Identification of microRNA differentially expressed in hepatocellular carcinoma versus non-tumoral liver tissue

MicroRNAs (miRNAs) are small, endogenously expressed, noncoding RNAs, which regulate gene expression through RNA interference by binding to a defined set of target transcripts. Recent data have shown that miRNAs are involved in cancer initiation and progression by affecting tumorigenic pathways with impact on cell proliferation and apoptosis. Since miRNA expression pattern can be used for the classification, prognosis and therapeutic approaches of human malignancies, we studied the miRNA expression profiles by quantitative PCR in hepatocelluar carcinoma (HCC).

Methods: From a total of 74 HCCs, tissues from HCV positive patients were selected and sections were prepared from paraffin-embedded, formalin-fixed material. By macrodissection 19 tumorous tissues representing dysplastic nodules of low and high grade, or carcinoma of G1, G2 and G3, were separated from non-tumorous tissue and used for RNA extraction by the Trizol method. Using the miRNA Archive Kit of Applied Biosystems, 80 miRNAs of each sample were reverse-transcribed and used for Real Time PCR. Relative quantification of miRNA was performed by the 2-ΔΔCt method. Normal liver was used as a calibrator. Values were accessed by global median normalisation and presented as log10 of relative quantity (RQ).

RESULTS and Discussion: By quantitative PCR we identified a subset of fourteen miRNAs dysregulated in dysplastic nodules and HCC. Log10 RQ values demonstrated upregulation of ten miRNA in all stages of hepatocellular carcinogenesis. Moderate overexpression of miRNA–200b, miRNA–326 or miRNA–194 was observed, but predominant upregulation was shown for miRNA–370, miRNA–299 and miRNA–125b, which putatively affect the expression of Bax /Akt, β-catenin or IGF R type I, respectively. Among the four downregulated miRNAs in HCC, miRNA–145 is a putative repressor of the TGF ß receptor II expression and was recently shown to be a prominent dysregulated miRNA in colon, lung and breast cancer.

Conclusion: Identification of a subset of dysregulated miRNA in a small number of HCC indicates, that certain miRNAs are involved in hepatocellular carcinogenesis. Further studies will shed light on miRNA function in HCC initiation and progression.

This work was supported by the BMBF KI 0405.

hepatocellular carcinoma - microRNA - regulation of gene expression