Single Nucleotide Mutation Changes the Capability of CCN3 in Osteosarcoma Cell Invasion and Differentiation

Abstract Background: This study is designed to identify the significant mutations in CCN3 gene in osteosarcoma and explore its influence in cell invasion and differentiation and the underlying mechanism. Methods: Sanger sequencing was applied to identify CCN3 gene sequence in human osteosarcoma cell lines, peripheral blood mononuclear cells (PBMC) and osteosarcoma tissue. Wildtype and mutant CCN3 (mCCN3) were ectopically expressed by lentivirus in human osteosarcoma cell lines. Tumor cell invasion was measured by Trans-well assay. Osteogenic differentiation was induced by osteogenic differentiating medium, and evaluated by alkaline phosphatase (ALP) activity, collagen type I alpha 1 chain (COL1A1) and osteocalcin expression. Western blotting was used to detect the protein level of CCN3 and mCCN3 in cytoplasmic, nuclear and secreted fractions of the cells. Results: A G to A single nucleotide mutation in coding region of CCN3 was found in both osteosarcoma cell and tissue. The frequency of this mutation in osteosarcoma tissue was much higher than that in para-carcinoma tissue and PBMC of healthy people. This nucleotide mutation decreased the nuclear glycosylated protein level of CCN3, and affected osteosarcoma cell invasion and differentiation. The lower nuclear ratio of glycosylated/non-glycosylated isoforms and the function change accounted for the different behavior of mCCN3 from that of CCN3. Conclusion: The G to A mutation identified in CCN3 resulted in differential glycosylated full length protein level and altered the function role of CCN3 in osteosarcoma cell invasion and differentiation.