inhibiting phosphorylation of GSK3β protein and enhancing the phosphorylation of MST1/MST2protein. The result of xenograft tumor nude mice experment may inhibit the tumor growth.CONCLUSIONS:Over-expression of RASSF1A in osteosarcoma cell lines MNNG/HOSC1can inhibit cell proliferation, migration and invasion, promote apoptosis and cause cell cycle arrest. RASSFIA inhibits the Wnt/β-catenin signaling pathway by promoting the degradation of β-catenin inhibition and inhibiting the phosphorylation of GSK3β.These results will provide a firm theoretical foundation for application of RASSFIA in gene therapy of osteosarcoma.