Inhibition of microRNA-21 upregulates the expression of programmed cell death 4 and phosphatase tensin homologue in the A431 squamous cell carcinoma cell line

Abstract

microRNA-21 (miRNA/miR-21) is a well-known oncogenic miRNA that is overexpressed in various carcinomas. The tumor suppressor genes, programmed cell death 4 (PDCD4) and phosphatase tensin homologue (PTEN), which target miR-21, are underexpressed in several types of cancer. However, the expression of miR-21 and its target genes, PDCD4 and PTEN, has not yet been reported in skin squamous cell carcinoma (SCC). In the present study, anti-miR-21 was transfected into the A431 cell line, and the expression of miR-21, PDCD4 and PTEN and the level of cell apoptosis were detected by quantitative polymerase chain reaction, immunocytochemistry and western blotting, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, respectively. The expression levels of PDCD4 and PTEN in the A431 cell line transfected with anti-miR-21 were significantly increased (P<0.05) and the apoptotic ratio was significantly increased (P<0.05). The data indicate that miR-21 may play an oncogenic role in the cellular processes of SCC and represent a novel target for effective therapies.

Keywords: apoptosis; carcinoma; microRNA-21; phosphatase tensin homologue; programmed cell death 4; squamous cell.

Figure 1

(A) Quantitative polymerase chain reaction (qPCR) result demonstrating mRNA expression of miR-21 in A431 cells, cells transfected with an unrelated fragment of control (NC) and cells transfected with ASO-miR-21. (B) The relative expression level of miR-21 in the A431 cells was significantly decreased by ASO-miR-21 compared with the control (P<0.05). NC, normal contol; ASO, antisense oligonucleotide; miR, microRNA.

Figure 2

Western blot analysis of PDCD4 and PTEN expression in A431 cells. PDCD4 and PTEN expression in the A431 cells was evidently increased by ASO-miR-21 compared with the control (P<0.05). PDCD4, programmed cell death 4; PTEN, phosphatase tensin homogue; ASO, antisense oligonucleotide; miR, microRNA; NC, negative control.

Figure 3

Expression of PDCD4 in the (A) A431 cells, (B) cells transfected with an unrelated fragment of control and (C) cells transfected with ASO-miR-21. Expression of PDCD4 in the (D) A431 cells, (E) cells transfected with an unrelated fragment of control and (F) cells transfected with ASO-miR-21. The expression of PDCD4 and PTEN in the A431 cells was evidently increased by ASO-miR-21 compared with the control, as detected by immunocytochemistry (P<0.05). PDCD4, programmed cell death 4; PTEN, phosphatase tensin homologue; ASO, antisense oligonucleotide; miR, microRNA.

Figure 4

Apoptosis in A431 cells stained with (A) DAPI and (B) TUNEL, cells transfected with an unrelated fragment of control stained with (C) DAPI and (D) TUNEL, and cells transfected with ASO-miR-21 stained with (E) DAPI and (F) TUNEL. The apoptotic ratio in the A431 cells was evidently increased by ASO-miR-21 transfection conpared with the control group (P<0.05). DAPI, 4′, 6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; ASO, antisense oligonucleotide; miR, microRNA.