Pulmonary metastases are the main cause of death in patients with osteosarcoma, however, the molecular mechanisms of metastasis are not well understood. To detect lung metastasis-related microRNA (miRNA) in human osteosarcoma, we compared parental (HOS) and its subclone (143B) human osteosarcoma cell lines showing lung metastasis in a mouse model. miR-143 was the most downregulated miRNA (P < 0.01), and transfection of miR-143 into 143B significantly decreased its invasiveness, but not cell proliferation. Noninvasive optical imaging technologies revealed that intravenous injection of miR-143, but not negative control miRNA, significantly suppressed lung metastasis of 143B (P < 0.01). To search for miR-143 target mRNA in 143B, microarray analyses were performed using an independent RNA pool extracted by two different comprehensive miR-143-target mRNA collecting systems. Western blot analyses revealed that MMP-13 was mostly protein downregulated by miR-143. Immunohistochemistry using clinical samples clearly revealed MMP-13-positive cells in lung metastasis-positive cases, but not in at least three cases showing higher miR-143 expression in the no metastasis group. Taken together, these data indicated that the downregulation of miR-143 correlates with the lung metastasis of human osteosarcoma cells by promoting cellular invasion, probably via MMP-13 upregulation, suggesting that miRNA could be used to develop new molecular targets for osteosarcoma metastasis.

Despite recent improvements in the therapy for osteosarcoma, 30–40% of osteosarcoma patients die of this disease, mainly due to its lung metastasis. We have previously reported that intravenous injection of miR‐143 significantly suppresses lung metastasis of human osteosarcoma cells (143B) in a mouse model. In this study, we examined the biological role and mechanism of miR‐143 in the metastasis of human osteosarcoma cells. We identified plasminogen activator inhibitor‐1 (PAI‐1) as a direct target gene of miR‐143. To determine the role of PAI‐1 in human osteosarcoma cells, siRNA was transfected into 143B cells for knockdown of PAI‐1 expression. An in vitro study showed that downregulation of PAI‐1 suppressed cell invasion activity, but not proliferation. Moreover, injection of PAI‐1 siRNA into a primary lesion in the osteosarcoma mouse model inhibited lung metastasis compared to control siRNA‐injected mice, without influencing the proliferative activity of the tumor cells. Subsequent examination using 143B cells revealed that knockdown of PAI‐1 expression resulted in downregulation of the expression and secretion of matrix metalloproteinase‐13 (MMP‐13), which is also a target gene of miR‐143 and a proteolytic enzyme that regulates tumor‐induced osteolysis. Immunohistochemical analysis using clinical samples showed that higher miR‐143 expressing cases showed poor expression of PAI‐1 in the primary tumor cells. All such cases belonged to the lung metastasis‐negative group. Moreover, the frequency of lung metastasis‐positive cases was significantly higher in PAI‐1 and MMP‐13 double‐positive cases than in PAI‐1 or MMP‐13 single‐positive or double‐negative cases (P < 0.05). These results indicated that PAI‐1, a target gene of miR‐143, regulates invasion and lung metastasis via enhancement of MMP‐13 expression and secretion in human osteosarcoma cells, suggesting that these molecules could be potential therapeutic target genes for preventing lung metastasis in osteosarcoma patients.

Abstract. cysteine-rich protein 61 (cYr61) is a member of the ccn (cYr61/cTGF/noV) family, which is associated with progression in a variety of human cancers. our previous study confirmed that the expression levels of CYR61 protein were decreased in gastric carcinoma compared to non-tumoral mucosa as determined by proteome analysis. Histological research also showed that the reduction in CYR61 expression was significantly correlated with cellular invasiveness and inversely correlated with matrix metalloproteinase-7 (MMP-7/ matrilysin) expression in human gastric carcinoma. We examined the cause of CYR61 down-regulation in a human gastric carcinoma cell line, MKN-45. Lower expression of cYr61, but no genetic or epigenetic alterations of the gene, were observed. We then examined the correlation between CYR61 protein and MMP-7 expression and cellular invasiveness in MKn-45 cells. cYr61 was secreted from CYR61 expression-vector-transfected 293T cells, and the supernatant was added to MKN-45 cells. The expression level of MMP-7 was reduced by treatment of the supernatant, including cYr61, in a dose-dependent manner. an invasion assay showed that the cellular invasiveness of MKn-45 was significantly suppressed by the transfection of CYR61 expression vector compared to transfection with a control vector. Taken together, these results raise the possibility that cYr61 suppresses cell invasion at least partly via the downregulation of MMP-7 expression in human gastric carcinoma cells.