MicroRNA-224, negatively regulated by c-jun, inhibits growth and epithelial-to-mesenchymal transition phenotype via targeting ADAM17 in oral squamous cell carcinoma

Abstract

Abnormal expression of miR-224 has been reported to promote cancer progression. However, the role of miR-224 is seldom reported in oral squamous cell carcinoma (OSCC). We reported that miR-224 expression was significantly down-regulated in OSCC tissues and cell lines. Restoration of miR-224 decreased OSCC cell growth and invasion. In addition, luciferase and Western blot assays revealed that ADAM17 protein was a downstream target of miR-224. The overexpression of ADAM17 dismissed miR-224's effect on cell growth and invasion. We concluded that miR-224 inhibited OSCC cell growth and invasion through regulating ADAM17 expression. Subsequently, we revealed that c-jun directly bind to miR-224 promoter and decreased miR-224 expression. Taken together, these findings demonstrated that miR-224 may function as a tumour-suppressive microRNA in OSCC and suggested that miR-224 may be a potential therapeutic target for OSCC patients.

Keywords: ADAM17; epithelial-to-mesenchymal transition; miR-224; oral squamous cell carcinoma.

Figure 1

miR‐224 expression was down‐regulated in OSCC tissues. A, miR‐224 expression was significantly decreased in OSCC tissues vs corresponding non‐tumour tissues, as determined by RT‐PCR. B, miR‐224 expression was negatively associated with OSCC clinical stage. C, OSCC patients with lower levels of miR‐224 had shorter overall survival when compared those with higher levels of miR‐224. D, Patients with higher miR‐224 expression had better disease free survival rate than those with lower miR‐224 expression

Figure 2

miR‐224 directly targeted ADAM17. A, ADAM17 wild‐type (WT) and mutant (MUT) 3′‐UTR as indicated. B and C, miR‐224 decreased ADAM17 expression at mRNA and protein level, respectively. D, miR‐224 decreased the luciferase activity of ADAM17 WT 3′‐UTR instead of MUT 3′‐UTR in OSCC cells

Figure 3

ADAM17 mediated miR‐224’s effect on cell growth and invasion. A, MiR‐224 decreased OSCC cell growth, while overexpression of ADAM17 counteracted this effect, as determined by MTT assay. B, MiR‐224 impaired OSCC cell colony formation ability, while ADAM17 restoration counteracted the effect. C, MiR‐224 delayed cell cycle progression from the G1 phase to S phase whereas this effect was dismissed by ADAM17 restoration. D, MiR‐224 negatively regulated G1/S phase checkpoint proteins and ADAM17 overexpression counteracted these effects. E, MiR‐224 decreased cell invasion ability, which was offset by ADAM17 overexpression. F, MiR‐224 inhibited the EMT phenotype, while the effect was neutralized by ADAM17 overexpression

Figure 4

c‐jun decreased miR‐224 expression through biding at its promoter. A, The putative transcription factor‐binding sites of c‐jun at miR‐224 promoter region. B, c‐jun down‐regulation increased miR‐224 expression. C, c‐jun down‐regulation increased miR‐224 promoter luciferase activity. D, The CHIP assay confirmed that c‐jun protein was recruited to all the four binding sites in the putative miR‐224 promoter. E, miR‐224 expression was negatively correlated with c‐jun expression in OSCC tissues

Figure 5

Restoration of miR‐224 decreased cell growth in vivo. A, LV‐miR‐224 cell‐derived xenograft tumours grew more slowly when compared with the LV‐ctrl cell‐derived xenograft tumours. B, The mean weight of LV‐miR‐224 cell‐derived xenograft tumours was less when compared with LV‐ctrl cell‐derived xenograft tumours. C, The Ki‐67 staining assay also revealed that LV‐miR‐224 cells had less proliferation index than the LV‐ctrl cells. D, The tunel assay revealed that the apoptosis rate was increased in LV‐miR‐224 cells, when compared with that in LV‐ctrl cells