PMC

Relative expression of miR-122 in human HCC-derived cell lines. Expression of miR-122 was evaluated in 21 human HCC-derived cell lines by Q-RT-PCR and normalized to the level of 5s RNA. Data are represented as fold induction relative to the lowest miR-122 expressing SNU-398 cell line using the 2^−ΔΔCt method. The great heterogeneity of miR-122 expression indicated that HCC-derived cell lines represent excellent natural models for functional studies of miR-122 in liver cancer.

The silencing of HNF1A, HNF3A, and HNF3B results in a decrease of miR-122 level. The high miR-122 expressing PLC/PRF/5 cell line was transfected with 40 nM of specific siRNA directed against HNF-1A, -3A, -3B, and -4A (siRNA, black column) vs. negative control (NC, white column). Forty eight hours after the transfection, the efficiency of the silencing was confirmed by the decreased level of respective mRNA (upper panel). As shown in the lower panel, the silencing of HNF-1A, -3A, and -3B significantly reduces the level of miR-122.

Repression of miR-122 correlates with clinically relevant parameters in human HCC. Expression of miR-122 was evaluated by Q-RT-PCR in 64 human HCC and 28 non-tumor surrounding liver tissues (ST). (a) Expression of miR-122 was evaluated in ST and HCC tissues as a function of experimentally well-defined gene expression signatures (described in ; ; ; ). Data indicated that miR-122 was specifically repressed in HCC with bad prognosis which harbor hepatoblast, c-Met and late TGF-β gene expression signatures. (b) The assessment of miR-122 expression as a function of biochemical parameters indicated that miR-122 was particularly repressed in HCC with high proliferation and ubiquitination index, and low apoptotic index (). (c) The evaluation of miR-122 expression as a function of etiology, Edmonson grade and tumor size indicated that miR-122 was repressed in HCC arising from HBV-infected livers and was associated with poorly differentiated and large size tumors.

miR-122 impacts the propensity of cancer cells to migrate and invade. (a) Ingenuity Pathway Analysis was performed on the genes up-regulated in HCC samples which exhibit a low miR-122 expression (Cluster B in ). This procedure revealed a network associated with cell motility and identified RAC1 and RHOA as two central regulators. (b) Inhibition of miR-122 results in the acquisition of migrating and invasive properties. The effect of miR-122 inhibition on cellular migration and invasion was assessed in Huh-1 by transfecting 20 nM of either anti-miR-122 (black column) or negative control (NC-Anti-miR, white column). Invasive and migrating activity was measured by using the BD BioCoat Matrigel Invasion Chambers. Data indicate that the loss of miR-122 increases the migrating and invasive properties of Huh-1 cells. (c) Conversely, in the miR-122 negative SK-Hep1 cell line the restoration of miR-122 by transfecting miR-122 precursor molecules results in a decreased migration and invasion (Pre-miR-122, black column) as compared to cells transfected with negative control (NC-Pre-miR, white column).

miR-122 expression defines specific gene expression profiles in human HCC. (a) Hierarchical cluster analysis of 509 genes differentially expressed in liver primary tumors as a function of miR-122 expression. Data are presented in a matrix format in which rows and columns represent genes and HCC samples, respectively. Differentially expressed genes are grouped in two clusters, A and B. HCC samples cluster in two groups, C1 and C2, which greatly differ in term of miR-122 level (C1: 2.7±0.4; C2: 0.6±0.4; mean±s.e.m; P<0.001). Genes included in cluster A and cluster B are up- and down-regulated in cluster C1 HCC, respectively. As expected cluster B includes several predicted miR-122 target genes (*). (b) Gene Set Enrichment Analysis using the genes embedded in the clusters A and B as signatures and the gene expression profiles derived from the experimental inhibition of miR-122 in mice (). Cluster A and B genes are significantly enriched in the profiles corresponding to control mice (Enrichment Score ES=0.578, P<0.001), and mice treated with antagomir-122 (ES=0.658, P<0.001), respectively.

High level of miR-122 in HCC defines a specific gene expression profile linked to HNF4A. (a–b) Ingenuity Pathway Analysis applied to the genes which expression was positively correlated with the expression of miR-122 in human primary HCC (a) and HCC-derived cell lines (b). Liver-enriched transcription factors, HNF4A in particular, were identified as the central regulators in the gene signature associated with a high miR-122 expression. (c–e) In HCC-derived cell lines the loss of miR-122 is associated with a loss of HNF4A. (c) The evaluation of HNF4A mRNA level in HCC-derived cell lines indicated a positive correlation between HNF4A and miR-122. (d) The assessment of HNF4A expression in protein extracts isolated from the cell lines which exhibit high vs. low miR-122 further indicates a positive correlation between miR-122 and HNF4A expression. High miR-122 expressing cell lines, such as PLC/PRF/5, Huh-1, Hep40 or Huh-6, exhibit a considerably higher HNF4A protein levels as compared to SNU-182, SNU-387, and SNU-398 lines which exhibit low miR-122levels. (e) Analysis of HNF4A expression in cytoplasmic (C) and nuclear (N) compartments indicates a nuclear enrichment of HNF4A in the high miR-122 expressing cell lines.