Dysregulation of MiR-519d Affects Oral Squamous Cell Carcinoma Invasion and Metastasis by Targeting MMP3

Abstract

MicroRNA-519d (miR-519d) has been reported to play important roles in tumor development and progression in multiple cancers, either as tumor suppressor or tumor promotor. However, the expression level, biological function and molecular mechanisms of miR-519d in oral squamous cell carcinoma (OSCC) remain unclear. Therefore, the aims of this study were to investigate the functional role of miR-519d in OSCC and the possible underlying regulatory mechanism. In this study, we found that miR-519d was significantly downregulated in OSCC tissues and cell lines compared with normal oral mucosae and normal oral epithelial cells. Importantly, downregulation of miR-519d was closely correlated with the lymph node metastasis, advanced tumor stage and poor overall survival of OSCC patients. Furthermore, miR-519d significantly inhibited the migration and invasion of OSCC cells. Using bioinformatics and biological approaches, we showed that miR-519d directly targeted matrix metalloproteinase-3 (MMP3), which might account for the underlying mechanism involved in the miR-519d mediated suppression of OSCC progression. What is more, miR-519d expression was inversely correlated with MMP3 expression in OSCC tissues, and high levels of MMP3 expression in OSCC tissues were also associated with the metastasis and poor prognosis of these patients. In addition, we further identified that miR-519d acted as a regulator of epithelial-mesenchymal transition (EMT) in OSCC cells. Overall, the present study highlighted miR-519d as a tumor suppressor in OSCC by targeting MMP3 and supported biological and clinical links between miR-519d-MMP3 and OSCC, thus indicating the potential therapeutic value of miR-519d for alleviating OSCC metastasis.

Keywords: MMP3; epithelial-mesenchymal transition (EMT); invasion; metastasis; miR-519d; oral squamous cell carcinoma.

Figure 1

Downregulation of miR-519d in OSCC indicates malignant transformation. (A) The expression level of miR-519d in 60 pairs of OSCC tissues and adjacent normal tissues (P<0.0001). (B, C) Low miR-519d expression was correlated with lymph node metastasis (P=0.037) and advanced TNM stage (P=0.001) in OSCC. (D) Kaplan-Meier analyses of overall survival. Patients with low miR-519d expression had a significantly lower overall survival rate than patients with high miR-519d expression (P=0.0006). (E) The miR-519d expression in normal oral epithelial cells, primary cultured cancer cells and 4 OSCC cell lines. (F) The methylation status of the DNA region located upstream of C19MC was detected in 5 OSCC cell lines and 24 pairs of OSCC samples and adjacent normal tissues by MSP. OSCC, oral squamous cell carcinoma; NC, matched adjacent normal tissue. (G, H) The expression of miR-519d in OSCC cells treated with or without the demethylating agent 5-Aza-dc (10 μM) for 72 h. **, P < 0.01. ***, P< 0.001. ****, P<0.0001.

Figure 2

miR-519d significantly inhibits the migration and invasion of OSCC cells. (A) The expression of miR-519d in OSCC cells transfected with miR-519d mimics or miR-NC. (B) MTT experiment demonstrated that overexpression of miR-519d had no significant effect on the proliferation of OSCC cells. (C) The expression of miR-519d in OSCC cells transfected with miR-519d inhibitor or NC inhibitor. (D) MTT assay showed that silencing of miR-519d exhibited less effect on the proliferation of OSCC cells. (E, F) Dysregulation of miR-519d influenced the migration of OSCC cells, suggested by wound healing assay. (G, H) Transwell invasion assay revealed the suppressive role of miR-519d in the invasive abilities of OSCC cells. The fields of migrated and invasive cells on the membrane were captured (magnification × 100). Statistical analysis was performed using the t-tests. The data represent the mean values of three independent experiments. *, P < 0.05, **, P < 0.01. ***, P< 0.001. ****, P<0.0001.

Figure 3

MMP3 is a direct target of miR-519d in OSCC. (A) Venn diagrams of the predicted candidate target genes of miR-519d by bioinformatic method (miRecords) and mRNA microarray (GSE30784). (B) qPCR was adopted to validate the 13 potential targets in HN4 or HN30 cells after the transfection of miR-519d mimics or miR-NC. (C) The mRNA expression level of miR-519d in OSCC cells transfected with or without miR-519d inhibitor. (D) The protein level of MMP3 in HN4 or HN30 cells at 72 h after the transfection of miR-519d mimics or miR-519d inhibitor, as suggested by western blotting. (E) The predicted binding site of miR-519d and MMP3 and detailed sequences of WT-3′-UTR or MUT-3′-UTR of MMP3. (F, G and H) Luciferase reporter activity revealed the relative MMP3 reporter activities in 293T, HN4 or HN30 cells co-transfected with miR-519d mimics plus luciferase reporters or miR-519d inhibitor plus luciferase reporters. *, P < 0.05, **, P < 0.01. ***, P< 0.001. ****, P<0.0001

Figure 4

Regulation of MMP3 influences the effect of miR-519d on the migration and invasion of OSCC cells. (A) The mRNA and protein expression of MMP3 in HN4 or HN30 cells after concurrent transfection with miR-519d or/and an exogenous MMP3 expression vector. (B) The mRNA and protein expression of MMP3 in OSCC cells were detected after the transfection of miR-519d inhibitor or/and MMP3 specific siRNA. (C) Wound healing assay revealed that miR-519d inhibited the migration of HN4 or HN30 cells while ectopic expression of MMP3 reversed this effect. (D) As presented by wound healing assay, inhibiting the expression of MMP3 by siRNA could rescue the miR-519d inhibitor-induced promotion of cell migration. (E) Transwell invasion assay performed in HN4 or HN30 cells showed that the miR-519d-induced inhibition of cell invasion was abolished by MMP3 transfection. (F) siRNA targeting MMP3 rescued the tumor promoting effect of miR-519d inhibitor on cell invasion in OSCC. *, P < 0.05, **, P < 0.01. ***, P< 0.001. ****, P<0.0001.

Figure 5

miR-519d acts as a EMT regulator in OSCC. (A) HN4 or HN30 cells were transfected with miR-NC, miR-519d or miR-519d plus MMP3 plasmid. The protein levels of MMP3, E-cadherin, N-cadherin and Vimentin in these cells were measured by western blotting at 72 h after transfection. (B) HN4 or HN30 cells were transfected with NC inhibitor, miR-519d inhibitor or miR-519d inhibitor plus MMP3 siRNA. Western blotting showed the protein expression of MMP3, E-cadherin, N-cadherin and Vimentin in these cells at 72 h after transfection. (C) HN4 or HN30 cells were transfected with miR-NC, miR-519d or miR-519d plus MMP3 plasmid. Then immunofluorescence experiment was performed to detect the protein expression of MMP3, E-cadherin, N-cadherin and Vimentin in these cells (Scare bar: 20 μm). (D) OSCC cells (HN4 and HN30) were transfected with NC inhibitor, miR-519d inhibitor or miR-519d inhibitor plus MMP3 siRNA, and the expression of MMP3, E-cadherin, N-cadherin and Vimentin in tumor cells were measured using immunofluorescence (Scare bar: 20 μm).

Figure 6

Inverse correlation between the expression of miR-519d and MMP3, and the clinical implications of MMP3 in patients with OSCC. (A) MMP3 mRNA levels were determined by qPCR in 60 pairs of OSCC tumor samples and adjacent normal tissues. (P=0.0009). (B, C) The relative high expression level of MMP3 was correlated with lymph node metastasis (P<0.0001) and advanced TNM stage (P=0.041) in OSCC patients. (D) Survival curves revealed an overall better prognosis of patients with low MMP3 expression. (P=0.0197). (E) A negative correlation between miR-519d and MMP3 was found in OSCC clinical samples. (P=0.0034). (F) The Kaplan-Meier survival curves indicated that patients with a low miR-519d level and a high MMP3 level had the worst survival rate. (P=0.004).