MicroRNA 744-3p promotes MMP-9-mediated metastasis by simultaneously suppressing PDCD4 and PTEN in laryngeal squamous cell carcinoma

Abstract

MicroRNA controls cancer invasion by governing the expression of gene regulating migration and invasion. Here, we reported a novel regulatory pathway controlled by miR-744-3p, which enhanced expression of matrix metallopeptidase 9 (MMP-9) in laryngeal squamous cell carcinoma (LSCC). We profiled the differential micoRNA expression pattern in LSCC cell lines and normal epithelial cultures derived from the head and neck mucosa using microRNA microarray. MiR-7-1-3p, miR-196a/b and miR-744-3p were expressed differentially in the LSCC cell lines. Subsequent validation using real-time PCR revealed that high miR-744-3p level was positively correlated with regional lymph node metastasis of LSCC. Real-time cellular kinetic analysis showed that suppressing miR-744-3p could inhibit migration and invasion of LSCC cell lines and reduce the number of lung metastatic nodules in nude mice modules. In silico analysis revealed that miR-744-3p targeted 2 distinct signaling cascades which eventually upregulated MMP-9 expression in LSCC. First, miR-744-3p could suppress programmed cell death 4 (PDCD4), a direct suppressor of NF-κB (p65). PDCD4 could also prevent AKT activation and suppress MMP-9 expression. Further, suppressing miR-744-3p expression could restore phosphatase and tensin homolog (PTEN) expression. PTEN could inhibit AKT activation and inhibit MMP-9 expression in LSCC cells. The results revealed that suppressing miR-744-3p was effective to inhibit LSCC metastasis by inactivating AKT/mTOR and NF-κB (p65) signaling cascade. Targeting miR-744-3p could be a valuable therapeutic intervention to suppress the aggressiveness of LSCC.

Keywords: PDCD4; PTEN; laryngeal squamous cell carcinoma; metastasis; miR-744-3p.

Figure 1. Deregulated microRNAs in LSCC

As shown in the heat map, 21 microRNAs overexpressed and 2 microRNAs were downregulated in LSCC cell lines respectively. The line charts showed the expression levels of the 8 differentially expressed microRNAs in LSCC tissues. MiR-196a, miR-196b and miR-744-3p were significantly overexpressed in LSCC compared with their paired normal counterparts (P < 0.05).

Figure 2. Influence of miR-744-3p suppression on LSCC metastasis

(A–D) Figures of LSCC cell lines with mock control vector or expressing miR-744-3p shRNA. The left part of each figure is the bright field image of each clone, while the right part is the green fluorescent image of cells. (A) SNU899 with mock control vector; (B) SNU899 stably expressing miR-744-3p shRNA; (C) SNU1076 with mock control vector; (D) SNU1076 stably expressing miR-744-3p shRNA. (E) Relative expression levels of miR-744-3p. Compared with the mock control, the stable LSCC clone compromising miR-744-3p shRNA exhibited significant reduction of miR-744-3p expression level. (F) Real-time measurement of LSCC cell migration and invasion. The reduction of miR-744-3p could significantly inhibit the LSCC cell migration and invasion. (G– J) Metastatic foci in lung of representative nude mice injected with miR-744-3p suppressing LSCC cell lines and mock control (H&E staining). G: SNU899 with mock control vector; (H) SNU899 stably expressing miR-744-3p shRNA; (I) SNU1076 with mock control vector; (J) SNU1076 stably expressing miR-744-3p shRNA. Compared with the mock control groups, less metastatic foci were observed in the mice injected with miR-744-3p silencing LSCC cells. White arrow: metastatic foci. *P < 0.05, **P < 0.01.

Figure 3. Expression of PTEN and PDCD4 transcripts and their interaction with miR-744-3p in LSCC

(A) Relative expression levels of PTEN mRNA and PDCD4 mRNA in LSCC. Compared with the normal epithelial counterparts, the tumor exhibited significantly lower PTEN mRNA and PDCD4 mRNA expression levels. (B) Relative expression levels of PTEN mRNA and PDCD4 mRNA in LSCC cell lines. By miR-744-3p suppression, expression levels of PTEN and PDCD4 were significantly increased in both of SNU899 and SNU1076. (C) Ago 2 protein immunoprecipitation. Compared with mock control group, the suppression of miR-744- 3p significantly reduced miR-744-3p, PTEN and PDCD4 binding to the Ago2 protein. (D, E) Relative luminenscent signal intensity in luciferase reporter assay. The suppression of miR-744-3p significantly enhanced luciferase expression from the reporter plasmid containing the wild- type PTEN 3′-UTR sequence or containing the wild- type PDCD4 3′-UTR sequence. Similar enhancement of luminenscent signal intensity due to miR-744-3p suppression was not observed in the groups with mutant PTEN 3′-UTR sequence or mutant PDCD4 3′-UTR sequence. *P < 0.05, **P < 0.01.

Figure 4. Expression alteration of PTEN, PDCD4, phosphorylated AKT, phosphorylated NF-κB and MMP-9 caused by miR-744-3p suppression in LSCC

(A) Western blot analysis on protein expression in LSCC cell lines. By miR-744-3p suppression, PTEN and PDCD4 protein levels were significantly increased, while phosphorylated AKT and phosphorylated NF-κB protein levels were significantly reduced. (B) Relative expression levels of MMP-9 mRNA in LSCC. Compared with the normal epithelial counterparts, the tumor exhibited significantly higher MMP-9 mRNA expression level. (C) Relative expression levels of MMP-9 mRNA in LSCC cell lines. By miR-744-3p suppression, MMP-9 mRNA expression level was significantly reduced in both of SNU899 and SNU1076. (D) MMP-9 protein expression levels in LSCC cell lines. By miR-744-3p suppression, MMP-9 protein level was significantly reduced. *P < 0.05, **P < 0.01.

Figure 5. Influence of PDCD4 restoration on LSCC

(A) SNU899 stably expressing PDCD4; (B) SNU1076 stably expressing PDCD4. The upper part of figure is the bright field image of each clone, while the lower part is the green fluorescent image of cells. (C) Real-time measurement of LSCC cell migration and invasion. PDCD4 restoration could inhibit LSCC cell migration and invasion. (D) Relative expression levels of PDCD4 and MMP-9 mRNA in LSCC cell lines. Compared with the mock control, the stable LSCC clone with PDCD4 restoration exhibited significant enhancement of PDCD4 and reduction of MMP-9 mRNA expression levels. (E) Protein expression levels of PDCD4, phosphorylated AKT, phosphorylated NF-κβ and MMP-9 in LSCC cell lines. Compared with the mock control, PDCD4 protein level was significantly increased, while phosphorylated AKT, phosphorylated NF-κβ and MMP-9 protein levels were significantly reduced.

Figure 6. MMP-9 expression in LSCC cell lines

Significant downregulation of MMP-9 mRNA expression level was observed in LSCC cell lines with miR-744-3p suppression. However, the reduction of MMP-9 mRNA was not significant in LSCC cell lines after administration of everolimus. *P < 0.05, **P < 0.01.

Figure 7. Illustration of potential pathways for miR-744-3p mediating metastasis in LSCC

MiR-744-3p promotes LSCC metastasis by simultaneously inhibiting PTEN and PDCD4. Both of PTEN suppression and PDCD4 suppression can activate AKT and mTOR, and then further promote transcription activation of MMP-9, resulting in enhancement of LSCC metastasis. Moreover, loss of PDCD4 leads to NF-κB (p65) activation, which also promotes MMP-9 expression. Hence, suppression of miR-744-3p can powerfully prevent LSCC metastasis by inhibiting MMP-9 through inactivation of those 2 pathways above.

Figure 8. The RNA quality of the cell lines for microRNA microarray profiling

All the samples had rRNA Ratio (28s/18s) over 1.8 and RNA Integrity Number (RIN) over 8.0.