MicroRNA let-7c Inhibits Cell Proliferation and Induces Cell Cycle Arrest by Targeting CDC25A in Human Hepatocellular Carcinoma

Abstract

Down-regulation of the microRNA let-7c plays an important role in the pathogenesis of human hepatocellular carcinoma (HCC). The aim of the present study was to determine whether the cell cycle regulator CDC25A is involved in the antitumor effect of let-7c in HCC. The expression levels of let-7c in HCC cell lines were examined by quantitative real-time PCR, and a let-7c agomir was transfected into HCC cells to overexpress let-7c. The effects of let-7c on HCC proliferation, apoptosis and cell cycle were analyzed. The in vivo tumor-inhibitory efficacy of let-7c was evaluated in a xenograft mouse model of HCC. Luciferase reporter assays and western blotting were conducted to identify the targets of let-7c and to determine the effects of let-7c on CDC25A, CyclinD1, CDK6, pRb and E2F2 expression. The results showed that the expression levels of let-7c were significantly decreased in HCC cell lines. Overexpression of let-7c repressed cell growth, induced cell apoptosis, led to G1 cell cycle arrest in vitro, and suppressed tumor growth in a HepG2 xenograft model in vivo. The luciferase reporter assay showed that CDC25A was a direct target of let-7c, and that let-7c inhibited the expression of CDC25A protein by directly targeting its 3' UTR. Restoration of CDC25A induced a let-7c-mediated G1-to-S phase transition. Western blot analysis demonstrated that overexpression of let-7c decreased CyclinD1, CDK6, pRb and E2F2 protein levels. In conclusion, this study indicates that let-7c suppresses HCC progression, possibly by directly targeting the cell cycle regulator CDC25A and indirectly affecting its downstream target molecules. Let-7c may therefore be an effective therapeutic target for HCC.

Fig 1. Let-7c is down-regulated in various HCC cell lines.

Let-7c expression levels in various HCC cell lines (HepG2, Hep3B, SMMC-7721,Huh-7, MHCC97-H, and MHCC97-L), the normal human liver cell line L-02, A549 lung cancer cells and HEL 299 cells (human embryonic lung cell) were determined by quantitative real-time PCR. Each sample was analyzed in triplicate and normalized to U6 expression.

Fig 2. Let-7c inhibits HCC cell proliferation, induces cell apoptosis and induces G1 cell cycle arrest in vitro.

(A) HepG2 and SMMC-7721 HCC cells were transfected with the let-7c agomir at a final concentration of 30 or 50 nM. Expression of let-7c was determined using quantitative real-time PCR 48 h post-transfection. (B-D) HepG2 and SMMC-7721 HCC cells were transfected as in (A). At the indicated time points post transfection, the cell growth rate was evaluated using the CCK-8 assay (B). Cells were stained using propidium iodide (PI) and Annexin V 72 h post transfection and analyzed by FACS. Annexin V-positive cells were regarded as apoptotic cells (D) and the cell cycle distribution was calculated (C).

Fig 3. Let-7c agomir inhibits tumor growth in a xenograft mouse model of HCC in vivo.

(A) Photographs of tumor-bearing mice in the fifth week after injection with let-7c agomir (Left) or negative control (Right). (B) From seventh day after the injection, measurements of tumor size were taken every 7 days for 5 weeks. Effects of let-7c agomir on the xenograft mouse model are shown. Data are shown as the mean ± S.D. The statistical difference was analyzed by the two-sample t test. (C) Photographs of tumors that developed in the mouse model of HCC treated with let-7c or the negative control are presented. The two smallest tumors with the let-7c treatment are indicated by red arrows, and the two smallest tumors in the negative control group are indicated by blue arrows.

Fig 4. pLenO-RFP-Let-7c inhibits tumor growth in a xenograft mouse model of HCC in vivo.

(A) Effects of pLenO-RFP-let-7c (Lv-let-7c) and pLenO-RFP (Lv-control, negative control) in the xenograft mouse model are shown. Data are shown as the mean ± S.D. The statistical difference was analyzed by the two-sample t test. (B) qRT-PCR assays of mature let-7c expression in tissues of the Lv-let-7c and Lv-control group. (C) Weight of tumors of mice in the Lv-let-7c and Lv-control groups. Data are shown as the mean ± S.D. The statistical difference was analyzed by the two-sample t test. (D) Photographs of tumors are presented. The three smallest tumors in the Lv-let-7c group are indicated by blue arrows.

Fig 5. Let-7c targets CDC25A in HCC cells.

(A) Firefly luciferase reporter vectors containing the CDC25A wild-type (pmiR-CDC25A-3′-UTR-wt) or mutant (pmiR- CDC25A-3′-UTR–mut) 3′-UTR were generated and co-transfected into HepG2 cells along with either the let-7c agomir or negative control to identify CDC25A targets. The 3′UTR of CDC25A mRNA contained two complementary sites for the seed region of let-7c. (b) Wild: wild-type; Mut: mutated. The seed sequence is underlined. (B) Relative luciferase activity was analyzed after the reporter plasmids or control reporter plasmid were co-transfected with let-7c into HEK-293 cells. Representative experiments are shown. (C) Western blot assays of the endogenous CDC25A protein level in HepG2 cells transfected with the let-7c agomir, negative control or let-7c inhibitor. (D) Real-time PCR assay of CDC25A mRNA expression in HepG2 cells transfected with the let-7c agomir or negative control.

Fig 6. CDC25A induces the G1-to-S phase transition in HCC cells.

(A,B) Western blotting of CDC25A protein expression in HepG2 cells infected/transfected with lenti-CDC25A, lenti-control, CDC25A-siRNA or negative control-siRNA. (C) Western blot analysis of CDC25A expression in SMMC-7721 cells and SMMC-7721-let-7c stable cells with or without CDC25A* reintroduction. (D,E) Infection/transfection of HepG2 cells with lenti-CDC25A, lenti-control, CDC25A-siRNA or negative control-siRNA was performed to investigate the effects of CDC25A on the HCC cell cycle. Representative images are shown. (F) Cell cycle assays of SMMC-7721 cells or SMMC-7721-let-7c stable cells with or without CDC25A* reintroduction. Restoration of CDC25A significantly induced the G1-to-S phase transition in SMMC-7721 cells. Representative images are shown. *CDC25A was reintroduced without its 3′-UTR to prevent the expression of CDC25A from being inhibited by let-7c.

Fig 7. Effect of Let-7c on CDC25A, CDK6, CyclinD1, pRb, Rb and E2F2 protein expression in HCC cells.

(A) HepG2 and SMMC-7721 cells were transfected with the let-7c agomir, let-7c inhibitor or negative control. Levels of CDC25A, CDK6, CyclinD1, pRb, Rb and E2F2 protein were detected by western blot. The value under each band indicates the relative expression levels of CDC25A, CDK6, CyclinD1, pRb, Rb and E2F2 compared to actin. (B) Immunohistochemistry was performed to determine CDC25A protein expression in HCC xenograft tumor tissues. Images were captured at 40×10 magnification. Positive CDC25A expression was observed as brown particles in the cytoplasm and the nucleus.