Matrix metalloproteinase 12 is induced by heterogeneous nuclear ribonucleoprotein K and promotes migration and invasion in nasopharyngeal carcinoma

Abstract

Background: Overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a DNA/RNA binding protein, is associated with metastasis in nasopharyngeal carcinoma (NPC). However, the mechanisms underlying hnRNP K-mediated metastasis is unclear. The aim of the present study was to determine the role of matrix metalloproteinase (MMP) in hnRNP K-mediated metastasis in NPC.

Methods: We studied hnRNP K-regulated MMPs by analyzing the expression profiles of MMP family genes in NPC tissues and hnRNP K-knockdown NPC cells using Affymetrix microarray analysis and quantitative RT-PCR. The association of hnRNP K and MMP12 expression in 82 clinically proven NPC cases was determined by immunohistochemical analysis. The hnRNP K-mediated MMP12 regulation was determined by zymography and Western blot, as well as by promoter, DNA pull-down and chromatin immunoprecipitation (ChIP) assays. The functional role of MMP12 in cell migration and invasion was demonstrated by MMP12-knockdown and the treatment of MMP12-specific inhibitor, PF-356231.

Results: MMP12 was overexpressed in NPC tissues, and this high level of expression was significantly correlated with high-level expression of hnRNP K (P = 0.026). The levels of mRNA, protein and enzyme activity of MMP12 were reduced in hnRNP K-knockdown NPC cells. HnRNP K interacting with the region spanning -42 to -33 bp of the transcription start site triggered transcriptional activation of the MMP12 promoter. Furthermore, inhibiting MMP12 by MMP12 knockdown and MMP12-specific inhibitor, PF-356231, significantly reduced the migration and invasion of NPC cells.

Conclusions: Overexpression of MMP12 was significantly correlated with hnRNP K in NPC tissues. HnRNP K can induce MMP12 expression and enzyme activity through activating MMP12 promoter, which promotes cell migration and invasion in NPC cells. In vitro experiments suggest that NPC metastasis with high MMP12 expression may be treated with PF-356231. HnRNP K and MMP12 may be potential therapeutic markers for NPC, but additional validation studies are warranted.

Figure 1

Identification of hnRNP K-targeted MMPs. (A) Affymetrix cDNA microarrays were used to examine the gene expression levels of MMP1, MMP12, MMP13 and MMP28 in hnRNP K-knockdown versus control siRNA-transfected NPC-TW02 cells. (B) Verification of hnRNP K-regulated MMP gene expression in NPC cells. NPC-TW02 cells were transfected with either hnRNP K-targeting (K) or control (C) siRNA. Cells were harvested post-transfection 48 h to detect the MMPs mRNA expression levels by quantitative RT-PCR analyses. All data are presented as the mean ± SD from three experiments. *P < 0.05. (C) Affymetrix cDNA microarrays were used to examine the gene expression levels of MMP1, MMP12, MMP13 and MMP28 in NPC tumor versus adjacent normal tissues. (D) Quantitative RT-PCR analyses of MMP gene expression in nine matched-pairs of NPC tumor and adjacent normal tissues.

Figure 2

Correlation of MMP12 and hnRNP K expression in NPC biopsy tissues. (A) Immunohistochemical staining of MMP12. A representative NPC biopsy tissue sample containing adjacent nontumor (N) and tumor (T) cells stained with a specific MMP12 antibody is shown at 200x magnification. (B and C) The N and T areas are shown at 400x magnification, respectively. (D-I) Consecutive NPC tissue sections were stained using anti-hnRNP K and anti-MMP12 antibodies, and subjected to immunohistochemical assessment. Representative NPC tissue sections with high-level expression of MMP12 (D and F) and total hnRNP K (E, N^Hi/C⁺; G, N^Hi/C⁻) and low-level expression of MMP12 (H) and hnRNP K (I, N^Lo/C⁻) are shown. Total^Hi, total hnRNP K high; Total^Lo, total hnRNP K low; N^Hi, nuclear hnRNP K high; N^Lo, nuclear hnRNP K low; C⁺, cytoplasmic hnRNP K positive; C⁻, cytoplasmic hnRNP K negative; scale bar in (A-C) = 100 μm, (D-I) = 50 μm.

Figure 3

Suppression of hnRNP K expression downregulates the expression and activity of MMP12 in NPC. (A) NPC-TW02 and -HK1 cells were transfected with hnRNP K-targeting (K) or control (C) siRNA. Twenty-four hours after transfection, cells were further cultured in serum-free medium for another 48 h. MMP12 mRNA levels were determined by quantitative RT-PCR, and MMP12 and hnRNP K protein levels in the culture supernatant (Sup) and in the cell extract (CE), respectively, were examined by Western blotting. PGK1 and Actin protein levels were used as the loading control for the secreted and the cytoplasmic proteins, respectively. (B) The enzymatic activity of MMP12 was analyzed by zymography. The supernatants from the NPC cells treated with either hnRNP K-targeting (K) or control (C) siRNA were collected after 48 h, and were subjected to zymographic analysis. The protein levels of hnRNP K and actin in the cell extracts were analyzed by Western blotting.

Figure 4

Transcriptional regulation of MMP12 by hnRNP K. (A) Schematic diagrams of the utilized reporter constructs, which contained 5' serial deletions of the MMP12 promoter. (B) NPC-TW02 cells were pretreated with control siRNA (C) or hnRNP K-targeting siRNA (K) for 24 h and then transfected with pGL3-basic (pGL3) with or without 5' serial deletions of the promoter sequence of the MMP 12. Firefly and Renilla luciferase activities were determined at 24 h post-transfection. *P < 0.05. (C) DNA pull-down assays were performed using nuclear extracts isolated from NPC-TW02 cells and 5' biotin-labeled probes corresponding to the −42/+97 (−42 to +97) or +2/+97 (+2 to +97) regions of the MMP12 promoter. The hnRNP K levels in the immunoprecipitates and 1% inputs were determined by Western blotting. (D) Chromatin immunoprecipitation was carried out using nuclear extracts from NPC-TW02 cells and an antibody against hnRNP K, followed by quantitative PCR of a sequence within the MMP12 promoter region (−95 to −20). Mouse IgG immunoprecipitation was done as a negative control. *P < 0.01.

Figure 5

MMP12 promotes cell migration and invasion in NPC-TW02 cells. (A) MMP12 mRNA expression was detected in stable NPC-TW02 cells of MMP12-knockdown (KD1 and KD2) and control (LacZ) by quantitative RT-PCR, and MMP12 protein levels in the culture supernatant were examined by Western blotting. PGK1 protein levels were used as the loading control for secreted proteins. Cell migration (B) and invasion (C) assays were performed in stable NPC-TW02 cells of MMP12-knockdown (KD1 and KD2) and control (LacZ). Images were captured at 24 h under 12.5× magnification. The relative fold-change in the number of migrated cells is shown, with the results from control cells given as 1.0. All data are presented as the mean ± SD from three independent experiments. *P < 0.05. (D) Cell growth in stable NPC-TW02 cells of MMP12-knockdown (KD1 and KD2) and control (LacZ) was determined by counting the cell numbers for 4 days.

Figure 6

MMP12-specific inhibitor, PF-356231, inhibits cell migration and invasion in NPC cells in a dose-dependent manner. Cell migration and invasion assays were performed in NPC-TW02 (A and B) and in NPC-HK1 (C and D) cells, respectively, in the presence of different concentration of MMP12 inhibitor PF-356231 (0, 25 and 50 nM). Images were captured under 12.5x magnification. The relative fold-change in the number of migrated cells is shown, with the results from control cells given as 1.0. All data are presented as the mean ± SD from three independent experiments. *P < 0.05.