Curcumin Inhibits LIN-28A through the Activation of miRNA-98 in the Lung Cancer Cell Line A549

Abstract

Metastasis is common in lung cancer and is associated with poor clinical outcomes and increased mortality. Curcumin is a natural anti-cancer agent that inhibits the metastasis of various cancers by modulating the expression of micro (mi) RNAs such as miR-98, which acts as a tumor suppressor. This study investigated the effect of curcumin on miR-98 expression and in vitro cell line growth and invasiveness in lung cancer. Curcumin treatment enhanced the expression of miR-98 and reduced that of the miR-98 target gene LIN28A as well as matrix metalloproteinase (MMP) 2 and MMP9 in vitro and in vivo. MiR-98 overexpression suppressed lung cancer cell migration and invasion by inhibiting LIN28A-induced MMP2 and MMP9 expression. Meanwhile, LIN28A level was downregulated by overexpression of miR-98 mimic. Induction of miR-98 by curcumin treatment suppressed MMP2 and MMP9 by targeting LIN28A. These findings provide insight into the mechanisms by which curcumin suppresses lung cancer cell line growth in vitro and in vivo and invasiveness in vitro.

Keywords: LIN28A; MMP; curcumin; lung cancer; metastasis; miR-98.

Figure 1

Curcumin inhibits tumor growth and MMP2/9 expression in a xenograft model. (A,B) Severe combined immunodeficiency mice were inoculated in the right flank with A549 cells. Tumor volume was measured every 3 days with slide calipers starting from day 7, and a growth curve was plotted. Tumors were weighed at the end of the experiment; each plot shows mean ± SEM of six mice per group. * p < 0.05 curcumin (CCM) vs. control (Con). (C–F) MMP2 and MMP9 mRNA expression was determined by qRT-PCR (C), and protein expression was evaluated by western blotting (D,E) and confirmed by immunohistochemistry (F).

Figure 2

Curcumin induces miR-98 and inhibits LIN28A expression in tumors. (A–E) MiR-98 level was increased in mice treated with curcumin relative to control mice (A), whereas LIN28A mRNA (B) and protein (C–E) levels showed the opposite trend. Protein levels detected by western blotting were semi-quantitatively analyzed by densitometry. Data are shown as mean ± SEM and are representative of three independent experiments.

Figure 3

Curcumin inhibits lung cancer cell migration and invasion by regulating miR-98 expression. (A,B) Curcumin increased miR-98 level in A549 cells in a dose- and time-dependent manner (100 μm curcumin), as determined by qRT-PCR. * p < 0.05 vs. untreated control group. (C–E) MiR-98 (15 nM) overexpression and curcumin (100 μm) treatment inhibits A549 cell migration (C,D) and invasion (E), effects that are abolished by miR-98 inhibitor (anti-miR-98; 15 nM). Data represent mean ± SEM of three independent experiments. * p < 0.05 vs. miR-NC group. ^$ p < 0.05 vs. miR-98 group.

Figure 4

LIN28A is a downstream targets of miR-98 in human lung cancer cells. (A) Putative miR-98 binding site in the 3′-UTR of the LIN28A gene. (B–D) LIN28A mRNA expression was evaluated by qRT-PCR, and protein levels were determined by western blotting and immunocytochemistry. Data represent the mean ± SEM of three independent experiments. * p < 0.05 vs. untreated control group.

Figure 5

Curcumin reduces LIN28A-induced MMP2/9 expression and blocks cancer metastasis. (A) Curcumin (100 μm) treatment decreased LIN28A expression. (B) Various curcumin (25, 50, 100 µm) treatment reduced LIN28A, MMP2/9 protein expression. (C,D) LIN28A silencing decreased LIN28A expression and MMP2/9 mRNA and protein expression in A549 cells, as determined by qRT-PCR and western blotting, respectively. LIN28A (30nM) knockdown significantly suppressed A549 cell migration (E,F) and invasion (G). Data represent mean ± SEM of three independent experiments. * p < 0.05 vs. untreated control group.