MicroRNA-205 directly regulates the tumor suppressor, interleukin-24, in human KB oral cancer cells

Abstract

MicroRNA (miRNA) is a form of small noncoding RNA that regulates the expression of genes either by inhibiting mRNA translation or by inducing its degradation. Small microRNA play important roles in regulating a large number of cellular processes, including development, proliferation and apoptosis. This study examined the biological functions of miR-205 as a tumor suppressor in KB oral cancer cells. The results showed that miR-205 expression was significantly lower in KB oral cancer cells than in human normal oral keratinocytes. Furthermore, the miR-205 over-expressed in KB oral cancer cells increased the cell cytotoxicity and induced apoptosis through the activation of caspase-3/-7. The transfection of miR-205 into KB oral cancer cells strongly induced IL-24, a well known cytokine that acts as a tumor suppressor in a range of tumor tissues. In addition, miR-205 targeted the IL-24 promoter directly to induce gene expression. Overall, miR-205 has significant therapeutic potential to turn on silenced tumor suppressor genes by targeting them with miRNA.

Fig. 1.

MicroRNA array in KB cells compared with NHOK cells. Total RNA from both KB cells and NHOK were isolated with miRNeasy mini kit following the manufacturer’s instructions. The concentration, purity, and amount of total RNA were quantified using the Nano-Drop^® ND-1000 ultraviolet Spectrophotometer (Thermo Scientific, USA). The miRNA array was scanned using an Affymetrix GeneChip Platefom (DNA Link, Korea) and stained on Fluidics Station 450 and scanned on a GeneChip^® Scanner3000 7G (Affymetrix). The image data were analyzed with the miRNA QC Tool software for quality control.

Fig. 2.

miR-205 notably decreased in KB oral cancer cells compared with normal human oral keratinocyte. (A) The relative content of miR-205 in both NHOKs and KB cells were accessed by miRNA array using affymetrix Genechip and Affymetrix software. (B) The relative expression of miR-205 was accessed by qRT-PCR according to methods described in “Materials and Methods”.

Fig. 3.

Over-expressed miR-205 increased the cell cytotoxicity via apoptotic cell death in KB oral cancer cells. (A) The measurement of cell cytotoxicity by up-regulated miR-205 as time-dependent (upper panel) and dose-dependent manner (low panel). Cell cytotoxicity was performed by MTT assay after transfection into KB cells as following defined treatment condition. (B) The observation of apoptotic cell death was performed by DAPI staining after transfection of 200 ng/ml pSuper-miR-205 and 200 ng/ml pSuper-miR-205mutation. The number of apoptotic dying cells were counted and presented as histogram. (C) Apoptosis executioner caspase-3/7 activity was determined using the cell-permeable fluorogenic substrate PhiPhiLux-G1D2 (OncoImmunin Inc., USA) according to the manufacturer’s instructions.

Fig. 4.

miR-205 downstream target gene array in KB cells. DNA samples isolated from KB cells with overexpressed miR-205 were run on the DNET microarray (DNA Link, Korea). Hybridized DMET arrays were washed and stained in the Affymetrix fluidic stations and scanned with the Affymetrix GeneChip^® Scanner 3000 7G. Data was generated with Affymetrix GeneChip^® Command console software and analyzed with the DMET Console software.

Fig. 5.

Over-expressed miR-205 increased tumor suppressor gene, IL-24, KB oral cancer cells. (A) The expression level of IL-24 was measured by qPCR after pSuper-miR-205. The amplified PCR productions of IL-24 were electrophorized on agarose gel and then presented as histogram after densitomatric analysis. (B) The expression level of IL-24 was accessed by qRT-PCR after transfection of pSuper-miR-205mutation and pSuper-miR-205 into KB cells. And then data analyzed by calculating method described in “Materials and Methods”. (C) The expressed IL-24 was quantified by Western blotting using IL-24 specific antibody.

Fig. 6.

IL-24 is a direct target of miR-205 in KB oral cancer calls. (A) The sequences of the miR-205 raget sites located at-131 bp is shown to the transcription start sites. (B) The luciferase activity of IL-24 promoter against miR-205. pGL3-basic-IL-24-555 bp was cloned as previously described in “Materials and Methods”. Luciferase activity was normalized to total protein.