Single nucleotide mutation changes the capability of CCN3 in osteosarcoma cell invasion and differentiation

Abstract

This study aimed to identify significant mutations in CCN3 gene in osteosarcoma, and to explore the influence of this gene on cell invasion and differentiation and the underlying mechanism. Sanger sequencing was used to identify CCN3 gene sequence in human osteosarcoma cell lines, peripheral blood mononuclear cells (PBMC), and osteosarcoma tissues. Wild-type 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 based on alkaline phosphatase activity and collagen type I alpha 1 chain and osteocalcin expression. Western blotting was used to detect protein levels of CCN3 and mCCN3 in cytoplasmic, nuclear and secreted fractions of cells. A G-to-A single nucleotide mutation in the coding region of CCN3 was found in both osteosarcoma cells and tissues. 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 nuclear glycosylated full length protein level of CCN3 and affected osteosarcoma cell invasion and differentiation. A lower nuclear ratio of glycosylated/non-glycosylated isoforms accounted for the different behavior of mCCN3 compared with CCN3. The G-to-A mutation identified in CCN3 resulted in differential glycosylated full-length protein levels and altered the functional role of CCN3 in osteosarcoma cell invasion and differentiation.