miR-34a-5p might have an important role for inducing apoptosis by down-regulation of SNAI1 in apigenin-treated lung cancer cells

Abstract

Apigenin is a flavonoid with antioxidant and anticancer effects. It has been reported that apigenin inhibits proliferation, migration, and invasion and induces apoptosis in cultured lung cancer cells. However, there is little information on the involvement of microRNAs (miRNAs) in its effects. miRNA microarray analysis and polymerase-chain-reaction analysis of miRNAs revealed that treatment of human lung cancer A549 cells with apigenin up-regulated the level of miR-34a-5p. Furthermore, mRNA microarray analysis and the results of three microRNA target prediction tools showed that Snail Family Transcriptional Repressor 1 (SNAI1), which inhibits the induction of apoptosis, had its mRNA expression down-regulated in A549 cells treated with apigenin. Our findings suggest that apigenin might induce apoptosis by down-regulation of SNAI1 through up-regulation of miR-34a-5p in A549 cells.

Keywords: Apigenin; Apoptosis; Human lung cancer cell A549; SNAI1 (SNAIL); miR-34a-5p.

Fig. 1

Effects of apigenin on viability and apoptosis of A549 cells. A A549 cells were treated by various concentrations of apigenin (n = 5) and cell viability was measured by using the Cell Counting Kit-8 (Dojindo, Japan). Data are representative of results from three separate experiments. *P < 0.05 vs. vehicle, and **P < 0.01 vs. vehicle. B Activity of caspase-3/7 in 100 μM apigenin-treated A549 cells for 72 h was visualized using the CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen, Japan) with a fluorescence microscope (KEYENCE BZ-X800, Osaka, Japan). Photographs a and b show the cells stained with Hoechst 33,342 to visualize nuclei and with CellEvent Caspase-3/7 reagents to detect apoptosis, respectively. Staurosporine at 10 μM was added to dish for 3 h. Bar 100 μm

Fig. 2

Up-regulation of miR-34a-5p by apigenin in A549 cells. A miRNA microarray analysis showed that miR-34a-5p was up-regulated by apigenin. A549 cells were treated with 50 µM apigenin for 48 h. B Up-regulation of miR-34a-5p was verified with real-time RT-qPCR (n = 3). **P < 0.01 vs. vehicle

Fig. 3

Down-regulation of SNAI1 mRNA by miR-34a-5p. A Venn diagram showing the overlap of mRNAs that were predicted to decrease by miR-34a-5p by alternative algorithms (TargetScan, DIANA TOOLS, and miRDB). B SNAI1 and FOXG1were chosen as targets of miR-34a-5p and were verified with real-time RT-qPCR (n = 3). *P < 0.05 vs. vehicle

Fig. 4

Schematic diagram by which the miR-34a-5p target SNAI1 regulates apoptosis with apigenin in A549 cells