MiR-138 induces cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma - PubMed (original) (raw)

MiR-138 induces cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma

Wen Wang et al. Carcinogenesis. 2012 May.

Abstract

The deregulation of microRNA (miRNA) is frequently associated with a variety of cancers, including hepatocellular carcinoma (HCC). In this study, we identified 10 upregulated miRNAs (miR-217, miR-518b, miR-517c, miR-520g, miR-519a, miR-522, miR-518e, miR-525-3p, miR-512-3p and miR-518a-3p) and 10 downregulated miRNAs (miR-138, miR-214, miR-214#, miR-27a#, miR-199a-5p, miR-433, miR-511, miR-592, miR-483-5p and miR-483-3p) by Taqman miRNAs array and quantitative real-time PCR (qRT-PCR) confirmation. Additionally, we investigated the expression and possible role of miR-138 in HCC. qRT-PCR results showed that miR-138 was downregulated in 77.8%(14/18) of HCC tissues compared with adjacent non-tumor tissues. Overexpression of miR-138 reduced cell viability and colony formation by induction of cell arrest in HCC cell lines and inhibited tumor cell growth in xenograft nude mice. The use of miR-138 inhibitor increased cell viability and colony formation in HCC cell lines and tumor cell growth in xenograft nude mice. Using TargetScan predictions, CCND3 was defined as a potential direct target of miR-138. Furthermore, CCND3 protein expression was observed to be negatively correlated with miR-138 expression in HCC tissues. The dual-luciferase reporter gene assay results showed that CCND3 was a direct target of miR-138. The use of miR-138 mimic or inhibitor could decrease or increase CCND3 protein levels in HCC cell lines. We conclude that the frequently downregulated miR-138 can regulate CCND3 and function as a tumor suppressor in HCC. Therefore, miR-138 may serve as a useful therapeutic agent for miRNA-based HCC therapy.

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Figures

Fig. 1.

Fig. 1.

The effect of miR-138 on cell viability (B), colony formation(C), xenograft tumor growth (D) and cell cycle (E). (A) The expression of miR-138 in HepG2 and Huh7 cells with or without transfection of miR-138 mimic, miR-138 inhibitor or NC duplex. (B) The effect of miR-138 on cell viability of HCC cell lines. (C) The effect of miR-138 on colony formation of HCC cell lines. (D) The effect of miR-138 on the tumor growth in xenograft nude mice. (E) The effect of miR-138 on cell cycle of HCC cell lines. Representative results (B, C and E) in HepG2 and Huh7 cells transfected with miR-138 mimic, miR-138 inhibitor, NC duplex or not. Column, mean of three independent experiments; bars, SD; *P < 0.01 and **P < 0.001. Representative results (D) in HepG2 transfected with miR-138 mimic, miR-138 inhibitor, NC duplex or not. *P < 0.01.

Fig. 2.

Fig. 2.

CCND3 is a direct target of miR-138. (A) The putative miR-138 binding sequence in the 3′UTR of CCND3 mRNA. Mutation was generated on the CCND3 3′UTR sequence in the complementary site for the seed region of miR-138. (B) The expression of CCND3 in 18 paired HCCs tumor tissues (T) and adjacent non-tumor tissues (N). (C) The negative correlation between the expression of miR-138 and CCND3 in paired HCC tissues. The relative quantity of miR-138 in HCC tumor tissues compared with adjacent non-tumor tissues (T/N) was showed after normalized to U6. The relative quantity of CCND3 protein in HCC tumor tissues compared with adjacent non-tumor tissues (T/N) was showed after normalized to glyceraldehyde-3-phosphate dehydrogenase. (D) Suppressed luciferase activity of wild-type 3′UTR of CCND3 by miR-138 mimic. HEK293T cells were cotransfected pGL3-CCND3-3′UTR or pGL3-CCND3-3′UTR-mut and miR-138 mimic or NC duplex. Firefly luciferase activity of each sample was measured 48 h after transfection and normalized to Renilla luciferase activity. (E) The expression of endogenous CCND3 regulated by miR-138. The expression level of endogenous CCND3 in HepG2 cells was analyzed 48 h after transfection with miR-138 mimic, miR-138 inhibitor or NC duplex by western blotting. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. Column, mean of three independent experiments; bars, SD; *P < 0.01.

Fig. 3.

Fig. 3.

CCND3 could induce cell cycle arrest. (A) CCND3 siRNA efficiently inhibits the expression of CCND3. The expression of endogenous CCND3 was analyzed 48 h after transfection with CCND3 siRNA or NC siRNA by western blotting. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. (B) CCND3 knockdown reduced cell viability of HCC cell lines. Representative results of cell viability in HepG2 cells transfected with CCND3 siRNA and NC siRNA or not. (C) CCND3 knockdown induced cell cycle of HCC cell lines. Representative results of cell cycle in HepG2 cells transfected with CCND3 siRNA NC siRNA or not. (D) CCND3 knockdown inhibited colony formation of HCC cell lines. Representative results of colony formation in HepG2 cells transfected with CCND3 siRNA and NC siRNA or not. Column, mean of three independent experiments; bars, SD; *P < 0.01.

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