miR-221 overexpression contributes to liver tumorigenesis - PubMed (original) (raw)

miR-221 overexpression contributes to liver tumorigenesis

Pascal Pineau et al. Proc Natl Acad Sci U S A. 2010.

Abstract

MicroRNA (miRNAs) are negative regulators of gene expression and can function as tumor suppressors or oncogenes. Expression patterns of miRNAs and their role in the pathogenesis of hepatocellular carcinoma (HCC) are still poorly understood. We profiled miRNA expression in tissue samples (104 HCC, 90 adjacent cirrhotic livers, 21 normal livers) as well as in 35 HCC cell lines. A set of 12 miRNAs (including miR-21, miR-221/222, miR-34a, miR-519a, miR-93, miR-96, and let-7c) was linked to disease progression from normal liver through cirrhosis to full-blown HCC. miR-221/222, the most up-regulated miRNAs in tumor samples, are shown to target the CDK inhibitor p27 and to enhance cell growth in vitro. Conversely, these activities can be efficiently inhibited by an antagomiR specific for miR-221. In addition, we show, using a mouse model of liver cancer, that miR-221 overexpression stimulates growth of tumorigenic murine hepatic progenitor cells. Finally, we identified DNA damage-inducible transcript 4 (DDIT4), a modulator of mTOR pathway, as a bona fide target of miR-221. Taken together, these data reveal an important contribution for miR-221 in hepatocarcinogenesis and suggest a role for DDIT4 dysregulation in this process. Thus, the use of synthetic inhibitors of miR-221 may prove to be a promising approach to liver cancer treatment.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Heat map of miRNA expression in HCC: Tree showing the miRNAs deregulated in HCC vs. cirrhotic tissues (paired t test, P < 0.01). Each column indicates the normalized log 2 ratio of the HCC/cirrhosis pairs. Clustering was performed with average linkage and uncentered correlation.

Fig. 2.

Fig. 2.

miRNA signatures in HCC. (A) Intersection of SAM analysis for three profiles. The intersection is shown among the deregulated miRNAs in the three classes of tissues: HCC, cirrhosis, and normal liver. Only the miRNA deregulated also in the HCC cell lines were considered. Red, up-regulated miRNAs; green, down-regulated miRNAs. (B) Validation of microarray by qPCR of differentially expressed miRNAs in nontumoral (NT) vs. tumoral (T) liver tissues and β-catenin mutated (β-catm) vs. wild-type (β-catwt) specimens. The relative expression fold of each miRNA is normalized to RNU6B and miR-140. P values were obtained by using a two-sided Student t test.

Fig. 3.

Fig. 3.

miR-221 inhibition impairs tumor cell growth. (A) HepG2 and HLE cells were plated at low density after transfection by premiR negative control (NC), premiR-221 or LNA scrambled antagomiR (Scr antimiR), or LNA antimiR-221. Cells were grown for 10 days, fixed, and stained by crystal violet. (B) Malhavu (miR-221 and miR-222 high expressor) and Huh6 (miR-221 and miR-222 low expressor) liver cancer cells were transfected with antimir-221, antimiR-222, or a negative control and then plated out in 96-well plates. Cell growth was measured by MTT assay at 72 h. Viability was identical for cells treated with transfection reagent alone or scrambled antimiR. The values are the means from three separate experiments done in triplicate. P values were obtained by using a two-sided Student t test. miR-221 levels are shown as measured by qPCR 24 h after transfection of LNA oligonucleotides.

Fig. 4.

Fig. 4.

miR-221 is protumorigenic in vivo. (A) Schematic representation of the transfer protocol using immortalized p53_−/−;myc_ liver progenitors infected by MSCV-derived retroviruses. (B) Kaplan–Meier curves showing the percentage of tumor-free injected mouse flank. Mice were injected s.c. with p53_−/−;myc_ hepatoblasts previously infected by retroviruses expressing miR-221 or sh-APC in the PIG-MSCV vector. Each group corresponds to 24 flanks/12 mice. Mice were monitored starting at 3 weeks after injection. The histogram corresponds to miR-221 expression levels as measured by qPCR in the three types of transduced liver progenitors. (C) Average latency for tumor appearance. Significant differences of tumor onset was observed between miR-221 and empty vector (*, P = 0.0142), sh-APC and empty vector (**, P < 1 10−4), and miR-221 and sh-APC (***, P = 0.0151). (D) Time required for tumor to reach 1,000 mm3. Values are mean ± SD. Each mouse was killed when tumor reached 1,000 mm3. Significant differences were observed between miR-221- and empty vector-infected cells (*, P < 0.05) and between sh-APC and empty vector (**, P < 0.005).

Fig. 5.

Fig. 5.

DDIT4 inhibition by miR-221. (A) Luciferase activity 72 h after cotransfection of 293T cells with psiCHECK-2-UTRs vectors, premiR-221, premiR negative control (NC), and LNA antimiR-221. Data are means ± SD of four independent experiments performed in triplicate. Renilla luciferase activity was significantly decreased for 3′ UTRs of positive controls (Kit, p27, CDKN1C) and for DDIT4, CREBZF, MYBL1, TBK1, DKK2, and BNIP3L (P < 0.05, Student’s t test). (B) Luciferase activity at 72 h in 293T cells cotransfected with premiR negative control (NC, black) or premiR-221 (gray) and luciferase reporters containing the wild-type (WT) or mutated miR-221 binding sites (mS1 and mS2) of human DDIT4 3′UTR or vector (V) (*, P < 0.01, Student’s t test). (C) 293T, Huh6, and NU.K.-1 cells were transfected with vector expressing miR-221, miR-222, or empty vector, and protein levels were checked by Western blotting with the indicated antibodies. One representative of three independent experiments is shown.

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