PAI-1, a target gene of miR-143, regulates invasion and metastasis by upregulating MMP-13 expression of human osteosarcoma.

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.

MicroRNA-143 regulates human osteosarcoma metastasis by regulating matrix metalloprotease-13 expression.

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.

Cysteine-rich protein 61 suppresses cell invasion via down-regulation of matrix metalloproteinase-7 expression in the human gastric carcinoma cell line MKN-45.

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 down-regulation of MMP-7 expression in human gastric carcinoma cells.