MMP-9 Knockdown Inhibits Oral Squamous Cell Carcinoma Lymph Node Metastasis in the Nude Mouse Tongue-Xenografted Model through the RhoC/Src Pathway

Abstract

Oral squamous cell carcinoma (OSCC) is one of the most common types of cancers in developing countries. A major contributor to the high mortality rate of OSCC is the tendency of oral cancer cells to metastasize to lymph nodes around the head and neck during the early stages of cancer development. Matrix metalloproteinase 9 (MMP-9), an endopeptidase, can degrade the extracellular matrix and basement membrane and plays a key role in tumor invasion and metastasis. In vitro, cell migration ability was conducted by scratching assays. We also investigated the interaction abilities between OSCC cells and vascular endothelial cells (ECs) by an adhesion assay and transendothelial migration assay. And we established a BALB/c nude mouse tongue-xenografted metastasis model to investigate the role of MMP-9 and explore its potential underlying mechanism in OSCC growth, lymph node metastasis, and angiogenesis in vivo. The results showed that knockdown of MMP-9 could significantly suppress OSCC cell migration, proliferation, interactions between endothelial cells, xenografted tumor growth, and angiogenesis and simultaneously markedly inhibited OSCC cell metastasis to mouse lymphonodi cervicales superficiales, axillary lymph nodes, and even distant inguinal lymph nodes. Mechanistic studies revealed that knockdown of MMP-9 also led to a decreased expression of RhoC, Src, and F-actin by RT-PCR, western blotting, and immunohistochemistry. And the bioinformatic analysis showed that MMP-9, RhoC, and Src mRNA expression was positively and linearly correlated in OSCC on TCGA database. Together, our findings indicated that MMP-9 plays a very important role in OSCC growth, migration, angiogenesis, and lymph node metastasis, and its potential mechanism may be mediated by RhoC and Src gene expression.

Figure 1

Knockdown of MMP-9 suppresses OSCC growth in the nude mouse tongue-xenografted model. (a, e) H&E staining of the tongue mucosa in CAL27- or SCC15-transfected cells of the nude mouse tongue-xenografted model, respectively. (b, c, e, f) H&E staining of tumor mass in the tongue of nude mice. ^∗P < 0.05, compared with the control group. (d, h) Mouse body weights in CAL27- or SCC15-transfected cells of the nude mouse tongue-xenografted model, respectively. ^∗P < 0.05, control group compared with the blank group; ^∗∗P < 0.01, control group compared with the blank group; ^#P < 0.05, MMP-9/shRNA group compared with the blank group; ^##P < 0.01, MMP-9/shRNA group compared with the blank group; ^☆P < 0.05, MMP-9/shRNA group compared with the control group.

Figure 2

Knockdown of MMP-9 suppresses OSCC cell proliferation in the nude mouse tongue-xenografted model. (a, b) Expression of MMP-9 in CAL27- or SCC15-transfected cells of nude mouse tongue-xenografted tumors by IHC, respectively. (c, d) Cell proliferation is suppressed by MMP-9/shRNA transfection as shown by Ki67-positive cells. All data are shown as the mean ± SD. ^∗∗P < 0.01, compared with the control group.

Figure 3

Knockdown of MMP-9 suppressed OSCC cell interactions between endothelial cells and xenografted tumor angiogenesis. (a) MMP-9/shRNA transfection suppresses microvascular density (MVD) by IHC. (b) Knockdown of MMP-9 could decrease cell adhesion to endothelial cells by the adhesion assay. (c) Knockdown of MMP-9 could decrease cell transendothelial migration between endothelial cells. All data are shown as the mean ± SD. ^∗∗P < 0.01, compared with the control group.

Figure 4

Knockdown of MMP-9 inhibited OSCC cell migration. (a) MMP-9/shRNA transfection inhibited CAL27 cell migration ability by the scratch migration assay. (b) MMP-9/shRNA transfection inhibited SCC15 cell migration ability by the scratch migration assay. ^∗∗P < 0.01, compared with the control group.

Figure 5

MMP-9 knockdown inhibits OSCC cell metastasis to lymphonodi cervicales superficiales in the nude mouse tongue-xenografted model. (a, b) Expression of CK by IHC in the lymphonodi cervicales superficiales in CAL27- and SCC15-transfected cells of the nude mouse tongue-xenografted model, respectively. (c, e) H&E staining of tumor mass in the lymphonodi cervicales superficiales (independent sample t-test). (d, f) The metastasis rates of lymphonodi cervicales superficiales. ^∗P < 0.05, ^∗∗P < 0.01, compared with the control group.

Figure 6

MMP-9 knockdown inhibits OSCC cell metastasis to axillary lymph nodes in the nude mouse tongue-xenografted model. (a, c) Expression of CK by IHC in axillary lymph nodes in CAL27- and SCC15-transfected cells of the nude mouse tongue-xenografted model, respectively (independent sample t-test). (b, d) The metastasis rates of axillary lymph nodes. ^∗P < 0.05, ^∗∗P < 0.01, compared with the control group.

Figure 7

MMP-9 knockdown inhibits OSCC cell metastasis to inguinal lymph nodes in the nude mouse tongue-xenografted model. (a, c) Expression of CK by IHC in inguinal lymph nodes in CAL27- and SCC15-transfected cells of the nude mouse tongue-xenografted model, respectively (independent sample t-test). (b, d) The metastasis rates of inguinal lymph nodes. ^∗P < 0.05, ^∗∗P < 0.01, compared with the control group.

Figure 8

Knockdown of MMP-9 suppresses RhoC, Src, and F-actin expression in vitro. (a) RNA expression of RhoC and Src by RT-PCR in CAL27-transfected cells. (b) Protein expression of RhoC and Src by western blotting in CAL27-transfected cells. (c) Knockdown of MMP-9 suppresses RhoC expression by immunocytochemistry. (d) MMP-9/shRNA transfection decreased phalloidin staining by immunofluorescence. All data are presented as the mean ± SD. ^∗∗P < 0.01, compared with the control group.

Figure 9

Knockdown of MMP-9 suppresses RhoC and Src expression in tongue-xenografted tumors. (a, b) MMP-9/shRNA transfection inhibits RhoC expression in the cells inoculated with CAL27 or SCC15-transfected cells of the nude mouse tongue-xenografted tumors by IHC, respectively. (c, d) MMP-9/shRNA transfection suppresses Src expression in the nude mouse tongue-xenografted tumors by IHC. All data are presented as the mean ± SD. ^∗∗P < 0.01, compared with the control group.

Figure 10

Correlation analysis of MMP-9, RhoC, and Src in OSCC. (a) Correlation analysis of MMP-9 and RhoC. (b) Correlation analysis of MMP-9 and Src. (c) Correlation analysis of RhoC and Src. All mRNA expressions in OSCC were based on TCGA database (including TCGA OSCC samples n = 341). P < 0.001.