SNPs at miR-155 binding sites of TYRP1 explain discrepancy between mRNA and protein and refine TYRP1 prognostic value in melanoma.

BACKGROUND: We previously demonstrated an inverse correlation between tyrosinase-related protein 1 (TYRP1) mRNA expression in melanoma metastases and patient survival. However, TYRP1 protein was not detected in half of tissues expressing mRNA and did not correlate with survival. Based on a study reporting that 3' untranslated region (UTR) of TYRP1 mRNA contains two miR-155-5p (named miR-155) binding sites exhibiting single-nucleotide polymorphisms (SNPs) that promote (matched miRNA-mRNA interaction) mRNA decay or not (mismatched), we aimed to investigate the role of miR-155 in the regulation of TYRP1 mRNA expression and protein translation accounting for these SNPs. METHODS: The effect of miR-155 on TYRP1 mRNA/protein expression was evaluated in two melanoma cell lines harbouring matched or mismatched miR-155-TYRP1 mRNA interaction after transfection with pre-miR-155. In parallel, 192 skin and lymph node melanoma metastases were examined for TYRP1 mRNA/protein, miR-155 and SNPs and correlated with patient survival. TYRP1 mRNA, SNPs at its 3'UTR and miR-155 were analysed by RT-qPCR, whereas TYRP1 protein was evaluated by western blot in cell lines and by immunohistochemistry in metastatic tissues. RESULTS: The miR-155 induced a dose-dependent TYRP1 mRNA decay and hampered its translation into protein in the line with the 'match' genotype. In melanoma metastases, TYRP1 mRNA inversely correlated with miR-155 expression but not with TYRP1 protein in the 'match' group, whereas it positively correlated with protein but not with miR-155 in the 'mismatch' group. Consequently, in the latter group, TYRP1 protein inversely correlated with survival. CONCLUSION: Polymorphisms in 3'UTR of TYRP1 mRNA can affect TYRP1 mRNA regulation by miR-155 and its subsequent translation into protein. These SNPs can render TYRP1 mRNA and protein expression nonsusceptible to miR-155 activity and disclose a prognostic value for TYRP1 protein in a subgroup of melanoma patients. These data support the interest in the prognostic value of melanogenic markers and propose TYRP1 to refine prognosis in patients with advanced disease.

Effects of epidermal growth factor on CYP inducibility by xenobiotics, DNA replication, and caspase activations in collagen I gel sandwich cultures of rat hepatocytes.

In this study, we investigated the combined effects of EGF and collagen I gel on the phenotype of cultured rat hepatocytes and we focussed our investigations on the regulation of xenobiotic-mediated induction of CYP, cell cycle progression and activation of capases 8 and 3. We found that EGF, added to basal culture medium or phenobarbital (3.2 mM) containing medium, provoked a moderate decrease of CYP1A1 and CYP2B1/2 activities. However, EGF did not exert any inhibitory effect on 3-methylcholantrene (5 microM) and beta-naphtoflavone (25 microM) induction of CYP1A1 activities. In collagen gel sandwich cultures, hepatocytes remained arrested in mid-G1 phase of the cell cycle, even in the presence of EGF. In conventional primary cultures, caspases 8 and 3 were activated at 3 and 5 days after plating respectively. In collagen gel sandwich cultures, we found that neither collagen I nor EGF prevented activation of caspase 8 while collagen I gel inhibited activation of caspase 3, preventing spontaneous apoptosis of cultured rat hepatocytes. In contrast, EGF transiently increased caspase 3 activity at day 1 after plating. Altogether, our data demonstrate that collagen I gel triggers intracellular signals which strongly affect cultured hepatocyte phenotype, leading to a cell cycle arrest in G1 phase and long-term survival through the inhibition of caspase 3 activation and that EGF-free medium improves survival and liver-specific gene expression in hepatocytes maintained in collagen I gel sandwich cultures.

Antiproliferative and apoptotic effects of O-Trensox, a new synthetic iron chelator, on differentiated human hepatoma cell lines.

We investigated the effects of a new iron chelator, O-Trensox (TRX), compared with desferrioxamine (DFO), on proliferation and apoptosis in cultures of the human hepatoblastoma HepG2 and hepatocarcinoma HBG cell lines. Our results show that TRX decreased DNA synthesis in a time- and dose-dependent manner and with a higher efficiency than DFO. Mitotic index was also strongly decreased by TRX and, unexpectedly, DFO inhibited mitotic activity to the same extent as TRX, thus there is a discrepancy between the slight reduction in DNA synthesis and a large decrease in mitotic index after DFO treatment. In addition, we found that TRX induced accumulation of cells in the G(1) and G(2) phases of the cell cycle whereas DFO arrested cells in G(1) and during progression through S phase. These data suggest that the partial inhibition of DNA replication observed after exposure to DFO may be due to a lower efficiency of metal chelation and/or that it does not inhibit the G(1)/S transition but arrests cells in late S phase. The effects of both TRX and DFO on DNA synthesis and mitotic index were reversible after removing the chelators from the culture medium. An apoptotic effect of TRX was strongly suggested by analysis of DNA content by flow cytometry, nuclear fragmentation and DNA degradation in oligonucleosomes and confirmed by the induction of a high level of caspase 3-like activity. TRX induced apoptosis in a dose- and time-dependent manner in proliferating HepG2 cells. In HBG cells, TRX induced apoptosis in proliferating and confluent cells arrested in the G(1) phase of the cell cycle, demonstrating that inhibition of proliferation and induction of apoptosis occurred independently. DFO induced DNA alterations only at concentrations >100 microM and without induction of caspase 3-like activity, indicating that DFO is not a strong inducer of apoptosis. Addition of Fe or Zn to the culture medium during TRX treatment led to a complete restoration of proliferation rate and inhibition of apoptosis, demonstrating that Fe/Zn-saturated TRX was not toxic in the absence of metal depletion. These data show that TRX, at concentrations of 20-50 microM, strongly inhibits cell proliferation and induces apoptosis in proliferating and non-proliferating HepG2 and HBG cells, respectively.