Correlation of MicroRNA-16, MicroRNA-21 and MicroRNA-101 Expression with Cyclooxygenase-2 Expression and Angiogenic Factors in Cirrhotic and Noncirrhotic Human Hepatocellular Carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression. METHODS: Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue. RESULTS: COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma. CONCLUSIONS: In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels

Use of selective serotonin reuptake inhibitors is associated with very low plasma free serotonin concentrations in humans

Background: Selective serotonin reuptake inhibitors (SSRIs) block the serotonin transporter on neurons, but also on platelets, thus decreasing platelet serotonin concentrations in users of SSRIs. Data on plasma free serotonin concentrations in SSRI users is lacking, while plasma free serotonin is available for receptor binding and plays a role in several pathophysiological processes We therefore measured the plasma free and platelet serotonin concentrations in users of SSRIs and age-matched healthy controls, and we analyzed plasma concentrations of the serotonin precursor tryptophan and serotonin metabolite 5-hydroxyindoleamineacetic acid (5-HIAA). Methods: For this cross-sectional single center case control study, participants were recruited at the departments of Psychiatry and General Medicine. High performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) was used to measure plasma free and platelet serotonin, plasma tryptophan and 5-HIAA concentrations. Pre-analytical conditions were optimized by careful blood collection, rapid sample handling, high speed centrifugation, drug and diet restrictions, and age-matched controls. Results: In 64 SSRI users, median concentrations of plasma free and platelet serotonin were 10-fold and 14-fold lower, respectively, than in 64 matched controls. Patients using higher dose SSRIs or those with higher affinity for the serotonin transporter had lower plasma free and platelet serotonin concentrations. Compared to controls, SSRI users had similar median plasma tryptophan concentrations, but slightly higher plasma 5-HIAA concentrations. Conclusion: SSRI users have low platelet serotonin and low plasma free serotonin. This could not be explained by lower concentrations of its precursor tryptophan, and only partially by increased breakdown to 5-HIAA