LincROR Mediates the Suppressive Effects of Curcumin on Hepatocellular Carcinoma Through Inactivating Wnt/β-Catenin Signaling

Abstract

As one of the leading causes of cancer-related death in the world, hepatocellular carcinoma (HCC) has continued to attract growing attention in recent decades. The use of traditional Chinese herbs in medicine has been practiced for thousands of years, and holds the potential of being a possible treatment for HCC. Curcumin, a bioactive ingredient derived from Curcuma longa, exhibits anti-tumor activity in various cancers. Although the effects of Curcumin on HCC have been elucidated, the underlying mechanism remains unclear. In the present study, Curcumin was demonstrated to inhibit the proliferation of HCC cells via inducing cell cycle arrest and apoptosis. Several previously reported lncRNAs related to tumorigenesis were chosen for examination of their expression profiles, and lincROR was found to be the most down-regulated in the Curcumin-treated HCC cells. Furthermore, Curcumin was found to decrease β-catenin expression and induce the inactivation of Wnt/β-catenin signaling. Therefore, Curcumin suppressed tumor growth through a lincROR/β-catenin regulatory pattern. In conclusion, our results demonstrated that Curcumin suppressed the cell proliferation via the down-regulation of lincROR and inactivation of Wnt/β-catenin signaling, suggesting that it may be a potential anti-cancer candidate for HCC patients with activated Wnt/β-catenin signaling.

Keywords: Wnt/β-catenin signaling; apoptosis; curcumin; hepatocellular carcinoma; lincROR.

Figure 1

Curcumin inhibited cell proliferation in HCC cells. A-C, SMMC-7721, and Huh-7 cells were treated with serial concentrations of Curcumin, and the effects of Curcumin on cell proliferation were measured by MTT assays at 24, 48, and 72 hours (A–C). (D) SMMC-7721 and Huh-7 cells were treated with Curcumin for about 14 days and the colony formation was examined. *P < 0.05, **P < 0.01, ***P < 0.001, compared with DMSO.

Figure 2

Curcumin induced cell cycle arrest in HCC cells. SMMC-7721 (A) and Huh-7 (B) cells were treated with 16μM Curcumin for 48 hours, then harvested and subjected to cell cycle analyses. **P < 0.01, ***P < 0.001, compared with DMSO.

Figure 3

Curcumin induced apoptosis in HCC cells. SMMC-7721 (A, B) and Huh-7 (C, D) cells were incubated with 16μM Curcumin for 48 hours, and the apoptotic cells were examined by Annexin V-FITC and PI double staining. (A, C), The results of one such assay; and (B, D), mean ± SD of three independent experiments. *P < 0.05, ***P < 0.001, compared with DMSO.

Figure 4

lincROR was the most changeable candidate in Curcumin-treated HCC cells. The two HCC cells were incubated with Curcumin for 48 hours, and several lncRNAs were chosen to examine their expression by qRT-PCR assays (A–D). LincROR was the most dramatic change candidate in these treated cells. *P < 0.05; **P < 0.01; ***P < 0.001, compared with DMSO.

Figure 5

Curcumin induced the inactivation of the Wnt/β-catenin signaling. (A–D) With Curcumin treatment for 48 hours, the TOPflash luciferase activity was examined in SMMC-7721 cells (A). The expression of β-catenin in SMMC-7721 cells was determined at mRNA level (B). The expression of total β-catenin (C) and intranuclear β-catenin (D) were examined in SMMC-7721 cells at protein level. Lamin A/C (nuclear expression) and GADPH (cytoplasmic expression) were used as the loading controls. (E) β-catenin was detected in SMMC-7721 cells with 48 hours' treatment by immunofluorescence staining (100×). (F) The downstream targets of the Wnt/β-catenin pathway in Curcumin-treated SMMC-7721 cells were examined by qRT-PCR assays. ***P < 0.001, compared with DMSO.

Figure 6

lincROR overexpression reversed Curcumin-induced growth inhibition and inactivated Wnt/β-catenin signaling. (A) The expression of lincROR in lincROR overexpressing SMMC-7721 cells was measured by qRT-PCR examination. ***P<0.001, compared with pVector. (B, C) The cell viabilities (B) and colony formation (C) was measured with Curcumin 48-hour treatment in lincROR overexpressing SMMC-7721 cells. (D) The protein level of β-catenin was examined by Western blotting after 48-hour treatment of Curcumin in lincROR overexpressing SMMC-7721 cells. (C) Other Wnt/β-catenin downstream target genes were examined by qRT-PCR with the same treatment in lincROR overexpressing SMMC-7721 cells. *P < 0.05; **P < 0.01; ***P < 0.001; compared with pVector+DMSO.