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MicroRNA-503 inhibits the G1/S transition by downregulating cyclin D3 and E2F3 in hepatocellular carcinoma

Xiao, Fenqiang, zhang, Wu, Chen, Liming, Chen, Fei, Xie, Haiyang, Xing, Chunyang, Yu, Xiaobo, Ding, Songming, Chen, Kangjie, Guo, Haijun, Cheng, Jun, Zheng, Shusen, Zhou, Lin·2013·Journal of Translational Medicine
DOI 10.1186/1479-5876-11-195PMC PMC3765277
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Abstract

Background: Increasing evidence indicates that deregulation of microRNAs (miRNAs) is involved in tumorigenesis. Downregulation of microRNA-503 has been observed in various types of diseases, including cancer. However, the biological function of miR-503 in hepatocellular carcinoma (HCC) is still largely unknown. In this study we aimed to elucidate the prognostic implications of miR-503 in HCC and its pathophysiologic role. Methods: Quantitative reverse transcriptase polymerase chain reaction was used to evaluate miR-503 expression in HCC tissues and cell lines. Western blotting was performed to evaluate the expression of the miR-503 target genes. In vivo and in vitro assays were performed to evaluate the function of miR-503 in HCC. Luciferase reporter assay was employed to validate the miR-503 target genes. Results: miR-503 was frequently downregulated in HCC cell lines and tissues. Low expression levels of miR-503 were associated with enhanced malignant potential such as portal vein tumor thrombi, histologic grade, TNM stage, AFP level and poor prognosis. Multivariate analysis indicated that miR-503 downregulation was significantly associated with worse overall survival of HCC patients. Functional studies showed miR-503 suppressed the proliferation of HCC cells by induction of G1 phase arrest through Rb-E2F signaling pathways, and thus may function as a tumor suppressor. Further investigation characterized two cell cycle-related molecules, cyclin D3 and E2F3, as the direct miR-503 targets. Conclusion: Our data highlight an important role for miR-503 in cell cycle regulation and in the molecular etiology of HCC, and implicate the potential application of miR-503 in prognosis prediction and miRNA-based HCC therapy.

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Figure 1 Expression pattern of miR-503 in HCC cell lines and tissues. (A) lower expression level of miR-503 was detected in tumors than in adjacent nontumorous tissues in 125 HCC patients. (B) relative expression levels of miR-503 in the indicated HCC cell lines (*P<0.05, **P<0.01, ***P<0.001).
Figure 1 Expression pattern of miR-503 in HCC cell lines and tissues. (A) lower expression level of miR-503 was detected in tumors than in adjacent nontumorous tissues in 125 HCC patients. (B) relative expression levels of miR-503 in the indicated HCC cell lines (*P<0.05, **P<0.01, ***P<0.001).CC BY · PubMed Central
Figure 2 Kaplan–Meier overall survival curves of HCC patients according to the level of miR-503 expression. Patients in the low miR-503 expression group had significantly a poorer prognosis than those in the high miR-503 expression group (P<0.01)
Figure 2 Kaplan–Meier overall survival curves of HCC patients according to the level of miR-503 expression. Patients in the low miR-503 expression group had significantly a poorer prognosis than those in the high miR-503 expression group (P<0.01)CC BY · PubMed Central
Figure 3 The effect of miR-503 on cell proliferation, clonogenicity in vitro. (A) The expression of miR-503 in MHCCLM3, HepG2 and Bel-7402 cells with transfection of miR-503 mimic, miR-503 inhibitor or respective NC. (B) The effects of miR-503 on the proliferation of HCC cell lines were measured by CCK-8 assay. (C) The effect of miR-503 on colony formation of HCC cell lines. **P< 0.01; ***P<0.001.
Figure 3 The effect of miR-503 on cell proliferation, clonogenicity in vitro. (A) The expression of miR-503 in MHCCLM3, HepG2 and Bel-7402 cells with transfection of miR-503 mimic, miR-503 inhibitor or respective NC. (B) The effects of miR-503 on the proliferation of HCC cell lines were measured by CCK-8 assay. (C) The effect of miR-503 on colony formation of HCC cell lines. **P< 0.01; ***P<0.001.CC BY · PubMed Central
Figure 4 The effect of miR-503 on clonogenicity in vivo and cell cycle. (A) Tumor formation in nude mice. Representative photographs and the curve of tumor growth are shown. (B) The effect of miR-503 on cell cycle of HCC cell lines. *P<0.05 **P< 0.01; ***P<0.001.
Figure 4 The effect of miR-503 on clonogenicity in vivo and cell cycle. (A) Tumor formation in nude mice. Representative photographs and the curve of tumor growth are shown. (B) The effect of miR-503 on cell cycle of HCC cell lines. *P<0.05 **P< 0.01; ***P<0.001.CC BY · PubMed Central
Figure 5 Cyclin D3 and E2F3 are direct targets of miR-503. (A) the putative miR-503 binding sequence in the 3′ UTR of Cyclin D3 and E2F3 mRNA. Mutation was generated on the Cyclin D3 and E2F3 3′ UTR sequence in the complementary site for the seed region of miR-503. (B) Analysis of luciferase activity. 293 T cells were cotransfected with 50 nM of either miR-503 or NC and 100 ng pmirGLO Vector containing Wt or Mut 3′UTR of Cyclin D3 or E2F3 (indicated as WT or MUT on the X axis). The relative firefly luciferase activity normalized with renilla luciferase was measured 48 h after transfection. (C) the effect of miR-503 on the expression of endogenous Cyclin D3 and E2F3 at mRNA levels. (D and E) Endogenous Cyclin D3 or E2F3 protein levels are down-regulated in LM3 cells transfected with miR-503 mimics and up-regulated in Bel-7402 cells transfected with the miR-503 inhibitor (F) the effect of siRNA on the expression of endogenous Cyclin D3 and E2F3 at protein level. (G) knockdown of CCND3 or E2F3 induced a significant accumulation of G1-phase cells and blocked G1/S transition. *** P < 0.001.
Figure 5 Cyclin D3 and E2F3 are direct targets of miR-503. (A) the putative miR-503 binding sequence in the 3′ UTR of Cyclin D3 and E2F3 mRNA. Mutation was generated on the Cyclin D3 and E2F3 3′ UTR sequence in the complementary site for the seed region of miR-503. (B) Analysis of luciferase activity. 293 T cells were cotransfected with 50 nM of either miR-503 or NC and 100 ng pmirGLO Vector containing Wt or Mut 3′UTR of Cyclin D3 or E2F3 (indicated as WT or MUT on the X axis). The relative firefly luciferase activity normalized with renilla luciferase was measured 48 h after transfection. (C) the effect of miR-503 on the expression of endogenous Cyclin D3 and E2F3 at mRNA levels. (D and E) Endogenous Cyclin D3 or E2F3 protein levels are down-regulated in LM3 cells transfected with miR-503 mimics and up-regulated in Bel-7402 cells transfected with the miR-503 inhibitor (F) the effect of siRNA on the expression of endogenous Cyclin D3 and E2F3 at protein level. (G) knockdown of CCND3 or E2F3 induced a significant accumulation of G1-phase cells and blocked G1/S transition. *** P < 0.001.CC BY · PubMed Central
Figure 6 Rb-E2F signaling is involved in miR-503-regulated G1/S transition. (A) phosphorylation of Rb protein on Ser780 and Ser811 is inhibited by miR-503. 48 h after transfection with the indicated RNA duplex, cells were subjected to Western blot analysis. Rb, total Rb protein; pRb, phosphorylated Rb. (B) effect of miR-503 on the expression of endogenous genes. miR-503 inhibited significantly the expression of CDK6 and the downstream genes of E2F3. Gene expression was examined by Western blot. β-actin, internal control. (C) signaling pathways that regulate the cell cycle control in HCC involving miR-503.
Figure 6 Rb-E2F signaling is involved in miR-503-regulated G1/S transition. (A) phosphorylation of Rb protein on Ser780 and Ser811 is inhibited by miR-503. 48 h after transfection with the indicated RNA duplex, cells were subjected to Western blot analysis. Rb, total Rb protein; pRb, phosphorylated Rb. (B) effect of miR-503 on the expression of endogenous genes. miR-503 inhibited significantly the expression of CDK6 and the downstream genes of E2F3. Gene expression was examined by Western blot. β-actin, internal control. (C) signaling pathways that regulate the cell cycle control in HCC involving miR-503.CC BY · PubMed Central

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Metadata

Authors
Xiao, F., zhang, W., Chen, L., Chen, F., Xie, H., Xing, C., Yu, X., Ding, S., Chen, K., Guo, H., Cheng, J., Zheng, S., & Zhou, L.
Venue
Journal of Translational Medicine
Publisher
Springer Science and Business Media LLC
Published
2013-08-22
Volume
11
Issue
1
ISSN
1479-5876
DOI
10.1186/1479-5876-11-195
PMC
PMC3765277
Cited by
71
References
2
Language
en

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