miR-24 functions as a tumor suppressor in Hep2 laryngeal carcinoma cells partly through down-regulation of the S100A8 protein

Abstract

microRNAs, a family of small non-coding RNAs, regulating approximately 30% of all human genes are deeply involved in the pathogenesis of several types of cancers, including laryngeal squamous cell carcinoma (LSCC). Here, we demonstrate that miR-24 is down-regulated in human LSCC tissues. Ectopic expression of miR-24 in Hep2 cells significantly induced cell morphology changes and inhibited cell proliferation and invasion ability in vitro by targeting S100A8 at the translational level. Meanwhile, miR-24 could significantly inhibit Hep2 cell invasion after S100A8 protein blockade. In conclusion, our results suggest that miR-24 may function as a tumor suppressor in LSCC through down-regulation of S100A8, which suggests that miR-24 could serve as a novel potential maker for LSCC therapy.

Figure 1

The expression of miR-24 is down-regulated in LSCC tissues. (A) We searched the database of miR-24 expression in normal and cancer tissues by miRNAMap-2.0, and found that miR-24 was usually up-regulated in normal cell lines/tissues while was down-regulated in cancer cell lines/tumor samples, as compared to the controls. (B) Quantitative analysis of the expression levels of miR-24 normalized to those of U6 by qRT-PCR. T, tumor tissues; R, matched non-tumor tissues. ^*P<0.05.

Figure 2

miR-24 induces morphological changes and impairs proliferation and invasion properties in Hep2 cells. (A) Amplification plot of miR-24. (B) Statistical analysis of miR-24 expression. a, vehicle group; b, control miR transfection group; c, pre-miR-24 transfection group. (C–E) Representative images showing the morphology of Hep2 cells transfected with vehicle, control miR and pre-miR-24, respectively. Scale bar, 100 μm. (F) Cell proliferation was measured by the MTT assay. Results are means of three independent experiments ± SD. (G–I) Representative images of invasive cells on the membrane by transfection vehicle, control miR and pre-miR-24 for 7 days, respectively (magnification, ×400). (J) Statistical analysis of average invasive cell number of three independent experiments ± SD (^*P<0.05). a, vehicle group; b, control miR group; c, pre-miR-24 group.

Figure 3

S100A8 is a validated target of miR-24. (A) 3'UTR of S100A8 is predicted to be a target of miR-24. (B) PCR screening result of Lut-S100A8-Wt construct. (C) Enzyme digestion result of Lut-S100A8-Wt construct. M, DL 2000 marker; 1, pGL3-promotor circular plasmid; 2, Lut-S100A8-Wt digested by XbaI. (D) Sequencing result of Lut-S100A8-Wt construct. (E) Sequencing result of Lut-S100A8-Mut construct. Rectangular box, mutated site. (F) The reporter assay, with each bar representing values from three independent experiments performed in quadruplicates. The transfection efficiency was normalized by co-transfected renilla luciferase and the light units were calculated by relative luciferase activity of firefly to renilla. ^*P<0.05.

Figure 4

S100A8 protein levels in Hep2 cells detected by Western blotting. (A) Representative image of the protein level of S100A8. β-actin was used as a reference control. (B) Quantitative analysis of the relative protein levels of S100A8 normalized to those of β-actin is shown. Data are the mean ± SD of three independent experiments. ^*P<0.05.