Role of microRNA-155 at early stages of hepatocarcinogenesis induced by choline-deficient and amino acid-defined diet in C57BL/6 mice - PubMed (original) (raw)

Role of microRNA-155 at early stages of hepatocarcinogenesis induced by choline-deficient and amino acid-defined diet in C57BL/6 mice

Bo Wang et al. Hepatology. 2009 Oct.

Abstract

MicroRNAs (miRs) are conserved, small (20-25 nucleotide) noncoding RNAs that negatively regulate expression of messenger RNAs (mRNAs) at the posttranscriptional level. Aberrant expression of certain microRNAs plays a causal role in tumorigenesis. Here, we report identification of hepatic microRNAs that are dysregulated at early stages of feeding C57BL/6 mice choline-deficient and amino acid-defined (CDAA) diet that is known to promote nonalcoholic steatohepatitis (NASH)-induced hepatocarcinogenesis after 84 weeks. Microarray analysis identified 30 hepatic microRNAs that are significantly (P < or = 0.01) altered in mice fed CDAA diet for 6, 18, 32, and 65 weeks compared with those fed choline-sufficient and amino acid-defined (CSAA) diet. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated up-regulation of oncogenic miR-155, miR-221/222, and miR-21 and down-regulation of the most abundant liver-specific miR-122 at early stages of hepatocarcinogenesis. Western blot analysis showed reduced expression of hepatic phosphatase and tensin homolog (PTEN) and CCAAT/enhancer binding protein beta (C/EBPbeta), respective targets of miR-21 and miR-155, in these mice at early stages. DNA binding activity of nuclear factor kappa B (NF-kappaB) that transactivates miR-155 gene was significantly (P = 0.002) elevated in the liver nuclear extract of mice fed CDAA diet. Furthermore, the expression of miR-155, as measured by in situ hybridization and real-time RT-PCR, correlated with diet-induced histopathological changes in the liver. Ectopic expression of miR-155 promoted growth of hepatocellular carcinoma (HCC) cells, whereas its depletion inhibited cell growth. Notably, miR-155 was significantly (P = 0.0004) up-regulated in primary human HCCs with a concomitant decrease (P = 0.02) in C/EBPbeta level compared with matching liver tissues.

Conclusion: Temporal changes in microRNA profile occur at early stages of CDAA diet-induced hepatocarcinogenesis. Reciprocal regulation of specific oncomirs and their tumor suppressor targets implicate their role in NASH-induced hepatocarcinogenesis and suggest their use in the diagnosis, prognosis, and therapy of liver cancer.

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Figures

Figure 1

MicroRNA expression was dysregulated at early stages of hepatocarcinogenesis. A. Clustering of the miRNA expression profiles at 4 time points (6, 18, 32 and 65 weeks) was performed by average linkage using correlation metrics. MicroRNAs were selected by class comparison using analysis of variance with randomized block design. The cluster tree with the fold change of 30 miRs (_P_≤0.01) that varied with the time course was constructed.

Figure 2

A. Validation of miRNA microarray data by real-time RT-PCR analysis of DNase I treated total RNA. RNAs from 5 mice were used for RT-PCR and each sample was analyzed in triplicate. Single and double asterisks denote _P_≤0.05 and ≤0.01 respectively. B. Localization of miR-122 in livers by LNA-ISH. Tissue sections were hybridized to biotin-labeled oligo (anti-miR-122), which was captured with alkaline phosphatase conjugated-streptavidin and the signal (blue color) was developed with NBT/BCIP. Cell body was stained with Nuclear fast red. Red arrows indicated mature miR-122. C. Representative photographs of H&E stained liver sections from mice. D. Localization of miR-155 in livers by LNA-ISH. Scrambled oligo probe was used as negative control. Green arrows indicated mature miR-155.

Figure 2

Figure 3

A. NF-κB was activated in the liver nuclear extract of mice fed CDAA diet. Identical amount (3 μg) of the extract was incubated with 32P-labed NF-κB oligo under optimal binding conditions. The protein DNA complex was resolved in a 5% polyacrylamide gel, dried and subjected to phosphorimager analysis. For competition and supershift assays, the extracts were preincublated with 100 fold molar excess of unlabeled oligos and antibodies, respectively for 30 minutes before adding labeled probe. B. Quantitative analysis of the data in A.

Figure 4

Downregulation of C/EBPβ and PTEN, respective targets of miR-155 and miR-21, in the livers of mice fed CDAA diet. A. Schematic representation of conserved miR-155 site in C/EBPβ 3'UTR. B. Western blot analysis of C/EBPβ in HCC cells. Hep3B and HepG2 cells were transfected with pre-miR-155 (50nM) followed by Western blot analysis after 48 hours. C. Real-time RT-PCR analysis of C/EBPβ in the liver of mice fed diet for 32 and 65 weeks. D and E. Western blot analysis of C/EBPβ, PTEN and GAPDH in the liver extracts. Equal amount of proteins were subjected to immunoblot analysis first with specific primary and secondary antibodies and the signal was developed with ECL reagent. The signal was quantified using Kodak Imaging software and the data was normalized to GAPDH.

Figure 4

Figure 5

A and B. Ectopic expression of miR-155 promoted growth of Hep3B and HepG2 cells in culture. Cells were transfected with miR-155 precursor or control RNA (50 nM) followed by MTT assay. C. Knockdown of endogenous miR-155 reduced SNU-182 cell growth. Cells were transfected with anti-miR-155 or control RNA (60 nM) followed by MTT assay. NC: negative control. The upper panels present real-time RT-PCR analysis of miR-155 in HCC cells at the last time point when cell growth was measured.

Figure 5

Figure 6

miR-155 and CE/BPβ levels were reciprocally regulated in primary hepatocellular carcinomas. A. Total RNA from 20 HCCs and pair-matched normal liver tissues was subjected to real-time RT-PCR analysis for miR-155 level. Expression of miR-155 in each sample presented in Supplementary Table 5 is depicted as dot plots. Horizontal bars indicate median expression value. B. Inverse correlation between C/EBPβ mRNA level and miR-155 expression in HCCs (r=−0.51, _P_=0.02), determined by real-time RT-PCR analysis in 20 HCC samples. C. Western blot analysis of C/EBPβ in whole tissue extracts (500 μg) from HCCs (T) and matching livers (N). Asterisks denote HCC samples in which C/EBPβ is reduced.

References

1. Bruix J, Boix L, Sala M, Llovet JM. Focus on hepatocellular carcinoma. Cancer Cell. 2004;5:215–219. - PubMed
1. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology. 2007;132:2557–2576. - PubMed
1. Yeh MM, Brunt EM. Pathology of nonalcoholic fatty liver disease. Am J Clin Pathol. 2007;128:837–847. - PubMed
1. Torres DM, Harrison SA. Diagnosis and therapy of nonalcoholic steatohepatitis. Gastroenterology. 2008;134:1682–1698. - PubMed
1. Eulalio A, Huntzinger E, Izaurralde E. Getting to the root of miRNA-mediated gene silencing. Cell. 2008;132:9–14. - PubMed

Role of microRNA-155 at early stages of hepatocarcinogenesis induced by choline-deficient and amino acid-defined diet in C57BL/6 mice - PubMed (original) (raw)