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Serum microRNA-21 as marker for necroinflammation in hepatitis C patients with and without hepatocellular carcinoma
(2011)
- Background: MicroRNA-21 (miR-21) is up-regulated in tumor tissue of patients with malignant diseases, including hepatocellular carcinoma (HCC). Elevated concentrations of miR-21 have also been found in sera or plasma from patients with malignancies, rendering it an interesting candidate as serum/plasma marker for malignancies. Here we correlated serum miR-21 levels with clinical parameters in patients with different stages of chronic hepatitis C virus infection (CHC) and CHC-associated HCC. Methodology/Principal Findings: 62 CHC patients, 29 patients with CHC and HCC and 19 healthy controls were prospectively enrolled. RNA was extracted from the sera and miR-21 as well as miR-16 levels were analyzed by quantitative real-time PCR; miR-21 levels (normalized by miR-16) were correlated with standard liver parameters, histological grading and staging of CHC. The data show that serum levels of miR-21 were elevated in patients with CHC compared to healthy controls (P<0.001); there was no difference between serum miR-21 in patients with CHC and CHC-associated HCC. Serum miR-21 levels correlated with histological activity index (HAI) in the liver (r = −0.494, P = 0.00002), alanine aminotransferase (ALT) (r = −0.309, P = 0.007), aspartate aminotransferase (r = −0.495, P = 0.000007), bilirubin (r = −0.362, P = 0.002), international normalized ratio (r = −0.338, P = 0.034) and γ-glutamyltransferase (r = −0.244, P = 0.034). Multivariate analysis revealed that ALT and miR-21 serum levels were independently associated with HAI. At a cut-off dCT of 1.96, miR-21 discriminated between minimal and mild-severe necroinflammation (AUC = 0.758) with a sensitivity of 53.3% and a specificity of 95.2%. Conclusions/Significance: The serum miR-21 level is a marker for necroinflammatory activity, but does not differ between patients with HCV and HCV-induced HCC.
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N-terminus of hMLH1 confers interaction of hMutL{alpha} and hMutL{beta} with hMutS{alpha}
(2003)
- Mismatch repair is a highly conserved system that ensures replication fidelity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutS{alpha} (hMSH2-hMSH6) and hMutL{alpha} (hMLH1-hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutS{alpha} recruits hMutL{alpha}, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutL{alpha} as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutS{alpha} using a novel interaction assay. We show that, although the interaction requires ATP, hMutL{alpha} does not need to hydrolyse this nucleotide to join hMutS{alpha} on DNA, suggesting that ATP hydrolysis by hMutL{alpha} happens downstream of complex formation. The analysis of the individual subunits of hMutL{alpha} demonstrated that the hMutS{alpha}–hMutL{alpha} interaction is predominantly conferred by hMLH1. Further experiments revealed that only the N-terminus of hMLH1 confers this interaction. In contrast, only the C-terminus stabilised and co-immunoprecipitated hPMS2 when both proteins were co-expressed in 293T cells, indicating that dimerisation and stabilisation are mediated by the C-terminal part of hMLH1. We also examined another human homologue of bacterial MutL, hMutL{beta} (hMLH1–hPMS1). We show that hMutL{beta} interacts as efficiently with hMutS{alpha} as hMutL{alpha}, and that it predominantly binds to hMutS{alpha} via hMLH1 as well.
