MMP-7 is upregulated by COX-2 and promotes proliferation and invasion of lung adenocarcinoma cells

Abstract

Matrix metalloproteinases (MMPs) have been implicated in a variety of pathophysiological conditions, of which MMP-7 is expressed by tumor cells of epithelial and mesenchymal origin. However, the function of MMP-7 in human lung adenocarcinoma (LAC) is unclear. In the present study the expression of MMP-7 in LAC was examined by immunohistochemical assay using a tissue microarray procedure. A loss-of-function experiment was performed to explore the effects and molecular mechanisms of lentiviral vector-mediated MMP-7 siRNA (siMMP-7) on cell proliferation and invasive potential in LAC A549 cells, measured by MTT and Transwell assays, respectively. It was found that, the expression of MMP-7 protein in LAC was significantly increased compared with that in adjacent non-cancerous tissues (ANCT) (76.0% vs 44.0%, P<0.001), and positively correlated with lymph node metastases of the tumor (P=0.014). Furthermore, targeted inhibition of cyclooxygenase-2 (COX-2) by siRNA downregulated the expression of MMP-7 and inhibited invasion of LAC cells, and knockdown of MMP-7 suppressed tumor proliferation and invasion in LAC cells. Taken together, our findings indicate that increased expression of MMP-7 is associated with lymph node metastasis and upregulated by COX-2, and promotes the tumorigenesis of LAC, suggesting that MMP-7 may be a potential therapeutic target for the treatment of cancer.

Figure 1.

The expression of MMP-7 protein in LAC tissues (magnification: 200×). LAC tissues were immunohistochemically stained with an anti-MMP-7 and COX-2 antibodies and classified as positive expression (A) and negative expression (C). Adjacent non-cancer tissues were immunohistochemically stained with an anti-MMP-7 antibody and classified as positive expression (B) and negative expression (D). COX-2 was highly expressed in LAC tissues (E) and lowly expressed in ANCT (F). Positive immunostaining of MMP-7 and COX-2 was mainly localized in the cytoplasm of tumor and tissue cells. Scale bars: A-C, E,F) 75 µm; D) 150 µm.

Figure 2.

The effect of COX-2 on the expression of MMP-7. After LAC A549 cells were transfected with siCOX-2 adenovirus for 24 h, the expression levels of COX-2 and MMP-7 were detected by Real-time PCR (A, B) and Western blot assays (C-F). The expression of COX-2 and MMP-7 was significantly decreased in siCOX-2 group compared with the CON and NC groups (each **P<0.01), suggesting that COX-2 might upregulate the expression of MMP-7 in LAC cells.

Figure 3.

The effect of COX-2 knockdown on cell invasion. A,B) Transwell assay was used to determine cell invasion; cell invasive potential was markedly inhibited in siCOX-2 group compared with the CON and NC groups (**P<0.01), suggesting that targeted inhibition of COX-2 might block invasion of LAC cells. Scale bars: A) 75 µm.

Figure 4.

Knockdown of MMP-7 expression in LAC A549 cells. After LAC A549 cells were transfected with siMMP-7 lentivirus for 24 h, the expression level of MMP-7 was detected by real-time PCR (A) and Western blot assays (B,C), indicating that the expression of MMP-7 could be knocked down in siMMP-7 group compared with CON and NC groups (**P<0.01).

Figure 5.

The effect of MMP-7 knockdown on cell proliferation. A) MTT assay was used to evaluate cell proliferative activity for consecutive 3 days; cell proliferative activity was remarkably diminished in a time-dependent manner in siMMP-7 group compared with the CON and NC groups (**P<0.01). B) [³H] thymi-dine incorporation into DNA was assessed by scintillation counting. Results are expressed as percentage of increase of [³H] thymi-dine incorporation in serum-stimulated cells over that of quiescent cells for each cell population, indicating that the increase of [³H]dT incorporation induced by serum was higher in CON and NC groups than in siMMP-7 group (**P<0.01). C,D) The expression level of PCNA protein, examined by Western blot assay, was downregulated in siMMP-7 group compared with the CON and NC groups (**P<0.01), suggesting that knockdown of MMP-7 might inhibit proliferation of LAC cells through downregulation of PCNA expression.

Figure 6.

The effect of MMP-7 knockdown on cell invasion. A,B) Transwell assay was performed to determine cell invasion; cell invasive potential was markedly weakened in siMMP-7 group compared with the CON and NC groups (**P<0.01), suggesting that knockdown of MMP-7 might inhibit invasion of LAC cells. Scale bar: A) 75 µm.