Altered microRNA Expression Patterns in Hepatoblastoma Patients

Abstract

Liver cancers in children are rare representing only 1.1% of malignancies, with an annual incidence rate of 1.5 cases per million. Hepatoblastoma and hepatocellular carcinomas are the most common malignancies of the liver occurring in young people aged 15 years or younger. Molecular basis of both tumors are still unclear, and common markers (i.e., CTNNB1, APC, IGF-2) are not always useful in the characterization of sporadic forms; in this respect, microRNA recently associated with carcinogenesis could play a pivotal role in their onset. CTNNB1 and APC were analyzed by sequencing, and IGF-2 promoter methylation status was assessed by methylation-specific polymerase chain reaction. MicroRNA expression was assayed by microarray and quantitative reverse transcription-polymerase chain reaction in hepatoblastoma samples. Although few genomic alterations were detected in ours samples, an altered expression of somemicroRNA in hepatoblastoma was observed. Unsupervised clustering shows that microRNA profile can distinguish tumor from nontumor tissues. Further analyses of microRNA contents in hepatoblastoma compared with hepatocellular carcinoma highlighted four upregulated microRNA (miR-214, miR-199a, miR-150 [P < .01], and miR-125a [P < .05]) and one downregulated microRNA (miR-148a [P < .01]). In conclusion, although our samples were poorly informative from a genetic point of view, they showed a peculiar microRNA expression pattern compared with nontumor tissues and hepatocellular carcinoma. MicroRNA could represent valid markers for the classification of pediatric liver tumors.

Figure 1

(A) Results of the unsupervised hierarchical clustering, in which NT tissues (blue) form a separate cluster from the HB tumors (gray). The heat map shows relative levels of microRNA expression in a green (low relative expression) to red (high relative expression) scale. (B) Volcano plot: a useful graphical that plots negative log₁₀ P values (obtained from t test) on the y-axis versus mean log₂ fold change on the x-axis, comparing altered microRNA expression in HB patients showing significant alteration of a small number of specific microRNA. Black dots indicate altered microRNA in HB versus NT tissues considered significant (P < .05).

Figure 2

Validation of microRNA showing different pattern compared with HCC in nine HB patients and in nonneoplastic tissues. Each histogram plots U6 normalized log₂ fold change (ratio of tumors to nontumors). Error bars, SD of three replicas.

Figure 3

PTEN and miR-214 correlation of three patients from HB frozen samples and relative nontumor tissues. (A) Quantitative reverse transcription-polymerase chain reaction microRNA analysis. Bars display relative values of miR-214 expression in HB compared with nontumor tissues (taken to 1). Error bars, SD of three replicas. (B) Western blot analysis of variations in the expression of PTEN protein and relative densitometry analysis. β-Actin was used as loading control.